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ViroSeq™

HIV-1 Genotyping System v2.0


For Use with the Applied Biosystems ® 3130/3130xl Genetic Analyzers and
ViroSeq® HIV-1 Genotyping System Software v2.8

Instructions For Use

0197
4J94-58

CDx 5002961 Rev. A

Celera Corporation
1401 Harbor Bay Parkway DRAFT
ABBOTT
Max-Planck-Ring 2
Alameda, CA 94502 September 16,65205
2009 11:45 am,
Wiesbaden
USA *+H3764J94580.* ViroSeq IFUGermany
3130 Title.fm
+ 49-6122-580
DRAFT
September 16, 2009 11:45 am, ViroSeq IFU 3130 Title.fm
Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Indications for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Summary and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Principles of the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
To Reorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Key to Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Sample Preparation and Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2


ViroSeq Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Warnings and Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Storage and Handling Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Required Materials Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Work Area Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Preventing RNA Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Run Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sequencing Runs on the Applied Biosystems® 3130 and 3130xl


Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

ViroSeq® HIV-1 Genotyping System Software v2.8 Analysis. . . . . . . . . 27


Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Computer Requirements for Running ViroSeq® Software v2.8 . . . . . . . . . . . . . . . . . . . . 27

ViroSeq™ HIV-1 Genotyping System v2.0 i


Installing, Uninstalling and Using the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Editing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Antiretroviral Drug Resistance Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Mutations Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Software Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
ViroSeq® Software and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Maintenance and Calibration of the Applied Biosystems® 3130 and


3130xl Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . 54
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Care of the Applied Biosystems® 3130 and 3130xl Genetic Analyzers . . . . . . . . . . . . . . 54
Capillary Array. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Flushing and Filling the Water Trap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Water Wash. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Polymer Block. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Spatial and Spectral Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Appendix A: Auditing and Software Access Control . . . . . . . . . . . . . . . 66


AB Navigator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Enabling Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

ViroSeq™ HIV-1 Genotyping System v2.0 ii


Section 1: Introduction Principles of the Procedure
Operator training is critical to performing the ViroSeq HIV-1 Genotyping assay
and achieving accurate results.
The ViroSeq™ HIV-1 Genotyping System is based on six major processes:
• Sample Preparation
Intended Use • Reverse Transcription (RT)
• Polymerase Chain Reaction (PCR)
The ViroSeq™ HIV-1 Genotyping System is intended for use in detecting HIV • Cycle Sequencing
genomic mutations that confer resistance to specific types of antiretroviral drugs, as • Automated Sequence Detection
an aid in monitoring and treating HIV infection. Specifically, the ViroSeq HIV-1
Genotyping System can be used to: • Software Analysis
• Detect HIV-1 Subtype B viral resistance in plasma samples collected in EDTA
with a viral load ranging from 2,000 to 750,000 copies/mL
• Genotype the entire HIV-1 protease gene from codons 1 to 99 and two-thirds of
To Reorder
the reverse transcriptase (RT) gene from codons 1 to 335
In order to use the ViroSeq HIV-1 Genotyping System: 4J94-58 ViroSeq™ HIV-1 Genotyping System v2.0 For Use with
the Applied Biosystems ® 3130/3130xl Genetic Analyzers
• The user (operator or technologist) must be trained in its use. and ViroSeq® HIV-1 Genotyping System Software v2.8
• Interpretation and application of the results should be done by a qualified
physician. Instructions for Use
The kit is not to be used as a screening test for HIV or as a diagnostic test to confirm
the presence of HIV infection.
Key to Symbols
Indications for Use
CDx = Celera Catalog Number = Biological Risks
Includes the following populations:
• HIV-1 infected individuals at drug therapy failure (with increased viral load)
before therapy switch Applied Biosystems
• HIV-1 infected individuals at initial presentation, before initial drug therapy ABI = Catalog Number = Temperature Limitation

Summary and Explanation = Contains 48 assays = Consult Instructions For


Use
Human Immunodeficiency Virus type 1 (HIV-1) is the etiologic agent responsible
for the Acquired Immunodeficiency Syndrome (AIDS) pandemic.1,2 Treatment of
HIV-1 infections with potent antiretroviral therapy can result in suppression of Authorized
Electrical Shock/Fire
= = Representative in the
HIV-1 replication.3,4 However, the eradication of HIV-1 is problematic since latent Hazard
European Community
infections provide a mechanism for lifelong persistence and re-emergence of the
virus.5,6 Frequently, treatment failures occur as a result of poor adherence to Xn
In Vitro Diagnostic
treatment regimens and/or the emergence of virus strains resistant to drugs.7 These = Harmful = Medical Device
HIV-1 mutant strains can be resistant to one or more drugs in each of the six classes
of antiretroviral drugs — nucleoside reverse transcriptase inhibitors, non-nucleoside
reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, entry
inhibitors, and HIV integrase strand transfer inhibitors.8,9,10 In addition, resistance to = Toxic = Manufacturer
a given drug can generate cross-resistance to other drugs of the same class.7,11
However, treatment failure does not always result in resistance to all drugs in a
regimen.12 Therefore, determining the best salvage therapy for patients failing
multiple regimens is a formidable challenge.
= Laser Hazard = Infection Risk
A key factor in identifying new treatment strategies is knowledge of the viral
resistance genotype as indicated by mutations present in the viral swarm in patient
plasma.13 Retrospective and prospective intervention-based studies have provided
evidence supporting the clinical utility of resistance testing.14-16 These studies
indicate that the presence of drug resistance is an independent risk factor for = Light Sensitive
treatment failure. Currently, resistance testing is recommended for persons with HIV
infections when they enter into care, regardless of whether therapy is immediately
initiated. Repeat testing at the initiation of antiretroviral therapy should be
considered. HIV drug resistance testing should be performed when changing
antiretroviral regimens following virologic failure. Resistance testing is also
recommended for pregnant women prior to the initiation of antiretroviral therapy
and for women who become pregnant while on therapy.17,18
The ViroSeq™ HIV-1 Genotyping System detects mutations in the RT and protease
regions of the pol gene and provides a report indicating genetic evidence of viral
resistance. It is a complete system that provides reagents for viral RNA isolation
from plasma, RT-PCR, and sequencing.19,20 The entire protease gene and two-thirds
of the RT gene are amplified to generate a 1.8 kb amplicon. The amplicon is used as
a sequencing template for seven primers that generate an approximately 1.3 kb
consensus sequence. The ViroSeq® HIV-1 Genotyping System Software
assembles, edits, and identifies mutations within this 1.3 kb sequence. The software
compares the consensus sequence with a known reference, HXB-2, to determine
mutations present in the sample. Finally, the ViroSeq software uses a proprietary
algorithm to analyze the mutations and generate a drug resistance report.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 1 of 68


Section 2: Sample ViroSeq™ HIV-1 RT-PCR Module
(continued)
Cap Color Quantity

HIV PCR Mix, tube Blue 1 x 1.42 mL


Preparation and <0.1% dATP, dCTP, dGTP, dTTP, dUTP
<0.1% non-infectious synthetic
oligonucleotide HIV-1 primers

Processing AmpliTaq Gold® DNA Polymerase, tube


5 U/L AmpliTaq Gold® DNA Polymerase
Gold 1 x 24 µL

AmpErase® UNG, tube Green 1 x 48 µL


1 U/L Uracil N-glycosylase
DTT (100 mM), tube Yellow 1 x 20 µL

ViroSeq Assay Kit 1.4% Dithiothreitol
 = 48
48 DNA Mass Ladder, tube Clear 1 x 36 µL
<0.1% DNA mass ladder
Item
Agarose Gel Loading Buffer, tube Clear 1 x 240 µL
RNA Diluent, tube Clear 1 x 1.6 mL
ViroSeq™ HIV-1 Genotyping System v2.0, 4J94-93
with:
• ViroSeq™ HIV-1 Sample Preparation
Module ViroSeq™ HIV-1 Sequencing Module Cap Color Quantity
• ViroSeq™ HIV-1 RT-PCR Module
• ViroSeq™ HIV-1 Sequencing Module
• ViroSeq™ HIV-1 8E5 Control Module

ViroSeq™ HIV-1 Sample Preparation Module Cap Color Quantity HIV SEQ Mix A, tube White 1 x 576 µL
BigDye® Terminator Ready Reaction Mix
HIV Viral Lysis Buffer, tube Clear 2 x 14.4 mL <0.1% non-infectious synthetic
43% Guanidine Thiocyanate oligonucleotide HIV-1 primers and
<2% Dithiothreitol nucleotides
<1% N-Lauroylsarcosine <0.1% AmpliTaq® DNA Polymerase, FS
<0.1% Magnesium Chloride
Contains Contact with Acids
Guanidine or Bleach releases a HIV SEQ Mix B, tube White 1 x 576 µL
Thiocyanate toxic gas.
BigDye® Terminator Ready Reaction Mix
<0.1% non-infectious synthetic
R 20/21/22 Harmful by inhalation, in contact with oligonucleotide HIV-1 primers and
skin and if swallowed. nucleotides
R 32 Contact with acids liberates very toxic gas. <0.1% AmpliTaq® DNA Polymerase, FS
R 36/38 Irritating to eyes and skin. <0.1% Magnesium Chloride
S 26 In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice. HIV SEQ Mix C, tube White 1 x 576 µL
S 35 This material and its container must be BigDye® Terminator Ready Reaction Mix
disposed of in a safe way. <0.1% non-infectious synthetic
oligonucleotide HIV-1 primers and
S 36/37/39 Wear suitable protective clothing, nucleotides
gloves and eye/face protection.
<0.1% AmpliTaq® DNA Polymerase, FS
S 46 If swallowed, seek medical advice <0.1% Magnesium Chloride
immediately and show this container or label.
RNA Diluent, tube Clear 3 x 1.6 mL HIV SEQ Mix D, tube White 1 x 576 µL
BigDye® Terminator Ready Reaction Mix
<0.1% non-infectious synthetic
oligonucleotide HIV-1 primers and
ViroSeq™ HIV-1 RT-PCR Module Cap Color Quantity
nucleotides
<0.1% AmpliTaq® DNA Polymerase, FS
<0.1% Magnesium Chloride
HIV RT Mix, tube Blue 1 x 384 µL
<0.1% dATP, dCTP, dGTP, dTTP HIV SEQ Mix F, tube Red 1 x 576 µL
<0.1% non-infectious synthetic
oligonucleotide HIV-1 primers BigDye® Terminator Ready Reaction Mix
<0.1% non-infectious synthetic
RNase Inhibitor, tube White 1 x 48 µL oligonucleotide HIV-1 primers and
20 U/L RNase Inhibitor nucleotides
<0.1% AmpliTaq® DNA Polymerase, FS
MuLV Reverse Transcriptase, tube Purple 1 x 48 µL <0.1% Magnesium Chloride
50 U/L Recombinant Murine Leukemia
Virus Reverse Transcriptase

Page 2 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


ViroSeq™ HIV-1 Sequencing Module Cap Color Quantity
Warnings and Precautions
(continued)
1. This test is for use with human plasma collected with EDTA anticoagulant
HIV SEQ Mix G, tube Red 1 x 576 µL only. Do not use plasma collected in heparin with this procedure as it has been
BigDye® Terminator Ready Reaction Mix shown to inhibit PCR.
<0.1% non-infectious synthetic 2. Do not pipette by mouth.
oligonucleotide HIV-1 primers and
nucleotides 3. Do not eat, drink, or smoke in laboratory work areas. Wear appropriate
personal protective equipment when handling specimens and kit reagents
<0.1% AmpliTaq® DNA Polymerase, FS (e.g., safety glasses, gloves, and protective clothing). Wash hands thoroughly
<0.1% Magnesium Chloride after handling specimens and kit reagents.
HIV SEQ Mix H, tube Red 1 x 576 µL 4. Avoid microbial and ribonuclease contamination of reagents when removing
aliquots from reagent bottles. We recommend the use of sterile disposable
BigDye® Terminator Ready Reaction Mix pipettes and pipette tips.
<0.1% non-infectious synthetic
oligonucleotide HIV-1 primers and 5. Do not pool reagents from different lots or from different bottles of the same
nucleotides lot.
<0.1% AmpliTaq® DNA Polymerase, FS 6. Do not use this kit after its expiration date.
<0.1% Magnesium Chloride 7. Minimize exposure to chemicals. Exposure includes contact, ingestion, or
inhalation of chemicals. Do not leave chemical containers open. Use only
with adequate ventilation.
ViroSeq™ HIV-1 8E5 Control Module 8. Dispose of unused reagents and waste in accordance with good laboratory
Cap Color Quantity
practices and local, state/provincial, or national environmental and health
regulations.

POTENTIAL BIOHAZARD.
Human source material. Use
Universal Precautions. 9. LASER HAZARD. Exposure to direct or reflected laser light at
40 mW for 0.1 seconds can burn the retina and leave permanent blind spots.
HIV-1 8E5 Positive Control, tube Red 5 x 500 µL Never look directly into the laser beam or allow a reflection of the beam to
enter your eyes. Follow the manufacturer’s recommendations for appropriate
The Positive Control was prepared by diluting protective eye-wear and clothing when using the Applied Biosystems ® 3130
cultured HIV-1 type B virus (8E5) in HIV RNA- or Applied Biosystems ® 3130xl Genetic Analyzers.
negative human plasma, non-reactive by US
FDA-licensed tests for antibody to HIV-1/2, HCV,
HTLV-I, and HBsAg. The 8E5 virus contains an
intact but defective viral genome that contains a 10. BIOLOGICAL RISKS. Biological samples such as tissues and
single base insertion at codon 219 of the RT gene.21 blood have the potential to transmit infectious diseases. Handle all specimens
Viral load is 50,000 to 100,000 copies/mL. as if they are infectious. Use safe laboratory procedures such as those outlined
in Biosafety in Microbiological and Biomedical Laboratories22 and in the
HIV-1 8E5 Negative Control, tube Clear 5 x 950 µL NCCLS Document M29-T.23
The Negative Control contains normal human
plasma tested to be free of HIV RNA, non-
reactive by US FDA-licensed tests for antibody to
HIV-1/2, HCV, HTLV-I, and HBsAg.
11. CAUTION: The controls contain human sourced and/or
potentially infectious components. Components sourced from human blood
have been tested and found to be nonreactive for HBsAg, anti-HCV, anti-
HIV-1/HIV-2 and HTLV-I by FDA licensed tests. No known test method can
offer complete assurance that products derived from human sources will not
transmit infection. Therefore, all human sourced materials should be
considered potentially infectious. The HIV-1 particles contained in the
positive control are defective, however, studies have shown a limited
capability to revert to an infectious form. This may need to be taken into
consideration in the event of a significant accidental exposure incident.
12. Components containing sodium azide may react with lead and copper
plumbing to form highly explosive metal azides. When disposing solutions
that contain sodium azide down laboratory sinks, flush the drains with large
volumes of water to prevent azide buildup.
13. Thoroughly clean and disinfect all work surfaces with a freshly prepared
solution of 0.5% sodium hypochlorite in deionized water. Do NOT use bleach
(0.5% sodium hypochlorite) with glassware or surfaces which have been
exposed to HIV Viral Lysis Buffer.
14. Information for European customers: for product not classified as dangerous
per European Directive 1999/45/EC  Safety data sheet available for
professional user on request. For those materials not provided by Celera, refer
to the manufacturer’s Safety Data Sheet for additional information.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 3 of 68


Storage and Handling Requirements C. Specimen Storage
Store plasma samples at –65 to –80°C. Do not store for greater than 6 months. Do
not freeze-thaw the plasma more than two times.
Note: When reagent storage recommendations are observed, both unopened and
open reagent tubes are stable until the expiration date. Do not use this kit or any of
its reagent components after the expiration date.
Required Materials Not Provided
ViroSeq Assay Kit
Item Storage Conditions
Module Storage Conditions Comments
PCR Cleanup Kit 4J94-73 < -15°C

ViroSeq HIV-1 Sample Store at Once opened, the following Sequencing Consumables Kit 4J94-92 15°C to 30°C
Preparation Module • –15 to –25°C components should be
• In a manual defrost freezer transferred to and stored in the
ViroSeq™ HIV-1 RT-PCR that is designated amplicon- amplified DNA area at 2 to 8°C:
Module free • DNA Mass Ladder
• Agarose Gel Loading
Buffer
Item ABI
Note: All other components
should be stored at –15 to –25°C
in the amplicon-free area. Applied Biosystems ® 3130xl Genetic Analyzer with: 3130XL
• Data Collection Software v.3.0 IMPORTANT! No software
• Firmware version 6286200-02 or 6286250-02 should be downloaded from the
ViroSeq™ HIV-1 8E5 Control Store at • Five vials each of • PC computer with: internet for this intended use.
Module • –15 to –25°C positive and negative – Microsoft® Windows ® XP Professional edition,
• In a manual defrost freezer plasma control. Service Pack 2
that is designated amplicon- • Thaw one vial each of – 2 GHz Intel® Pentium® 4 processor
free the positive and negative – 1 GB RAM
plasma control. – Dual 36 GB hard drives
• Use with each sample
run.
• Do not refreeze. Applied Biosystems ® 3130 Genetic Analyzer with: 3130
• Data Collection Software v.3.0 IMPORTANT! No software
• Firmware version 6278250-02 should be downloaded from the
ViroSeq™ HIV-1 Sequencing Store in the Post-Amplification These materials are • PC computer with: internet for this intended use.
Module area at: light sensitive. – Microsoft® Windows® XP Professional edition,
• –15 to –25°C Avoid prolonged Service Pack 2
• In a manual defrost freezer exposure to light. – 2 GHz Intel® Pentium® 4 processor
that is designated for – 1 GB RAM
amplified DNA only – Dual 36 GB hard drives

Applied Biosystems ® 3130xl Capillary Array, 50 cm 4315930

Specimen Applied Biosystems ® 3130 Capillary Array, 50 cm 4333466

Lower Polymer Block Cleaning Kit 4359572

A. Specimen Collection Sequencing Analysis Software v5.3.1 with


KB™ Basecaller v1.4
4360967
To obtain additional licenses, please
contact your Applied Biosystems
The ViroSeq HIV-1 Genotyping System is intended for use with plasma samples representative.
only. Collect 5 mL of whole blood in a sterile EDTA anticoagulant tube IMPORTANT! Do not download
(Vacutainer™ PPT™ Brand tubes, Becton-Dickinson #362788 or equivalent) and software from the internet for this
immediately invert the tube 8 to 10 times to mix. intended use.

IMPORTANT! GeneAmp® PCR System 9600 Thermal Cycler N801-0001


• This assay has been validated only for use with patient samples collected in or or
GeneAmp® PCR System 9700 Thermal Cycler N805-0001
EDTA.
• Specimens collected with heparin are not suitable for this assay.
MicroAmp® 96-Well Tray (to hold Reaction Tube with N801-0541
• Plasma specimens containing the following have been shown to not interfere attached cap)
with test results:
– Lipids up to 30 mg/mL MicroAmp® 96-Well Support Base N801-0531
– Bilirubin up to 0.6 mg/mL
– Hemoglobin up to 5 mg/mL Reservoir Septa 4315932
We recommend that plasma be separated from whole blood within 30 minutes of
collection, but no later than 120 minutes after collection, if using PPT tubes or their 96-Well Plate Base 4317237
equivalent.
96-Well Plate Retainer 4317241
• Centrifuge the tubes at 1,000 to 2,000 x g at room temperature (15 to 25°C) for
15 minutes.
96-Well Plate Septa 4315933
• As soon as possible, transfer the plasma from the PPT tubes (or equivalent) to
sterile, 1.5 mL polypropylene tubes and store them at –65 to –80°C until used.
MicroAmp® Optical 96-Well Reaction Plate N801-0560

B. Specimen Transport POP-6™ Polymer for 3130/3130xl Genetic Analyzers 4363783 (3500 µL) or
4352757 (7000 µL)

The transportation of human whole blood products, including plasma, must comply Running Buffer, 10X 402824
with country and local regulations for the transport of etiological agents.
You may ship the tubes with the plasma at 2 to 8°C for delivery within 24 hours, or
ship the tubes with the plasma at –70°C or less on dry ice.

Page 4 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Item ABI
Work Area Setup
Different parts of the ViroSeq HIV-1 Genotyping System procedure should be
Hi-Di™ Formamide, 25 mL 4311320
performed in different work areas. Each work area must have dedicated pipettors,
equipment, and supplies in order for the assay to be routinely successful. There are
three suggested work areas:
Contains Formamide. • Area 1: Sample Preparation (Pre-Amplification) — used for performing Sample
Preparation and working with HIV-1 samples.
R 61 May cause harm to the unborn child.
R 36/38 Irritating to eyes and skin.
• Area 2: Pre-Amplification — used for performing the Reverse Transcription and
S 53 Avoid exposure - Obtain special instructions before use.
PCR setup steps.
S 24/25 Avoid contact with skin and eyes. • Area 3: Post-Amplification (Amplified DNA) — dedicated to PCR
S 35 This material and its container must be disposed of in a amplification, Cycle Sequencing and Automated Sequence Detection, and other
safe way. activities that require the handling of amplified DNA.
S 36/37/39 Wear suitable protective clothing, gloves and
eye/face protection. Ideally, Areas 1 and 2 should be separate from each other to prevent the potential
S 45 In case of accident or if you feel unwell, seek medical transfer of exogenous RNA and DNA into the RT and PCR setup work area.
advice immediately (show the label where possible). However, if Areas 1 and 2 are located in the same room, they must be clearly
delineated. Benchtop biological safety cabinets may serve to isolate the areas in this
BigDye® Terminator v1.1 Sequencing Standard 4336791 room.
The pipettors and other equipment used in Area 1 are routinely exposed to
bloodborne human pathogens and should not be used in any other area outside of
Area 1.
Materials Not Provided When possible, use a dedicated space such as a biological safety cabinet with a UV
The user must supply the following equipment, materials, and reagents. source for the reagent setup of the RT and PCR steps. The UV germicidal lamps in
most biological safety cabinets damage DNA left on exposed surfaces, making it
(Use according to Manufacturer’s instructions unless otherwise specified.) unsuitable for subsequent amplification.
a. Aerosol-resistant tips, RNase-free, 10 to 1,000 µL Maintain strict physical isolation between the amplified DNA workspace (Area 3)
b. Agarose gel, containing ethidium bromide and the other two areas to avoid the transfer of amplified DNA out of Area 3.
c. Agarose gel electrophoresis system (gel box with power supply capable of Because of the type of equipment used in the amplified DNA workspace (Area 3), a
10 V/cm of agarose gel) relatively large space is required. This space requirement generally exceeds the
d. Aluminum foil tape space requirement for Areas 1 and 2.
e. BioSafety hood, laminar flow, clean hood
f. Bleach (for cleaning – make a 10% solution prior to use)
g. Centrifuge, microtube quick-spin (2,000 x g) Preventing RNA Degradation
h. Centrifuge, refrigerated (21,000 to 25,000 x g), with hermetically-sealed rotor For genotyping success, it is critical that you prevent RNA degradation in your test
for 1.5 mL tubes samples.
i. Centrifuge with microtiter plate rotor
RNA is degraded by RNases. These enzymes occur naturally in cells and are
j. Centrifuge with vacuum pump (if using CENTRI•SEP 96™ Well Plates) must liberated during cell lysis. RNases can remain in samples derived from cells if
have: purification is not complete. Also, because RNases are secreted by your skin,
– Capacity to create a vacuum RNases can be introduced into samples by contact with surfaces that you have
touched. You should wear gloves at all times when handling specimens, reagents,
– Swinging-bucket rotor equipment, and disposables.
k. CENTRI•SEP 96™ Well Plates RNases are very stable and do not need any cofactors, so they linger on surfaces and
l. EDTA, 0.5 M, pH 8.0 (Molecular Biology grade) remain functional under a wide range of environmental conditions. They have a high
m. Ethanol, 70% (for cleaning) activity, so only a small amount of RNase contamination can cause significant loss
in a sample of RNA. Sources of RNase contamination include:
n. Ethanol, 100%, non-denatured (Molecular Biology grade)
o. Ethidium bromide • General laboratory glassware and plasticware
p. Gloves, powder-free • Skin and hair
q. Isopropanol 100%, anhydrous (ACS grade) • Contaminated solutions
r. Lab coat/gowns
s. Lint-free tissue
t. Pipettors, capable of pipetting from 0 to 1,000 L
Workflow
u. Screwtop tubes, 1.5 mL (sterile, RNase/DNase-free), must withstand 21,000 You may complete the procedure over a period of two or more days. When stopping
to 25,000 x g at the specified points, you must store the prepared samples as specified in the table
v. Sodium Acetate, 3.0 M, pH 5.2 below until you are ready to continue.
w. TBE buffer, 10X IMPORTANT! Keep all samples, controls, and reagents cold (2 to 8°C) prior to
x. Transfer pipets, fine-tip making the mastermix. Once the mastermix is made, store it at room temperature,
y. Ultraviolet light box (300 nM UV lamp) but keep the original reagents on ice.
z. Vortex mixer, platform head (2500 rpm)
.

At the completion of. . . Store the samples at. . . For no longer than. . .
aa. Water, sterile, deionized, RNase/DNase-free
ab. Freezer (manual defrost), capable of achieving storage conditions cited A. Sample Preparation –65 to –80°C 2 weeks
throughout the product labeling
B. Reverse Transcription –15 to –25°C 2 weeks
ac. Refrigerator, capable of achieving storage conditions cited throughout the
product labeling C. PCR –15 to –25°C 2 weeks
D. Cycle Sequencing –15 to –25°C 3 days
E. Purified Sequences –15 to –25°C 1 week

ViroSeqTM HIV-1 Genotyping System v2.0 Page 5 of 68


Run Size 6. Insert the samples in the rotor with the orientation mark facing the outside
rim of the rotor.
The ViroSeq HIV-1 Genotyping System Kit contains enough reagents to perform
48 tests. We recommend that individual runs be performed in batches of 12 samples, Note: Use a hermetically sealed rotor with an O-ring to prevent splashing
which allows four runs per kit. Runs with fewer samples increase the number of and aerosols.
freeze-thaw cycles and reagent manipulations, and may result in decreased
performance or contamination. 7. Centrifuge at 21,000 to 25,000 x g for 60 min. at 2 to 8°C.
IMPORTANT! Do not freeze-thaw the reagents more than five (5) times.
8. While the samples are centrifuging:
• Thaw the Viral Lysis Buffer at room temperature.
Controls Contains Contact with Acids
Note: A positive and a negative control must be included in every run. Guanidine or Bleach releases a
Thiocyanate toxic gas.
The positive (8E5) control is a non-infectious24 HIV-1 virus at a concentration of
50,000 to 100,000 copies per mL. A 1:10 dilution of the positive control (5,000 to R 20/21/22 Harmful by inhalation, in contact with skin and if
10,000 copies/mL) must be used with every run to ensure the appropriate swallowed.
performance of the reagents. It is designed to mimic patient samples throughout all R 32 Contact with acids liberates very toxic gas.
aspects of the ViroSeq System testing procedure, from Section A, Sample
Preparation, through Section 4, ViroSeq® HIV-1 Genotyping System Software v2.8 R 36/38 Irritating to eyes and skin.
Analysis. S 26 In case of contact with eyes, rinse immediately with plenty of
water and seek medical advice.
The negative control is normal human plasma that has been tested to be free of
HIV-1 RNA. It must be used from Section A, Sample Preparation, through Section S 35 This material and its container must be disposed of in a safe way.
D1, To Quantify and Purify the PCR Products, to ensure that no contamination is S 36/37/39 Wear suitable protective clothing, gloves and eye/face
present. Use of a negative control is not necessary when performing the cycle protection.
sequencing procedure. S 46 If swallowed, seek medical advice immediately and show this
container or label.
Note: If precipitate is observed, heat the Viral Lysis Buffer to 37°C to
Protocol dissolve the precipitate, then cool to room temperature before use.

• Thaw the RNA Diluent and store it at 2 to 8°C.


A. Sample Preparation • Prepare the 70% ethanol, and store it at 2 to 8°C for use in step 20.
Note: Use Molecular Biology grade, 100% ethanol and RNase/DNase-free
Note: Time to complete 12 samples is a total of 2.5 to 3.0 hours; hands-on time is water only.
1.5 hours.
IMPORTANT! Perform this procedure in Pre-Amplification Area 1. CHEMICAL HAZARD. Ethanol is a flammable liquid and
vapor. Exposure may cause eye, skin, and upper respiratory tract
A1. To Set Up irritation. Prolonged or repeated contact may dry the skin. Exposure
may cause central nervous system depression and liver damage. Keep
away from heat, sparks, and flame. Wear appropriate protective
1. Precool a refrigerated centrifuge and rotor to 2 to 8°C, according to the eyewear, clothing, and gloves. Refer to the manufacturer’s Safety
manufacturer’s instructions. Data Sheet for additional information.

2. Thaw plasma samples and one tube each of the positive and negative 9. Remove the tubes immediately after the rotor stops moving.
controls at room temperature (15 to 25°C).
10. Carefully remove the supernatant from the tubes, using a fine-tip transfer
3. Keep only one reagent or sample tube open at a time. pipet. Do not disturb the pellet (which may not be visible).
TIP: When aspirating, start with the tip of the transfer pipet just below the
meniscus line and move the tip down, along the wall opposite the orientation
A2. To Isolate the HIV-1 Viral RNA mark, as you aspirate. Remove as much supernatant as possible without
disturbing the pellet.
1. Vortex the plasma samples 3 to 5 seconds to mix.
11. Add 600 L of Viral Lysis Buffer to each pellet.
2. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of
the tube. 12. Vortex 3 to 5 seconds to mix.

3. Label tubes according to your laboratory protocol, then aliquot 0.5 mL of the 13. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of
plasma into a fresh, RNase- and DNase-free screw-top tube. the tube.

4. Prepare a low viral RNA control. 14. Let the samples sit at room temperature (15 to 25°C) for 10 minutes to
Combine in a microfuge tube: ensure complete lysis of the virus.
– 50 L HIV-1 8E5 Positive Control
– 450 L HIV-1 8E5 Negative Control (negative plasma)
and label appropriately.

5. Place an orientation mark on the side of the tube and cap.

Page 6 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


15. Add 600 L of room temperature (15 to 25°C) isopropanol to each sample. 30. When the procedure is complete:
• Proceed to the Reverse Transcription steps, or
• Stop and store the samples in a –65 to –80°C freezer.
CHEMICAL HAZARD. Isopropanol is a flammable liquid IMPORTANT! Do not freeze-thaw the RNA more than two (2) times. Do
and vapor. Exposure may cause eye, skin, and upper respiratory tract not store the RNA for more than two weeks.
irritation. Prolonged or repeated contact may dry skin and cause
irritation. Exposure may cause central nervous system effects such as
drowsiness, dizziness, and headache. Wear appropriate protective
eyewear, clothing, and gloves. Refer to the manufacturer’s Safety B. Reverse Transcription
Data Sheet for additional information.
IMPORTANT! Perform this procedure in Pre-Amplification Area 2.
16. Vortex each tube for 3 to 5 seconds to mix.
B1. To Set Up
17. Insert the samples in the rotor with the orientation mark facing the outside
rim of the rotor.
1. If necessary, thaw the RNA samples. Be sure to also thaw the positive and
negative control RNAs.
18. Centrifuge the samples 12,500 to 15,000 x g at room temperature for 15 min.
2. Vortex for 3 to 5 seconds to mix.
19. Carefully remove the supernatant with a fine-tip transfer pipet. Do not
disturb the pellet (which may not be visible).
3. Label one 0.2 mL MicroAmp® Reaction Tube for each RNA sample.
IMPORTANT! Do not discard the supernatant with bleach waste because
toxic gas will form. 4. Thaw the HIV RT Mix and DTT.
Vortex for 3 to 5 seconds to mix.
20. Add 1 mL of cold (2 to 8°C) 70% ethanol (prepared at step 8) to each tube.
5. Remove the RNase Inhibitor and MuLV Reverse Transcriptase from the
21. Vortex each tube for 3 to 5 seconds to mix.
kit.

22. Insert the tubes in the rotor with the orientation mark facing the outside rim
6. Centrifuge all the reagent tubes and samples for 1 to 2 seconds at 2,000 x g
of the rotor.
to collect the contents at the bottom.

23. Centrifuge 12,500 to 15,000 x g at room temperature for 5 min.


7. Store all reagents and samples at 2 to 8°C or on ice until use.

24. Carefully remove the supernatant with a fine-tip transfer pipet. Do not
disturb the pellet (which should now be visible). B2. To Run the RT Reactions
Remove as much ethanol as possible.
Note: Use only the GeneAmp® 9600 or 9700 thermal cycler located in
Work Area 2.
25. Centrifuge 1 to 2 seconds at 2,000 x g to collect the residual fluid at the
Note: Program the thermal cycler prior to the reaction setup (refer to step 5). Please
bottom of the tube. refer to the thermal cycler operation manual for detailed programming instructions.

26. Carefully remove any residual ethanol with another fine-tip transfer pipet. 1. Prepare the RT Mastermix according to the following table. When finished,
Air dry the tubes, with the caps off, for 1 to 5 min, or until no ethanol is return the stock solutions to –15 to –25°C.
visible.
IMPORTANT! It is critical to remove all visible ethanol. Residual ethanol Volume for 1 Volume for 5 Volume for 15
inhibits the RT-PCR reaction. Reagent
Reaction (µL) Reactions (µL) Reactions (µL)
HIV RT Mix 8 40 120
27. Resuspend each pellet in cold (2 to 8°C) RNA Diluent according to the
RNase Inhibitor 1 5 15
guidelines below.
MuLV Reverse 1 5 15
Transcriptase
DTT, 100 mM 0.4 2.0 6.0
Then add this quantity of RNA
If the viral load is. . .
Diluent. . . Final volume 10.4 52.0 156.0
>15,000 copies per mL 100 L
2,000 to 15,000 copies per mL 50 L
Note: Prepare sufficient volume for 1 to 2 extra reactions to compensate for
pipetting loss.
unknown 50 L
Controls
2. Vortex the RT Mastermix for 2 to 3 seconds to mix.
Undiluted positive 100 L
Low positive 50 L 3. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of
Negative 50 L the tube.
IMPORTANT! The RT Mastermix must be at room temperature (15 to
25°C) when you add it to the reaction tubes. Do not leave it at room
28. Vortex vigorously for 10 seconds to resuspend the pellet. Some insoluble temperature for more than 30 min.
material may remain.
4. Add 10 L of the viral RNA to 0.2 mL MicroAmp® Reaction Tubes and
29. Centrifuge 1 to 2 seconds at 2,000 x g to collect contents at the bottom of the return the stock viral RNA to ice.
tube. IMPORTANT! Use a clean tip for each addition.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 7 of 68


5. Immediately cap and place the MicroAmp® Reaction Tubes into a thermal
C2. To Perform the PCR Reaction
cycler that has been set to the conditions below, and begin the program.
Allow the samples to ramp, with the instrument, to 65°C. While the RNA is 1. Prepare the PCR Mastermix.
heating, leave the RT Mastermix at room temperature. Combine in a sterile 1.5 mL microcentrifuge tube:
Note: Pause the run after the 42°C, 5 min. step and proceed to step 6
immediately. Volume for 1 Volume for 5 Volume for 15
Reagent
Reaction (µL) Reactions (µL) Reactions (µL)
HIV PCR Mix 29.5 147.5 442.5
Temperature (°C) Time Process
AmpliTaq Gold DNA 0.5 2.5 7.5
65 30 seconds Relaxes the RNA secondary structure Polymerase
42 5 minutes Cools to the optimal enzyme activity temperature AmpErase UNG 1 5 15
Manually Pause and perform step 6, then press Resume Final volume 31 155 465
42 60 minutes Reverse transcription
99 5 minutes Inactivates MuLV Reverse Transcriptase
4 Hold Holds until you are ready to proceed
Note: Prepare sufficient volume for 1 extra reaction to compensate for
(>10 minutes) pipetting loss.

2. Vortex the PCR Mastermix for 3 to 5 seconds to mix.


Note: Set the volume on the thermal cycler to 20 L when
prompted. 3. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of
IMPORTANT! The GeneAmp® 9600 and 9700 instruments stay the tube.
paused for only 10 minutes. Step 6 must be performed within 10
minutes. 4. Add 30 L of PCR Mastermix to each RT reaction tube containing HIV-1
cDNA.
6. While the instrument is paused, perform the following: The final volume is now 50 L.
a. Immediately remove the RNA samples from the thermal cycler.
b. Add 10 L of the room temperature RT Mastermix to each reaction tube, 5. Proceed with the samples to the thermal cycler in Area 3.
and cap the tubes. Do not return to Area 2 for the remainder of the day.
c. Vortex the tray with samples for 3 to 5 seconds to mix.
d. Centrifuge the sample tray for 1 to 2 seconds at 2,000 x g to collect the
contents at the bottom. 6. Note: Use only the GeneAmp® 9600 or 9700 thermal cycler
located in Work Area 3.
7. Return the samples to the thermal cycler and press Resume. Note: Please refer to the thermal cycler operation manual for
detailed programming instructions.
Set the thermal cycler program as follows:
8. After completion of the RT program, hold the samples in the thermal cycler
for at least 10 minutes at 4°C, but do not exceed 18 hours.
Temperature
Time Cycles
(°C)
9. When the program is complete:
50 10 min 1
• Proceed to the PCR steps, or
93 12 min 1
• Stop and store the samples at –15 to –25°C until you are ready to perform
the PCR, but do not store for more than two weeks. 93 20 sec
64 45 sec 40

66 3 min
C. PCR 72 10 min 1
4 Hold —

C1. To Set Up
IMPORTANT! Perform this procedure in Pre-Amplification Area 2. Note: Set the volume on the thermal cycler to 50 L when
prompted.
1. If the samples are frozen, thaw them at room temperature (15 to 25°C).
7. Transfer the tubes to the thermal cycler, and start the program.
2. Centrifuge the sample tray or tubes for 1 to 2 seconds at 2,000 x g to collect
the contents at the bottom. 8. When the program is complete:
• Proceed to the purification steps, or
3. Thaw the HIV PCR Mix. • Stop and store the samples at –15 to –25°C.
IMPORTANT! Do not leave tubes on hold for more than 24 hours. The
4. Vortex for 3 to 5 seconds to mix. residual UNG activity may destroy your amplified DNA.

5. Remove the AmpliTaq Gold® DNA Polymerase and AmpErase® UNG


reagents from the kit. Thaw and vortex for 1 to 2 seconds.

6. Centrifuge all reagents for 1 to 2 seconds at 2,000 x g to collect the contents


at the bottom.

Page 8 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


D. Cycle Sequencing 13. Evaluate the quantity of PCR products in each sample by comparing the
intensity of each band to the intensities of the DNA Mass Ladder bands.
IMPORTANT! Perform this procedure in Post-Amplification Area 3.
Note: Do NOT dilute at this step.
D1. To Quantify and Purify the PCR Products
14. Proceed to purification of PCR products with PCR Cleanup Reagent for
samples determined to have an intensity equal to or greater than the 20 ng
To run the agarose gel of unpurified PCR product:
mass ladder band.
Note: The RT-PCR product band must have an intensity equal to or greater
1. Thaw the DNA Mass Ladder at room temperature (15 to 25°C). This will than the 20 ng mass ladder band to ensure a high quality sequence.
take at least 30 minutes.
Vortex 10 to 15 seconds to mix before using.
Enzymatic Method for PCR Product Clean-up
2. Thaw the Agarose Gel Loading Buffer at room temperature. This will take
at least 30 minutes. To purify the PCR products:
Note: If crystals form, heat the solution to 65°C for 10 minutes. Note: Use only the GeneAmp® 9700 thermal cycler.
Vortex 10 to 15 seconds to mix before using. Note: Program the thermal cycler prior to the reaction setup (refer to step 3). Please
refer to the thermal cycler operation manual for detailed programming instructions.
3. Prepare a 1% agarose gel using TBE and containing 0.5 µg/mL of ethidium
bromide (or use a purchased 1% agarose gel). 1. Carefully pipette 3 µL of PCR Cleanup Reagent into each tube that now
contains 45 µL of PCR product with a band intensity greater than or equal
to 20 ng/5 µL.
CHEMICAL HAZARD. Ethidium bromide causes eye, skin, and
respiratory tract irritation and is a known mutagen (that is, it can
change genetic material in a living cell and has the potential to cause 2. a. Cap the tubes.
cancer). Wear appropriate protective eyewear, clothing, and gloves. b. Vortex the tubes or tray with sample for 3 to 5 seconds to mix.
Refer to the manufacturer’s Safety Data Sheet for additional
information. c. Centrifuge the tubes or sample tray for 1 to 2 seconds at 2,000 x g to
collect the contents at the bottom.
The final volume is now 48 µL.
4. Prepare a 1X TBE gel buffer solution containing 0.5 µg/mL of ethidium
bromide.
3. Program the thermal cycler using the following program:
5. Place the prepared gel in the gel box.
Temperature Time Cycles
6. Add enough 1X TBE gel buffer to cover the gel. (°C)
37 15 min 1
7. For each sample, mix: 80 15 min 1
• 5 µL of Agarose Gel Loading Buffer 4 Hold —
• 5 µL of unpurified PCR product
Note: Set the reaction volume on the thermal cycler to 48 L when
8. Load the DNA Mass Ladder solution. prompted.
a. 6 µL in Lane 1
b. 3 µL in Lane 2 4. Transfer tubes or tray with samples to the thermal cycler and start the
program.
This gives the following results:
5. When the program is complete, proceed to the dilution steps.

Bands 6 µL Lane 3 µL Lane Dilution of purified PCR product for Cycle Sequencing:
Location Size (kb) (ng) (ng)

Top 2.0 100 50 1. Dilute the purified PCR product for each sample with deionized, distilled
water (ddH2O) according to the following table.
Second 1.2 60 30

0.8 40 20 If the band intensity was. . . Then. . .


Third
20 to 40 ng adjust the sample volume to 60 L.
40 to 60 ng make a 1:2 dilution of your sample with ddH2O
(1 part sample and 1 part water).
9. Load the remaining lanes with 10 µL each of the samples 60 to 100 ng make a 1:4 dilution of your sample with ddH2O
(1 part sample and 3 parts water).
IMPORTANT! Be sure to record which sample is loaded in which lane.
>100 ng make a 1:10 dilution of your sample with ddH2O
(1 part sample and 9 parts water).
10. Electrophorese at 10 V/cm until the bromphenol blue has migrated at least
5 cm into the gel.

11. Examine the gel with UV light.

12. Photograph your gel using an exposure time that does not saturate the film
and shows the differences in intensity of the mass ladder fragments.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 9 of 68


Illustration: 3. In each tube or well, combine as follows:

Volume for One


Component
Reaction (L)
Add one of the following HIV-1 SEQ mixes to each tube
or plate well:
HIV SEQ Mix A
HIV SEQ Mix B 12
HIV SEQ Mix C
HIV SEQ Mix D
HIV SEQ Mix F
HIV SEQ Mix G
HIV SEQ Mix H
Diluted, purified PCR product 8
2. Cap and vortex each diluted sample for 3 to 5 seconds to mix. Final Volume 20

3. Centrifuge 1 to 2 seconds at 2,000 x g to collect the contents at the bottom of


the tube. 4. Close the tube caps or place a MicroAmp® Optical 96-Well Reaction Plate
cover over the plate.
4. When the procedure is complete, proceed to Cycle Sequencing.
5. Centrifuge at room temperature for 5 to 10 seconds.

D2. To Set Up Cycle Sequencing 6. Transfer the samples to the thermal cycler.
Note: Use only the GeneAmp® 9600 or 9700 thermal cycler located in
Work Area 3. 7. Set the thermal cycler program as follows:
Note: Program the thermal cycler prior to the reaction setup (refer to step 7). Please
refer to the thermal cycler operation manual for detailed programming instructions. Temperature
Time Cycles
Note: Do not set up sequencing reactions for the negative control. (°C)

IMPORTANT! The sequencing mixes are light sensitive. Do not expose the mixes 96 10 sec
to light for extended periods. 50 5 sec 25

60 4 min
1. Thaw the HIV-1 sequencing mixes at room temperature (15 to 25°C).
4 Hold —

2. Set up the reaction format. You can use one of the following options.
• One MicroAmp® Reaction Tube for each HIV SEQ Mix Note: Set the reaction volume on the thermal cycler to 20 L when
• MicroAmp® 8-Tube Strip(s) in a MicroAmp® tray prompted.
• A MicroAmp® Optical 96-Well Reaction Plate IMPORTANT! Do not leave your samples on hold for more than 24 hours.
IMPORTANT! If you are using a MicroAmp® Optical 96-Well Reaction Store your samples at –15 to –25°C.
Plate, you must consider the layout of the samples as well as the instrument
platform you use. 8. Start the thermal cycler.

9. When the program is complete:


• Proceed to the purification steps, or
• Stop and store the samples at –15 to –25°C.
Do not store the samples for more than 3 days at –15 to –25°C.

D3. To Purify the Sequences


There are three methods recommended for the purification of BigDye® Terminator
A 96-well Reaction Plate loaded with sample 1 (S1) beginning in column 1 sequence reactions. Use one of the following methods.
of the 96-well plate, S2 in column 2, and so forth. Row H is not used unless
you want to run 13 samples on one plate. D3. a To Purify the Sequences With Ethanol/EDTA

If using a 3130 Genetic Analyzer for the automated sequence detection step, 1. Remove the MicroAmp® tray from the thermal cycler, and remove the caps
only 7 columns of samples may be analyzed at one time. The remaining from each tube or the cover from the plate.
columns of samples and 10 µL from each well containing the positive
control must be transferred to a second plate. Refer to To Prepare Purified 2. To each sequencing reaction, add 5 µL of 125 mM EDTA (prepared from the
Sequencing Reactions for Analysis on the 3130 Genetic Analyzer. 0.5 M EDTA).

Page 10 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


3. To each sequencing reaction, add 60 µL of 100% ethanol. 2. Add 52 L of the sodium acetate/ethanol solution to each sequencing
reaction.
CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor.
Exposure may cause eye, skin, and upper respiratory tract irritation. 3. Seal the tubes with strip caps or the plate with adhesive aluminum foil tape.
Prolonged or repeated contact may dry the skin. Exposure may cause central
nervous system depression and liver damage. Keep away from heat, sparks, 4. Mix by either inverting three times or vortexing.
and flame. Wear appropriate protective eyewear, clothing, and gloves. IMPORTANT! Mix thoroughly at this step. This is essential for efficient
Prepare enough for one reaction more than you need. Refer to the precipitation.
manufacturer’s Safety Data Sheet for additional information.
5. Centrifuge at 2,000 x g for 20 minutes.
4. Seal the tubes with strip caps or the plate with adhesive aluminum foil tape.
6. As soon as the centrifuge stops, remove the caps or foil tape without
5. Mix thoroughly by vortexing. disturbing the pellets.

6. Centrifuge the tray/plate at 2,000 x g for 20 minutes at room temperature. 7. Immediately place an absorbent paper towel or lint-free tissue on top of the
tray/plate and invert.
7. As soon as the centrifuge stops, carefully remove the caps or foil tape
without disturbing the pellets. 8. Centrifuge 150 x g for 1 minute.

8. Immediately place an absorbent paper towel or lint-free tissue on top of the 9. Add 150 L of 70% ethanol to each well.
tray/plate and invert.
10. Centrifuge at 2,000 x g for 5 minutes.
9. Place the tray or plate in the centrifuge in the inverted position, on top of a
lint-free tissue or paper towel, and centrifuge at 150 x g for 30 seconds. 11. Immediately place an absorbent paper towel or lint-free tissue on top of the
tray/plate and invert.
10. Add 150 µL of 70% ethanol to each well, then seal with caps or foil tape.
12. Centrifuge 150 x g for 1 minute.
11. Centrifuge at 2,000 x g for 5 minutes at room temperature.
13. When the centrifuge stops and drying is complete, remove the tray/plate and
12. As soon as the centrifuge stops, carefully remove the caps or foil tape seal with strip caps or with adhesive aluminum tape, and proceed to:
without disturbing the pellets. • Section 3, Sequencing Runs on the Applied Biosystems® 3130 and
3130xl Genetic Analyzers, or
13. Immediately place an absorbent paper towel or lint-free tissue on top of the • Store at –15 to –25°C in the dark. Analyze the samples within one week.
tray/plate and invert. IMPORTANT! Store in the dark. The products of sequencing reactions are
light-sensitive.
14. Place the tray or plate in the centrifuge in the inverted position, on top of a
lint-free tissue or paper towel, and centrifuge at 150 x g for 30 seconds.
D3.c To Purify With CENTRI•SEP 96™ Well Plates
15. When the centrifuge stops and drying is complete, remove the tray/plate and IMPORTANT! CENTRI•SEP 96 plates are temperature sensitive. Do not store
seal with strip caps or with adhesive aluminum foil tape, and proceed to: plates below 4°C. Plates must be at room temperature prior to use.
• Section 3, Sequencing Runs on the Applied Biosystems® 3130 and
3130xl Genetic Analyzers, or 1. Make sure the CENTRI•SEP 96 plates are at room temperature.
• Store at –15 to –25°C in the dark. Analyze the samples within one week.
IMPORTANT! Store in the dark. The products of sequencing reactions are 2. Remove the adhesive-foil sealing film from the bottom of the
light-sensitive. CENTRI•SEP 96 plate, and then from the top.

3. Prepare the CENTRI•SEP 96 plate.


D3.b To Purify With Ethanol/Sodium Acetate
a. Stack the CENTRI•SEP 96 plate on top of a MicroAmp® Optical 96-Well
Reaction Plate, and tape the two plates together with a base.
1. Prepare the sodium acetate/ethanol solution by combining the following per b. Centrifuge the plate at 700 x g for 2 minutes to pack the column.
reaction: c. Remove the tape and separate the plates.
2 L of 3.0 M sodium acetate, pH 5.2 d. Discard the remaining liquid in the wash plate by shaking it vigorously.
e. Wash and save the MicroAmp® Optical 96-Well Reaction Plate for
50 µL of 100% ethanol repeated use as a wash plate.

4. Place a 96-well collection plate on a 96-well base, then stack the


CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. CENTRI•SEP 96 plate on top.
Exposure may cause eye, skin, and upper respiratory tract irritation.
Prolonged or repeated contact may dry the skin. Exposure may cause central
5. Tape the plates to the 96-well base, making sure that you align the
nervous system depression and liver damage. Keep away from heat, sparks,
alphanumeric indices on all the plates.
and flame. Wear appropriate protective eyewear, clothing, and gloves.
Prepare enough for one reaction more than you need. Refer to the
manufacturer’s Safety Data Sheet for additional information.
IMPORTANT! Prepare a fresh solution for each set of precipitations.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 11 of 68


6. Load the sequencing reactions. Using a multichannel pipettor, transfer the Limitations
20-µL sequencing reactions to the individual wells in the CENTRI•SEP 96
plate.
• Carefully place samples on the centers of the gel beds. Do not place the
samples on the side of the wells.
Reagents and Chemistry
• Do not touch the gel bed with the pipette tips. • Only trained users are allowed to perform the ViroSeq assay.
• Accurate and reliable results are dependent on proper sample collection and
7. Centrifuge the plate at 700 x g for 2 minutes. storage prior to testing.
• Blood collected using heparin tubes is unsuitable for use with PCR and this
8. Discard only the CENTRI•SEP plate (you should have approximately 20 µL assay.
in each well of the collection plate). • Testing for antiretroviral drug resistance has only been validated on patient
samples with viral loads between 2,000 and 750,000 copies per mL. In a typical
9. Dry the samples in a centrifuge with vacuum pump equipped with the HIV positive patient population in the U.S. that is on HIV highly active
antiretroviral therapy (HAART), about 25% of the patients can be expected to
appropriate rotor. Do not overdry the samples. have suppressed viral loads. These samples may not always generate
interpretable results with the ViroSeq HIV-1 Genotyping System if the viral
10. When the centrifuge stops and drying is complete, remove the tray/plate and load is below 1,000 copies/mL. Users should test samples with a viral load of
seal with strip caps or with adhesive aluminum tape, and proceed to: 2,000 copies/mL or greater.
• Section 3, Sequencing Runs on the Applied Biosystems® 3130 and • Regardless of viral load, the agarose band intensity of the RT-PCR product must
3130xl Genetic Analyzers, or be equal to or greater than the intensity of the 20 ng mass ladder band to produce
• Store at –15 to –25°C in the dark. Analyze the samples within one week. a high-quality sequence.
• The presence of AmpErase® UNG in the ViroSeq HIV-1 Genotyping System
IMPORTANT! Store in the dark. The products of sequencing reactions are reduces the risk of contamination with previously amplified product only.
light-sensitive. Sample to sample contamination, or contamination from the positive control, can
still occur. Careful adherence to work area setup and the protocol reduces the
possibility of contamination.
• Only Applied Biosystems thermal cyclers and automated DNA sequencers can
Quality Control be used with this assay.

The ViroSeq™(8E5) Control Module, containing both positive and negative


controls, is provided with the ViroSeq HIV-1 Genotyping System. A positive and
negative control must be used with every run. The HIV-1 8E5 Positive Control is
required to monitor both RT-PCR success and Genotyping success. The HIV-1 8E5
Negative Control is required to monitor RT-PCR success only.

RT-PCR Success
RT-PCR success is determined in section “D1. To Purify and Quantify the PCR
Products.” To ensure a high quality sequence, the intensity of the RT-PCR band must
be equal to or greater than the intensity of the 20 ng mass ladder band in the agarose
gel. Plasma samples with viral loads above 2,000 copies per mL should provide at
least 20 ng of RT-PCR product at this step. As the viral load decreases below 1,000
copies per mL, the chance for a successful PCR decreases. However, any sample
with a DNA quantitation of 20 ng or greater can be sequenced to provide a genotype,
regardless of the starting concentration in plasma. The HIV-1 8E5 Positive Control
has a starting viral load of 50,000 to 100,000 copies/mL; the low positive control
(1:10 dilution) has a viral load of 5,000 to 10,000 copies/mL. The negative control
has no detectable HIV-1 RNA.
The expected RT/PCR results for the ViroSeq controls are:

Positive/Low Positive Control Negative Control


Expected Value >40 ng/5 L (undiluted Expected Value 0 ng
positive)
>20 ng/5 L (low
positive)
If. . . Then. . . If. . . Then. . .
the band intensity is: run is invalid. Repeat any band is present on sample to sample
• Positive, testing. the agarose gel contamination has
undiluted: occurred.
<40 ng • All sample results
• Low positive: are invalid
<20 ng • Repeat from “A.
Sample
all of the other sample a system failure has Preparation”
reactions failed (less occurred. through “D1. To
than 20 ng/5 µL). • Repeat all samples Purify and Quantify
and controls the PCR Products”
repeated failure occurs contact Abbott
Molecular support

Page 12 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


• For illustrations and diagrams that explain how to conduct a specific task on the
Section 3: Sequencing Applied Biosystems ® 3130/3130xl Genetic Analyzer, refer to the Applied
Biosystems® 3130/3130xl Genetic Analyzer Getting Started Guide (PN
4352715) and the Maintenance, Troubleshooting, Reference Guide (PN

Runs on the Applied 4352716).

Required Materials
Biosystems® 3130 and Item ABI
3130xl Genetic Analyzers ABI PRISM ® 3130xl Genetic Analyzer with:


Data Collection Software v.3.0
Firmware version 6286200-02 or 6286250-02
3130XL
IMPORTANT! No software
should be downloaded from the
• PC computer with: internet for this intended use.
– Microsoft® Windows® XP Professional edition, Service
Pack 2
– 2 GHz Intel® Pentium® IV processor
– 1 GB RAM
Introduction – Dual 36 GB hard drives
Applied Biosystems ® 3130 Genetic Analyzer with: 3130
The ViroSeq™ HIV-1 Genotyping System v2.0 has been approved for in vitro • Data Collection Software v.3.0 IMPORTANT! No software
• Firmware version 6278250-02 should be downloaded from the
diagnostic use with the ViroSeq® HIV-1 Genotyping System Software v2.8 on the • PC computer with: internet for this intended use.
Applied Biosystems ® 3130 Genetic Analyzer and the Applied Biosystems ® 3130xl – Microsoft® Windows XP Professional edition, Service
Genetic Analyzer. Both instruments are fully automated, fluorescence-based Pack 2
– 2 GHz Intel® Pentium® IV processor
capillary electrophoresis platforms that simultaneously analyze samples within a – 1 GB RAM
single run. As summarized below, key differences between the two platforms – Dual 36 GB hard drives
include the number of 96-well plates that can be loaded on the instrument and the
number of capillaries in the capillary array. Applied Biosystems ® 3130xl Capillary Array, 50 cm 4315930

Note: The number of capillaries in the instrument's capillary array determines run Applied Biosystems ® 3130 Capillary Array, 50 cm 4333466
size. Lower Polymer Block Cleaning Kit 4359572
Parameter 3130 Genetic Analyzer 3130xl Genetic Analyzer Sequencing Analysis Software v5.3.1 with the 4360967
KB™ Basecaller v1.4 To obtain additional licenses,
Number of 96-Well Plates 1 2 please contact your Applied
Biosystems representative.
Number of Capillaries in Array 4 16 IMPORTANT! Do not
download software from the
Number of Runs/One 96-Well Plate 24 6 internet for this intended use.
GeneAmp® PCR System 9600 Thermal Cycler N801-0001
Together, these parameters define the throughput capacity of the instrument and or or
allow sites to determine the system configuration that is most compatible with GeneAmp® PCR System 9700 Thermal Cycler N805-0001
current and future sequencing volume. MicroAmp® 96-Well Support Base N801-0531
Reservoir Septa 4315932
96-Well Plate Map
96-Well Plate Base 4317237
For each 3130xl Genetic Analyzer run, injections are made from each well of 16
96-Well Plate Retainer 4317241
wells of two consecutive plate columns, starting with the column that contains well
A1 (e.g., wells A1 though H2, A3 through H4, etc.). A full plate requires six runs to 96-Well Plate Septa 4315933
inject all 96 wells.
MicroAmp® Optical 96-Well Reaction Plate N801-0560
For each 3130 Genetic Analyzer run, injections are made from each well of 4
consecutive wells within a plate column starting with the column that contains well POP-6™ Polymer for 3130/3130xl Genetic Analyzers 4363783 (3500 µL) or
A1 (e.g., wells A1 through D1, E1 through H1, A2 through D2, etc.). It takes four 4352757 (7000 µL)
runs to inject 16 wells, and 24 runs to inject a full plate of 96 wells. Running Buffer, 10X 402824
COLUMNS Hi-Di™ Formamide, 25 mL 4311320

1 2 3 4 5 6 7 8 9 10 11 12
A

B
Contains Formamide.
R 61 May cause harm to the unborn child.
C R 36/38 Irritating to eyes and skin.
R S 53 Avoid exposure - Obtain special instructions before use.
D
O S 24/25 Avoid contact with skin and eyes.
W
S E S 35 This material and its container must be disposed of in a safe
way.
F S 36/37/39 Wear suitable protective clothing, gloves and eye/face
protection.
G S 45 In case of accident or if you feel unwell, seek medical advice
immediately (show the label where possible).
H

: Single Run on 3130 Genetic Analyzer (4-capillary array)

: Single Run on 3130xl Genetic Analyzer (16-capillary array)

• Ensure that the instrument is calibrated before proceeding with the run.
Refer to Maintenance and Calibration of the Applied Biosystems® 3130 and
3130xl Genetic Analyzers.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 13 of 68


Protocol 3. Fill and/or change the buffer and water reservoirs before each run.
• Anode reservoir. Fill to the fill line with fresh 1X Running Buffer
(16 mL).
To Prepare for Automated Sequence Detection • Cathode reservoir. Fill to the fill line with fresh 1X Running Buffer
(16 mL).
• Water reservoirs. Fill to the fill line with fresh deionized water (16 mL
To Start the Computer Workstation each).

IMPORTANT! You must start the computer workstation before starting the
4. Check for bubbles in the pump block, lower polymer block, interconnect
instrument.
tube, polymer supply tube, and channels.
Remove all bubbles with the Bubble Remove Wizard.
1. Power on the computer and monitor.

2. In the Log-in to Windows dialog box, enter the user name and, if 5. Check:
applicable, enter a password, then click OK. • That the polymer block fits securely on the instrument.
• For dried polymer around the polymer block, and clean as necessary.
• That the capillary tips are not crushed or damaged.
To Start the Instrument
6. Perform a spatial calibration if you have just installed a new or used capillary
1. On the instrument, ensure that the: array on the instrument.
• Oven door is closed and locked
• Instrument doors are closed 7. Perform a spectral calibration if needed. Refer to To Perform A Spectral
Note: If the doors are open during power on, a yellow warning light will Calibration.
continue to blink until the doors are closed.

2. Press the on/off button on the front of the instrument. Data Collection Software
Note: While the instrument is booting up and performing self-checks, the
yellow status light blinks. IMPORTANT! Do not rename the computer. The instrument computer was
assigned a unique name before the 3130/3130xl Genetic Analyzers Data
Collection software was installed. Doing so may cause the Data Collection software
3. Ensure the green status light is on and not blinking before proceeding. to malfunction.
Note: If the green status light does not come on, start the Data Collection To determine the 3130 or 3130xl firmware and Data Collection software versions
software and view the event log at: installed on your system, click Help > About from the menu or select GA
E:\AppliedBiosystems\UDC\DataCollection\Log\Instrument Name Instruments from the Data Collection window.
IMPORTANT! Use only the following versions of software:
To Set Up the Instrument for the Run ABI 3130 Data Collection software v.3.0 with Firmware v. 6278250-02
or
Refer to the Applied Biosystems® 3130/3130xl Genetic Analyzer Getting Started ABI 3130xl Data Collection software v.3.0 with Firmware v. 6286200-02 or
Guide and the Maintenance, Troubleshooting, Reference Guide for detailed 6286250-02
installation, maintenance, and instructions for general use of the instruments. Never move or delete any system file or folder unless specifically directed to do so
Note: After opening the instrument door, you must allow the autosampler homing by an Applied Biosystems representative or by the Applied Biosystems®
sequence to complete before issuing further commands. The instrument is not 3130/3130xl Genetic Analyzer Getting Started Guide. Doing this could render the
available until all commands are completed, including steps within wizards and software inoperable.
manual control commands, and the autosampler is in the home position.
To Start the Data Collection Software
1. Prepare and have the following reagents and consumables on hand:
• POP-6™ Polymer for 3130/3130xl Genetic Analyzers
1. Select Start > All Programs > Applied Biosystems > Data Collection>
• A 50 cm, 16-capillary array or a 50 cm, 4-capillary array Run 3130 Data Collection v3.0 or Run 3130xl Data Collection v3.0.
Note: Replace the capillary array after 100 runs and check that the capillary
array information is correct in the Data Collection database. The Service Console is displayed.
• Running Buffer, 10X As each application activates, the red circles (off) change to yellow triangles
Note: To prepare 1X Running Buffer, dilute 5 mL of 10X Running Buffer, (activating), and then to green squares (on) when they are fully functional.
with 45 mL of deionized water.
When all the applications are running (all green squares—this could take
several minutes), the Foundation Data Collection Viewer window is
displayed.
CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and
respiratory tract irritation. Wear appropriate protective eyewear, clothing, Enter the user login and password. The login ID should be a user with
and gloves Instrument Protocols access (refer to Appendix A: Auditing and Software
Access Control).
2. Ensure that there is sufficient POP-6 polymer for the run, and add polymer if
needed.
Note: If adding more polymer, ensure that the polymer is from the same lot.
Note: Change the polymer if it has been on the instrument for 7 days. Do
not use expired polymer or polymer containing precipitated material.

Page 14 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


2. Click the + to expand subfolders in the
left window pane. All application folders
are now visible and ready to access.

Default Run Module 3130xl Default Run Module 3130

To Create Required Files for the ViroSeq HIV-1


Genotyping System v2.0 To Create a ViroSeq Instrument Protocol
The following three files, Instrument Protocol, Analysis Protocol and Results Group,
must be created one time by the user. These files are used for all subsequent plate An instrument protocol contains all the settings needed to run the instrument. An
records. Refer to To Create a Plate Record. instrument protocol contains the protocol name, type of run, run module, and dye
set.
To View the Default Run Module for a Sequencing Run 1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.
The Run Module specifies information about how the sample is run (e.g., the
duration of the run, the run temperature, and the injection time). 2. In the Instruments Protocol section, click New. The Protocol Editor opens.

1. Select GA Instruments > ga3130 or ga3130xl > Module Manager. 3. Complete the Protocol Editor
a. Name:
2. To view the default sequencing run parameters, double-click ViroSeq_Instrument_Protocol
StdSeq50_POP6_1 from the Run Module list. All the parameters for the b. Description: Optional
default module are displayed. c. Type: Regular
The default module installed with 3130/3130xl Data Collection software d. Run Module:
cannot be edited. StdSeq50_POP6_1
e. Dye Set: E-BigDyeV1
3. Click Cancel to close the default Run Module.

4. Click OK.

To View an Instrument Protocol

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

2. In the Instrument Protocols section, double-click the instrument protocol


you want to view. The protocol editor dialog box displays all the parameters
for the instrument protocol.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 15 of 68


To Edit an Instrument Protocol
4. Basecalling tab:
• Basecaller: KB.bcp
1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.
• DyeSet / Primer: KB_3130_POP6_BDTv1.mob
2. In the Instrument Protocols section, click on the instrument protocol to • Processed Data: True Profile
highlight and click Edit. • Ending Base: At PCR Stop
• Quality Threshold: Do not assign N’s to Basecalls.
3. In the Protocol Editor dialog box, edit the parameters that you want to
change.

4. Click OK.

5. In the Warning dialog box, click Yes to overwrite the previous settings.

To Create a ViroSeq Analysis Protocol

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

2. In the Analysis Protocol section, click New. The Sequencing Analysis


Protocol Editor opens. Complete the Sequencing Analysis Protocol Editor
as described in the following steps.

3. General tab:
• Name: ViroSeq_Analysis_Protocol
• Description: optional
• Sequence File Formats: uncheck all check boxes.

5. Mixed Bases tab:


• Uncheck the Use Mixed Base Identification check box.

Page 16 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


6. Clear Range tab: 3. Complete the General tab:
• Uncheck all check boxes. a. Results Group Name: enter ViroSeq_Results_Group.
b. Results Group Owner: the owner name can be used in naming and
sorting sample files.
c. Results Group Comment: enter a comment (optional).

4. Complete the Analysis tab:


a. Analysis Type: SequencingAnalysis.
b. Analysis Actions: select the check box Do Autoanalysis.
To View an Analysis Protocol

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

2. In the Analysis Protocols section, double-click the analysis protocol you


want to view. The protocol editor dialog box displays all the parameters for
the analysis protocol.

To Edit an Analysis Protocol

1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

2. In the Analysis Protocols section, click on the analysis protocol to highlight


and click Edit.

3. In the Sequencing Analysis Protocol Editor dialog box, edit the parameters
that you want to change.

4. Click OK. 5. Select the Destination tab to define a data storage location. Use the default
Root Destination path for data storage:
E:\AppliedBiosystems\udc\datacollection\Data
To Create a ViroSeq Results Group
A Results Group is a component of Data Collection software that is used to name,
analyze, and organize sample files.

1. Select GA Instruments > Results Group.

2. Click New. The Results Group Editor window displays.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 17 of 68


6. Select the Naming tab to customize sample file and run folder names.
To Create a Plate Record
1. Select GA Instruments > ga3130 or ga3130xl > Plate Manager.

2. Click New to open the New Plate dialog box.

a. Complete the Sample File Name Format pane:


– Click the Name Delimiter list and select the underscore symbol (_)
to separate the Format elements in the file name. 3. Complete the information in the New Plate dialog box:
– Click the Format list and then select the following format elements a. Name: enter a name for the plate.
for the file name in this order: Sample Name, Well Position. As you
select the elements for the file name, they are reflected in the IMPORTANT! The new Plate Name must be unique.
Example line. The names of the Format elements eventually Note: A useful way to name a Plate is to use the current date as the initial
truncate, but the Example field remains visible (up to 72 characters). identifier, followed by an underscore character and additional information,
b. Complete the Run Folder Name Format pane: (e.g., Date [DDMMYY]_TEXT).
– Click the Format list and then select the format element Plate Name. b. Description: enter a description for the plate (optional).
This will group all samples within a plate (across runs) into a single c. Application: SequencingAnalysis.
folder.
d. Plate Type: 96-Well
IMPORTANT! Sample name, run folder name, and path name, combined, e. Owner Name: enter an owner name.
cannot exceed 250 characters. f. Operator Name: enter an operator name.
g. Click OK. The SequencingAnalysis Plate Editor opens.
7. Click OK to save the Results Group.

To Duplicate the ViroSeq Results Group (Optional)

1. Select GA Instruments > Results Group.

2. Click the ViroSeq_Results_Group to highlight.

3. Click Duplicate.

4. When the Duplicate Results Group dialog box opens, rename the ViroSeq
Results Group.
Note: A useful way to name a Results Group is to use the current date as
the initial identifier, followed by an underscore character and additional
information, (e.g., Date [DDMMYY]_TEXT).
IMPORTANT! The duplicate Results Group name must be unique.

5. Click OK to save the duplicate ViroSeq Results Group.

Page 18 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


4. Complete the information in the SequencingAnalysis Plate Editor
To Prepare Purified Sequencing Reactions for Analysis
on the 3130xl Genetic Analyzer
a. In the Sample Name column of a row, enter the appropriate sample
name, then click the next cell. Sample names are limited to a maximum Note: For Instructions on preparing purified sequencing reactions for analysis on
of 32 characters. the 3130 Genetic Analyzer, refer to the next section.
– When naming the samples, use letters, numbers, and the following
punctuation only:-_(){} # +. DO NOT USE SPACES. 1. If your samples are frozen, bring them to room temperature.
– The sample name must be identical for all seven primer sequences of
a sample. IMPORTANT! This must be done immediately before loading the samples.
– The sample name and primer identification must be separated by two
consecutive underlines (Shift plus_). 2. Resuspend your samples by adding 20 L of Hi-Di formamide to each
– The primer identification contains the primer name and any other sample well; use a new aliquot of Hi-Di formamide for each experiment.
information unique to the specific sequence. For example:
Patient196__A, Patient196__B, Patient196__C, Patient196__D,
Patient196__F, Patient196__G, Patient196__H.
b. Priority: the value 100 automatically displays in the column. Contains Formamide.
c. In the Results Group 1 column, select the ViroSeq Results Group R 61 May cause harm to the unborn child.
created specifically for the plate from the drop-down list.
d. In the Instrument Protocol 1 column, select the R 36/38 Irritating to eyes and skin.
ViroSeq_Instrument_Protocol. S 53 Avoid exposure - Obtain special instructions before use.
e. In the Analysis Protocol 1 column, select the S 24/25 Avoid contact with skin and eyes.
ViroSeq_Analysis_Protocol.
S 35 This material and its container must be disposed of in a safe way.
Note: Left-click in the column header and press Ctrl+D to fill down
whenever a field is the same for all samples. S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.
f. Click OK. S 45 In case of accident or if you feel unwell, seek medical advice
immediately (show the label where possible).
Note: Samples resuspended in Hi-Di formamide are stable at room
temperature for up to 35 hours. Do not leave these samples at room
temperature for a longer period of time.

3. Vortex for 10 to 15 seconds.

4. Centrifuge the plate for 30 to 40 seconds at 2,000  g to collect the liquid at


the bottom of the plate.
Note: Heat denaturation of the samples is not required.

5. Prepare and install the plate assembly.

To Prepare Purified Sequencing Reactions for Analysis


on the 3130 Genetic Analyzer

1. If your samples are frozen, bring them to room temperature.


IMPORTANT! This must be done immediately before loading the samples.

2. Resuspend your samples by adding 20 L of Hi-Di formamide to each


sample well; use a new aliquot of Hi-Di formamide for each experiment.
To Edit a Plate Record

1. Select GA Instruments > ga3130 or ga3130xl > Plate Manager. Contains Formamide.
R 61 May cause harm to the unborn child.
2. Select the plate record to edit and click Edit. R 36/38 Irritating to eyes and skin.
S 53 Avoid exposure - Obtain special instructions before use.
3. Edit the parameters in the Plate Editor. S 24/25 Avoid contact with skin and eyes.
S 35 This material and its container must be disposed of in a safe way.
4. Click OK.
S 36/37/39 Wear suitable protective clothing, gloves and eye/face protection.
S 45 In case of accident or if you feel unwell, seek medical advice
immediately (show the label where possible).
Note: Samples resuspended in Hi-Di formamide are stable at room
temperature for up to 35 hours. Do not leave these samples at room
temperature for a longer period of time.

3. Vortex for 10 to 15 seconds.

4. Centrifuge the plate for 30 to 40 seconds at 2,000  g to collect the liquid at


the bottom of the plate.
Note: Heat denaturation of the samples is not required.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 19 of 68


5. IMPORTANT! Signal degradation may occur for samples left on 2. Place the plate assembly on the autosampler. Use position A or B for the
the 3130 Genetic Analyzer for more than 14 runs (1 run = 3130xl Genetic Analyzer or position B for the 3130 Genetic Analyzer.
approximately 2.5 hours).
Position the plate with well A1 in the upper right corner, with the notched-
If the plate contains 7 or fewer columns of samples (including end of the plate base away from you.
control), prepare and install the plate on the plate assembly as
Note: The plate will only fit into the autosampler when properly positioned.
described in the section below.
IMPORTANT! Ensure the plate assembly fits flat in the autosampler.
OR Failure to do so may allow the capillary tips to lift the plate assembly off of
If the plate contains more than 7 columns of samples, continue with the autosampler.
step 6 below.
3. When the plate is correctly positioned, the plate position indicator on the
6. Transfer the remaining columns of samples from plate 1 to a new Plate View page changes from gray to yellow.
MicroAmp® Optical 96-Well Reaction Plate (plate 2). Verify that this has happened before proceeding.
Note: Additional plates may be purchased from Applied Biosystems. Refer
to Required Materials Not Provided on page 4. 4. Close the instrument doors.
Note: Closing the doors returns the autosampler to the home position,
7. Transfer 10 µL of positive control (one-half of the volume from the placing the tips of the capillaries in buffer.
sequencing setup) from plate 1 to plate 2. Perform this step for all 7 wells
containing the positive control.
To Load Samples and Link a Plate to the Plate Record
8. Prepare and install plate 1 on the plate assembly as described in the section
below. 1. Search for the plate record:
Select GA Instruments > ga3130 or ga3130xl > instrument name > Run
9. Seal plate 2 with aluminum foil tape and store at –15 to –25°C in the Scheduler > Plate View.
dark. Analyze the samples within one week. There are two search options:
Note: Load plate 2 after plate 1 is completed. Follow steps 10 to 13 below. • Find All
• Advanced
10. Bring plate 2 to room temperature. Under Find All:
IMPORTANT! This must be done immediately before loading the samples. • Select Barcode in the Type of Search drop-down list.
• If you have a limited number of plates in the database, click Find All. All
plates in the database will display in the plate record section.
11. Vortex for 10 to 15 seconds.
Under Advanced:
12. Centrifuge the plate for 30 to 40 seconds at 2,000  g to collect the liquid at a. Select Advanced in the Type of Search drop-down list.
the bottom of the plate. b. Use the drop-down list to define search conditions for a category or
multiple categories (Run Name, Results Group Name, Plate Name,
Note: Heat denaturation of the samples is not required. etc.)
c. Use the Plate Name for the Plate ID category.
13. Prepare and install plate 2 on the plate assembly as described in the section d. For each category with a condition selected, type a value (primary search
below. string) in the Value 1 column.
• Click Search. All plates in the database that match the search criteria
display in the plate record section.
To Prepare a Plate Assembly
2. Select the plate record, then click the plate position indicator that
corresponds to the plate being linked. The plate position indicator changes
1. Secure a clean and dry plate septa on the sample plate.
from yellow to green when linked and the green Run Instrument button is
IMPORTANT! Never use warped plates. active.
IMPORTANT! Make sure the plate septa lies flat on the plate. The holes in
the plate retainer must align with the holes in the septa or the capillary tips 3. After a plate is linked, use the Run View window to verify that the runs are
will be damaged. scheduled correctly.
a. Select GA Instruments > ga3130 or ga3130xl > instrument name > Run
2. Place the sample plate into the plate base. Scheduler > Run View.
b. Select a row for any run. The corresponding wells to be injected for that
3. Snap the plate retainer onto the plate and plate base. run are highlighted in the plate diagram.
IMPORTANT! If the well is not highlighted, it will not be injected.
4. Ensure that the plate retainer holes are aligned with the holes in the septa
strip.
To Unlink a Plate Record
IMPORTANT! Damage to the array tips will occur if the plate retainer and
septa strip holes do not align correctly.
1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Run
Scheduler > Plate View.
To Place the Plate onto the Autosampler
2. Select the plate record to be unlinked.
1. Press the Tray button.
3. Click Unlink. The plate position indicator changes from green to yellow
when unlinked.

Page 20 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


To Start the Run 3. The Error Messages lists:
a. Errors that have occurred during the current run
1. Verify that the active spectral calibration matches your dye set, capillary b. Information for service engineers. A “fatal” error usually requires that
array length, and polymer type for all scheduled runs. you restart the Data Collection software.
a. Select GA Instruments > ga3130 or ga3130xl > instrument name >
Spectral Viewer. 4. The EPT Chart displays the real time chart of EPT status.
b. In the Dye Set drop-down list, select E-BigDyeV1.
c. In the list of Calibrations for the Dye Set drop-down list, select a
spectral calibration. 5. System Status changes from green to flashing red when errors occur.
d. Click Set.
Monitor the System Messages from a Run in Progress
2. Click the green Run Instrument button in the toolbar.
IMPORTANT! Do not open the door of the genetic analyzer once the run 1. Select GA Instruments > ga3130 or ga3130xl > instrument name >
has begun. Instrument Status > Event Log.
Do not simultaneously perform data analysis or use other software during the
run.

3. The Processing Plates dialog box opens, then click OK.

4. The software automatically performs a run validation:


• If the validation passes, the run will start.
• If any of the validation test fails, the run will not start. Check the event
log for information.

Do not use data in the process of collection if electrical power fails in the
middle of a run.

To Monitor a Run

Monitor a Run in Progress

1. Select GA Instruments > ga3130 or ga3130xl > instrument name >


Instrument Status. Instrument Status window displays the real time
instrument status, including the EP Voltage, EP Current, Laser Power,
Laser Current and Oven Temperature.
2. Clear error messages by clicking Clear Errors. The System Status light
flashes red until all errors are cleared. Take corrective action based upon the
error message.

3. The Event Log itemizes events such as errors and general information for all
data collection steps. Clear Errors changes the System Status from red to
green (ready state).

2. The Event Messages lists:


a. The instrument's recent actions
b. The status of each capillary as passed or failed at the end of a spectral
calibration
c. The calibration data at the end of a spatial calibration
d. Information for service engineers.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 21 of 68


Use the Capillary Viewer to View Data During a Run
2. Zoom in and out:
IMPORTANT! Always exit from the Cap/Array Viewer and the Capillaries a. Select a rectangular area over the area of interest by holding down the
Viewer windows. During a run, do not leave these pages open for extended periods. mouse button. Release the mouse button to zoom-in.
This may cause unrecoverable screen update problems. Leave the Instrument b. Click the Zoom-out icon button in the toolbar to return to full view
Status window open.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > To View Raw Data from a Completed Run
Capillaries Viewer. Use the Capillaries Viewer to examine the quality of
the raw data during a run for several capillaries at once There are two formats for viewing data within the Data Collection software under
the Run History icon:
• the Cap/Array Viewer window, and
• the Capillary Viewer window (capillary-by-capillary).

1. Select GA Instruments > ga3130 or ga3130xl > Run History to select a


run to view.

2. Search for a run using Barcode or Advanced search. After choosing a run,
click the Capillary Viewer or the Array Viewer (under the Run History
icon) from the left Viewer window.

2. Select the check boxes of the desired capillaries to be viewed. The capillaries
are displayed in the order in which the boxes are checked.

3. Zoom in and out:


a. Select a rectangular area over the area of interest by holding down the
mouse button. Release the mouse button to zoom-in.
b. Click the Zoom-out button in the toolbar to return to full view.

Use the Cap/Array Viewer to View Data During a Run


IMPORTANT! Always exit from the Cap/Array Viewer and the Capillaries
Viewer windows. During a run, do not leave these pages open for extended periods.
This may cause unrecoverable screen update problems. Leave the Instrument
Status window open.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name >


Cap/Array Viewer. View this window during a run in progress to examine
the data quality, which is depicted as color data for the entire capillary array.

Page 22 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


3. Note: EPT Viewer - The electrophoresis data of the run, such as 2. In the Analysis Defaults window, under Add Samples Settings, select
voltage, current, and oven temperature profiles can be seen in the Analysis Protocol as None. Check the Base Calling (BC) check box.
EPT viewer under Run History. Uncheck the Post Processing (PP) and Print (P) check boxes.

3. In the Analysis Defaults window, under Sequence File Formats, select


“Use settings in the sample’s Analysis Protocol”. Click OK.

4. Click OK on the Warning Analysis Defaults dialog.

To View Analyzed Data


When a run is finished, the sample files are automatically extracted into a run folder,
along with a run log, to a location defined in your preferences or the following
default location:
E:\AppliedBiosystems\UDC\Data Collection\Data\ga3130xl\<run folder
name>
or Create a ViroSeq Analysis Protocol
E:\AppliedBiosystems\UDC\Data Collection\Data\ga3130\<run folder name>
1. From the Main menu, select Analysis > Analysis Protocol Manager. The
If the data has been analyzed, the data is in the location defined in the following
default location: Analysis Protocol Manager - DataStore window opens.
E:\AppliedBiosystems\UDC\Data Collection\Data\plate folder\ab1 files
2. Click New in the Analysis Protocol Manager - DataStore window and
create the ViroSeq Analysis Protocol by clicking on the appropriate tabs as
Sequencing Analysis Software v5.3.1 with KB described in the following steps.

Basecaller v1.4 3. General tab:


• Name: ViroSeqAnalysisProtocol_580Trim
To Use the Sequencing Analysis Software v5.3.1 and • Description: optional
Analyze the Data • Sequence File Formats: uncheck all check boxes.
The Sequencing Analysis Software v5.3.1 will be installed on the hard drive of the
instrument computer workstation. This software is used to:
• Review basecalled sequences
• Reanalyze data to trim to 580

Launch the Sequencing Analysis Software

1. Start the Sequencing Analysis Software v5.3.1 by double-clicking the


Sequencing Analysis v5.3.1 desktop shortcut or select Start > Programs >
Applied Biosystems > Sequencing Analysis > Sequencing Analysis 5.3.1.

2. In the Login window, enter a valid User Name and password. Click OK.
Note: To check the software name and version number, from the main menu
select Help > About Sequencing Analysis. Click OK to close the About
dialog.

Set the Analysis Defaults

1. From the Main menu, select Analysis > Analysis Defaults. The Analysis
Defaults window opens.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 23 of 68


4. Basecalling tab: 6. Clear Range tab:
• Basecaller: KB.bcp • Uncheck all check boxes,
• DyeSet / Primer: KB_3130_POP6_BDTv1.mob
• Processed Data: True Profile
• Ending Base: check the box and enter 580 in the Bases field.

• Quality Threshold: Do not assign N’s to Basecalls.

7. Click OK to return to the Analysis Protocol Manager - Data Store window.

8. Click Done.

Add Sample Files to the Sample Manager


5. Mixed Bases tab:
• Use Mixed Base Identification: uncheck the check box. Sample files may be added or removed from the Sample Manager window at any
time, except for files that are currently being processed by the program. Refer to
Additional Sample Manager Functions.

1. Click the Add Samples icon in the toolbar or select File > Add
Sample(s).

2. Navigate to the location of the sample folders. The individual sample


files are located within the sample folders.

3. To add files to be analyzed, choose one of the steps below:

To add … Then …

All samples within a single folder Select the folder, then click Add
Selected Samples.

A single file Open the desired sample folder,


select the file, then click Add
Selected Samples.

Continuous multiple files Open the desired sample folder, use


the Shift key to select sample files,
then click Add Selected Samples.

Discontinuous multiple files Open the desired sample folder, use


the Ctrl key to select samples, then
click Add Selected Samples.

Continuous multiple sample folders Use the SHIFT key to select


contiguous sample folders, then
click Add Selected Samples.

Discontinuous multiple sample folders Use the Ctrl key to select sample
folders, then click Add Selected
Samples.

4. When the desired sample files have been added, click OK in the Add
Samples dialog box to close the dialog box.

Page 24 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


You can remove a sample file from the Sample Manager window at any time Show the Sample Data
except when the program is currently processing that file. You can also rearrange the
order that the sample files appear in the Sample Manager window.
1. Use the Show check box to show data for one or more samples files or
double-click on the sample file name.
Analyze Sample Files Using the Sequencing Analysis
Software with 580 Trim 2. To show the data in the Sample Navigator view, click the Toggle Show
Sample Navigator/Sample Manager icon in the toolbar.
1. From the Main menu, select Analysis > Analysis Protocol Manager.
There are seven views available in the Sample Manager or Sample
Navigator panes:
2. Select the ViroSeqAnalysisProtocol_580Trim. Click Apply to All
a. Annotation
Samples.
b. Sequence
c. Features
3. Click Done. d. Electropherogram
e. Raw (Data)
4. Verify the following settings for each sample: f. EPT
g. Audit
BC Check Box Box is checked
To change the view, select the tab for the desired view.
PP Check Box Box is not checked View Description
Annotation Summary of the sample information written by the data
P Check Box Box is not checked collection and analysis software
Sequence The nucleotide (base) sequence text called for the data. Gray-
colored sequence text represents the trimmed bases.
Basecaller KB.bcp The view is available only after basecalling is done.
Feature The features that were found in the sequence by the post
DyeSet/Primer KB_3130_POP6_BDTv1.mob processing (clear range).
Electropherogram A four-color picture of analyzed data with peaks representing
the bases. The original bases, edited bases or complementary
Matrix File None bases can be displayed.
This is the default view that is displayed when a sample files
are shown and is available only after basecalling is done.
Spacing 0
Raw The raw data collected by the instrument.
0 EPT A plot of run voltage, current, power and temperature values.
Peak 1 Location
Audit Information about modifications to the data (base change,
deletion, inserting, change in analysis setting, sample name
Start Point 0 change). This window contains data only if the Audit Trail
feature is activated in the Authentication and Audit tab of the
Options dialog box.
Stop Point 0

5. Click the Start Analysis icon or select Analysis > Start Analysis.
Review Sequencing Analysis Data
IMPORTANT! Review the Sequencing Analysis data using the checklist below
6. After analysis, visually check: before proceeding. to the ViroSeq® HIV-1 Genotyping System Software analysis:
• that the Spacing, Peak 1 Location, Start Point and Stop Point have been
defined by the software, and
• the color of the BC check box. The color indicates the analysis status. Item Check for / Action
The color status is cleared at the start of each new sample processing.
Review or Correct Verify that the Ending Base for a subset of sample
Ending Base electropherograms are at 580 bases. If not, be sure to
Check Box Color Indication
select the ViroSeqAnalysisProtocol_580Trim and re-
analyze data.
Green The basecaller analyzed the sample file correctly.
Review or Correct Inspect a subset of samples to verify that the Start Point
Blue The basecaller analyzed the sample file successfully, but detected
some anomalies that may or may not be serious. Review the error
Start Point begins after the initial dye blob.
message, the sample score, and the data. If not, reset the Start Point value to begin after the initial
dye blob in a sample or determine the average Start
Yellow The basecaller analyzed the sample file successfully, but with poor Point for a subset of samples.
quality data. Some anomalies that may or may not be serious.
Review the error message, the sample score, and the data. a. Enter the new start point value in the Peak 1 column.
Verify that the value in the Start Point column
Red A software failure occurred. Check the software error messages. matches Peak 1.
Note: For a subset of samples, use Fill Down to enter
values for all samples.
b. Check the BC check box(es) for the sample(s).
c. Click the Start Analysis icon or select Analysis >
Start Analysis.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 25 of 68


Item Check for / Action
To Print Sample Data

Sample names Verify sample file names are correct. Use the same 1. To temporarily change the page orientation, paper type, panels/page, and so
sample name for all samples of a project. Sample file forth, select File > Page Setup to open a special Page Setup dialog box.
names:
• Must be less than 59 characters 2. Adjust the settings as needed. Then click OK to close the dialog box.
• Should include any information needed to identify a
sample. 3. In the row number column, select one or more samples to print.
• Must have two consecutive underscores to separate
the sample name and the sequence primer. The
4. Select File > Print to open the Print dialog box.
ViroSeq software requires the double underscore to
assemble and create projects.
• IMPORTANT! The information to the left of the 5. Select the appropriate options for printing. Click Print to close the Print
double underscore must be identical for all six or dialog box and open the standard Print dialog box for the printer.
seven sequences of a sample. The information to the
right of the double underscore is unique to the 6. Make any required changes in the Print dialog box, and then click Print to
individual sequence and should include the primer
letter. start printing.

DyeSet/Primer file KB_3130_POP6_BDTv1.mob

BaseCaller KB.bcp Users and Authentication Set-up


Sufficient signal Review the signal strength numbers for each base.If the
strength total A, C, G, and T signal strength value is below 400, To Set-up New Users
inspect the raw and analyzed data and discard any noisy IMPORTANT! Only Administrators can set up and change information in the
data. Users, Authentication, and Audit tabs.

Sequence Segments IMPORTANT! Six of the seven segments must be 1. Select Tools >Options to open the Options dialog box.
successfully sequenced in order to proceed with the
analysis in the ViroSeq software. One of the missing
segments can be A or D. 2. Select the Users tab, then click New.

3. Fill-in the appropriate user name, password, first and last names, then select
Save the Analyzed Data the level of user from the User Group drop-down list.
Select File > Save All Samples or click the Save All Samples icon from the toolbar
to save the Analyzed sample files. 4. Admin - has complete access to manage User accounts and Audit set up,
create/edit Analysis Protocol and Settings, Reports, Sample Manager,
and Sequencing Analysis.
Additional Sample Manager Functions Scientist - has access to create/edit Analysis Protocol and Settings,
Reports, Sample Manager, and Sequencing Analysis.
Analyst - has access to Reports, Sample Manager, and Sequencing
To Remove a Single File From Sample Manager Analysis.

1. Click on the sample row number.The entire row will be highlighted.

2. Click Remove Samples icon or select File > Remove Samples or use the
Delete key. The Sequencing Analysis software removes the selected file
from the list.

To Remove Multiple Files From Sample Manager

1. To remove all files, select File > Remove All Samples from the Main menu.

2. To remove several adjacent files:


a. Click on the sample row number of the first file in the group.
b. Hold down the Shift key and click on the sample row number of the last
file in the group.
c. Click the Remove Samples icon or select File > Remove Samples or use
the Delete key.

3. To remove multiple files that are not next to each other:


a. Hold down the Ctrl key while clicking on the sample row number of
each file to be removed.
b. Click the Remove Samples icon or select File > Remove Samples or use
the Delete key.

Page 26 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Section 4: ViroSeq® Materials Required
Description

HIV-1 Genotyping ViroSeq® HIV-1 Genotyping System Software v2.8 4J94-22

System Software v2.8 Computer Requirements for Running


Analysis ViroSeq® Software v2.8
The following requirements must be met to run ViroSeq software on an offline,
stand-alone computer system. The software cannot be installed on Windows NT.
• Java™ Runtime Environment (JRE)
Microsoft® Windows® 2000 - JRE version 1.4.2_10,
Introduction or
Microsoft® Windows® XP - JRE version 1.4.2_10.
ViroSeq® software processes the six or seven primer sequence files that correspond Note: JRE is included on the ViroSeq software installation CD and is
to a single plasma sample to generate a project. A project is an assembly of the automatically installed with the ViroSeq software.
sample files containing all the sequencing information required to produce a
genotype. The project format supports manual review and editing of the Configuration 1 Configuration 2 Configuration 3
electropherogram data to generate a final consensus sequence for the HIV-1 protease
and reverse transcriptase (RT) genes. Operating system requirements: Operating system requirements: Operating system requirements:
IMPORTANT! For information about known limitations and anomalies in the • Microsoft® Windows® • Microsoft® Windows® XP, • Microsoft® Windows® XP,
ViroSeq® HIV-1 Genotyping System Software v2.8, refer to the Release Notes 2000, service pack 4 service pack 2 service pack 2
included with the ViroSeq Software v2.8 CD. Computer Platform: Computer Platform: Computer Platform:
• Intel® Pentium® IV (Single • Intel® Pentium® M (Single • Intel® Core™ 2 Duo (Dual
Terminology Used in This Section Core) Core) Core)
• 2.0 GHz or above • 1.86 GHz or above • 2.4 GHz or above
Term Definition • 1 GB RAM or above • 512 MB RAM or above • 2 GB or above
ASCII American Standard Code for Information Interchange. A simple text • 40 GB hard drive or above • 40 GB hard drive or above • 40 GB hard drive or above
format that is recognizable across many platforms and computers.
Basecall The nucleotide identity assigned to a signal peak in an • Keyboard, mouse • Keyboard, mouse • Keyboard, mouse
electropherogram.
• Monitor supporting screen • Monitor supporting screen • Monitor supporting screen
Consensus Sequence A sequence developed from several other aligned sequences. resolution of 1280 x 1024 resolution of 1280 x 1024 resolution of 1440 x 900
Edit Cursor A graphical element that highlights a single nucleotide in the
consensus sequence and that is the focus for an edit operation.
Electropherogram The processed signals of the fluorescence of sequencing dyes
computed by the collection and analysis software provided with Installing, Uninstalling and Using the
sequencing instrumentation.
FASTA An ASCII-formatted text file that contains file name information and a
sequence.
Software
IUB International Union of Biochemists Before installing or uninstalling ViroSeq® software v2.8, you must log on to the
Java A programming language designed to generate applications that can Windows system using an account with Administrator privileges. Do not attempt to
run on different computer operating systems without modification. install or uninstall ViroSeq software using a Standard User or Restricted User
Navigation Bar A graphical element showing POIs and providing a means to navigate
account.
to the corresponding data.
POI Position of Interest. A nucleotide position that warrants inspection by
the user. To Install the Software On a Windows 2000 or XP
Primer Identification The matching of a sequence to a known primer. Computer
Project The software entity that comprises the data, editing history, trim
positions, assembled sequences, and so forth, associated with
processing the sequence segments from a single HIV-1 sample. Administrative privileges are required for the installation to run properly.
Project File The primary file created and used by the ViroSeq software. It contains
all the data necessary to produce a genotype analysis. 1. Close all other desktop applications.
Reference Sequence The fixed sequence against which all comparisons are made. The
ViroSeq reference sequence is based on HXB-2 (GenBank K03455). 2. Insert the ViroSeq software v2.8 CD into the CD drive.
Sample File A file containing the sample sequence data, generated in the
Sequencing Analysis Software.
3. The installer should start automatically. If it does not, perform the following
Sequence A string of nucleotide bases.
actions:
Sequence Segment The sequence data resulting from one primer.
a. Double-click My Computer on the desktop.
Trimming Marking data for exclusion from use in deriving the consensus b. Using the right-mouse button, select the drive that contains the CD.
sequence.
c. Select Explore.
VM Virtual Machine. The Windows Java™ interpreter that allows the
computer to understand the Java language. d. In Windows Explorer, open the ViroSeq software folder.
Double-click Setup.exe.
XML Extensible Markup Language. A flexible text format used in exchange
of data on the Web and elsewhere.
4. Accept the terms of the License Agreement.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 27 of 68


5. Follow the instructions on the installer screen. 3. Type your password in the New Password box and in the Re-Enter
Note: The software is installed in the C: directory. A different directory may Password box.
be selected, if desired. The ViroSeq v2.8 icon is automatically installed on Note: Your password is case sensitive and must be at least six but not more
the desktop. than 14 characters long.

4. Click OK.
To Uninstall the Software On a Windows 2000 or
XP Computer
To Add New Users
The software can be easily removed from the computer using the Uninstall program.
The uninstaller will remove all program files and the desktop icon, but it will not Only trained operators are allowed to use the ViroSeq software.
remove any user-modified files from the system.
1. In the Navigation Window, select Edit > Change Passwords.
1. From the Windows Start menu, select Start > Programs >ViroSeq v2.8 >
Uninstall. Note: A ViroSeq project must be opened in order for the Navigation
Window to appear.

2. The InstallShield Wizard dialog is displayed. 2. In the User Information panel, type the new user name you want to add
into the User Name box.
3. Click Remove.
3. In the Employee ID box, type an employee identification name or number.
4. Click Next.
4. Type a password in the New Password box and in the Re-Enter
5. A confirmation dialog is displayed. Password box.

6. Click Yes to remove the application. 5. Click Add User.


The new user is entered in the Registered Users panel.
7. The uninstall process will complete automatically, and then display the
Maintenance Complete dialog window. 6. You can continue adding users as needed, then click OK when you have
finished.

To Start the Software


There are three ways to start the software: To Remove a User
1. Double-click the ViroSeq v2.8 desktop icon. 1. In the Navigation Window, select Edit > Change Passwords.
The Administrator Access dialog box opens.
WARNING! Do not open more than one instance of the ViroSeq
software v2.8 application concurrently.
2. In the Registered Users panel, select the name you want to remove.
2. Launch ViroSeq software from the Start menu. The Remove button becomes enabled.
a. Click the Start menu.
b. Select Programs > ViroSeq v2.8 > ViroSeq v2.8. 3. Click Remove to delete the selected user, then click OK.

3. Launch the software from C:\ViroSeq v2.8.


a. Click the C:\ViroSeq v2.8 directory. To Change User Passwords
b. Double-click the ViroSeq icon.
The Administrator may change user passwords in the Administrator Access dialog
window after selecting Edit > Change Passwords. Non-administrators may change
user passwords in the User Access dialog after selecting Edit > Change Passwords.
To Log Into the Software When the User Access dialog appears, enter the Current Password, New Password,
and Re-Enter Password fields, then click OK.
For security purposes, the software requires each user to log on to the software. The The following rules apply to user names and passwords:
User Login dialog displays when the software is launched. Enter a valid user name • User names must be unique. They are not case sensitive.
and password, then click OK. The user name as well as the system date and time are
recorded in the History Window. Refer to Software Error Messages. • Spaces are allowed in the user name and password.
If logging in for the first time, the default user name is Admin and the password is • Passwords are case sensitive.
hiv1. • Passwords cannot be blank.
• Passwords must be at least 6 characters long, but not longer than 14 characters.
• Passwords are encrypted as asterisks when displayed on screen.
To Change the Administrator Password • Passwords can be made up of letters, numbers, and special (printable) characters.
• The default password hiv1 cannot be used when adding a new user.
1. Type your location name into the Installation Site box.
Note: Abbreviations and numerical identifications are acceptable.

2. For Default Password, type hiv1.

Page 28 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


To Verify the Software Installation 2. Navigate to the folder containing your Sequencing Analysis software files
and open it.
Verify that the ViroSeq HIV-1 Genotyping System Software v2.8 was installed
correctly by analyzing the QA10 demonstration sample files (.ab1) located in the
C:\ViroSeq v2.8 directory. 3. Select one sequence data file and click Open.
The software assembles and aligns the primer sequence files from a sample
1. Click New in the Project Status window. The Open dialog box opens. to generate a consensus sequence and creates a project.
A Progress window opens while the software is assembling the project.
2. Navigate to C:\ViroSeq v2.8 > Projects > Demo > QA10 folder. Note: The software creates separate projects for each unique sample name
given to the data in the Sequencing Analysis software. There is a limit to the
3. Select one sequence data file and click Open. number of projects that can be assembled at one time. A conservative limit is
14 projects.
The software assembles and aligns the data to generate a consensus sequence
When complete, the Progress window closes and the Navigation and
using .ab1 files found in the QA10 folder. The Navigation and View*Edit
View*Edit windows open, displaying one of your assembled projects.
windows open, displaying the completed assembled project for QA10.

4. Select File > Generate Resistance Report to preview the Drug Resistance ViroSeq software displays existing, assembled projects in the Project Status
Mutations listed on page 1 of the Antiretroviral Drug Resistance report. window. View the Project Status window by going to the Navigation Window and
selecting File > Open Project. The Status column indicates whether or not the
software found all the input segments.
Verify that the resulting drug resistance mutations match the information in the table
below:
If the Status is . . . Then . . .
QA10 RT Protease
OK All of the expected segments were found and the project assembled
Resistance M41L L10I successfully.
Mutations A62V L24I
T69ins M46I
? ViroSeq software encountered assembly issues.
V118I F53L
M184V I54V Possible causes include:
T215Y A71V • One or more segments could not be identified
• Fewer than 6 segments were assembled
V82A
Note: Do not proceed if fewer than six segments are assembled into the
project. One of the missing segments can be A or D.

Project Overview
To Open an Existing Project
Main Steps in a Project
1. In the Project Status window, select the project you want.
If the project you want is not in the Project Status window:
DNA Sequencing Data Files Check for:
• Correct sequence quality a. Click Find to open the Open dialog box.
• Defined boundaries b. Navigate to the folder where the project is stored.
• Correct sample names c. Select the project you want.
• Appropriate DyeSet/Primer file
2. Click Open. The Navigation and View*Edit windows for the project open.
Create a New Project • Find and select sequencing files
• Assemble the project
IMPORTANT! Do not open and edit the same project file in more than one
Review Project Data Verify that: instance of the ViroSeq software. Editing the same data segments in multiple
• A sufficient number of sequencing files are assembled instances of ViroSeq software at the same time will result in unexpected software
• Sample names are correct behavior.
• Sequencing files are aligned in the correct order and
orientation

Edit Data • Enter patient, lab, and sample-specific information


The Navigation Window
• Distinguish peaks and noise
After assembling a project, ViroSeq software graphically presents the consensus
• Trim segment ends
sequence in the Navigation window. The Navigation window has two parts:
• Verify and reconcile mixed basecalls and mismatched
bases • Navigation bar
• Compare with reference sequence • Segment alignment section

Prepare the Resistance Report • Print or save it in XML format


• Save the FASTA file

To Create a New Project

1. Launch ViroSeq software and click New in the Project Status window.
The Open dialog box is displayed.
If ViroSeq software is already open, select File > New Project.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 29 of 68


About the Navigation Bar About the View*Edit Window
The Navigation bar is in the top portion of the Navigation window. It provides an
overview of the number and locations of positions of interest (POIs). These appear The View*Edit window displays the consensus sequence generated by the software.
as vertical lines in a gray bar. It also contains navigation and editing tools.

Numbers on the horizontal scale represent codon positions for each gene. POIs are
color coded as shown in the following table.
Color Coding for Positions of Interest (POIs)

Color Meaning

Gray = background color No difference relative to the reference


Red Selected POIs
Black Mismatches between segments
Green Known or unknown difference between reference and consensus
sequence
Blue Multibase position
Yellow Shows areas covered by only one segment during assembly
White User-confirmed basecall
Note: You must save your project first.

About the Segment Alignment Section Parts of the View*Edit Window and Their Descriptions

The segment alignment section is displayed in the lower part of the Navigation
window. This portion graphically shows how the sequence segments overlap. Item Name Description

1 Jump left button Jumps to the leftmost POI.

2 Reverse jump button Jumps to the POI to the left of the active POI.

3 Trim button Trims and untrims the selected end of a segment in the
The label on each segment consists of the project name and the primer that was used. electropherogram.
The segments point in the direction of polymerization.
Verify that the sequences are positioned as expected. 4 Revert button Reverts the current POI to its original base assignment.
• Forward direction - primers A, D, B, C
5 Editing palette Edits the active POI.
• Reverse direction - primers F, G, H Buttons represent letter codes for bases and mixes of bases.

In rare cases, segment D may be aligned at the wrong location. After 6 Auto jump on edits When checked, causes an automatic jump to the next POI after
assembling the project, check the position of segment D. It should completely check box each edit.
cover the protease gene and the beginning of the RT gene. If segment D is not
positioned as specified, please assemble the project again without segment D. 7 Reverse jump check Used after editing the current position, jumps to the next POI to
box the left.
This box is only active when the Auto jump on edits check box
is selected.

8 Navigation Options Opens the Navigation Options dialog box. In this dialog box
button you select the active POIs.
Active POIs are those POIs visited by the jump buttons.

9 Forward jump button Jumps to the POI to the right of the active POI.

10 Jump right button Jumps to the rightmost POI.

11 Labels panel Labels each line in the sequence panel. Also gives the name of
primer and direction of polymerization for each sequence.

12 Codon number Gives the codon number for the reference sequence.

13 Reference Translation Gives the one-letter code for the amino acid translation of the
code for the reference sequence.

14 Consensus Translation Gives the one-letter code for the amino acid translation of the
code for the consensus sequence.

15 Reference Sequence Bases of the reference sequence.

Page 30 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Parts of the View*Edit Window and Their Descriptions (continued) Amino Acid Codes

Item Name Description Amino Acid Three-Letter Code One-Letter Code

16 Consensus Sequence Bases of the consensus sequence. Lysine Lys K

17 Box around base Current POI. Methionine Met M

18 Sequence peaks Electropherogram for a sequence segment. ViroSeq software Phenylalanine Phe F
shows all sequence segments that overlap at a position.
Proline Pro P
19 Scroll bar Horizontal scroll bar for the sequence panel.

Serine Ser S
20 Position information Explains why a position has been identified as a POI.
box
Threonine Thr T
21 Close View Edit Closes the View Edit window.
Tryptophan Trp W

Editing Palette Tyrosine Tyr Y

Valine Val V

File Menu

IUB Codes

IUB Codes

A = adenosine S = G or C (Strong—3 H bonds)


C = cytidine W = A or T (Weak—2 H bonds)
G = guanosine Y = C or T (pYrimidine)
T = thymidine B = C, G, or T
U = uracil D = A, G, or T File Menu Options
K = G or T (Keto) H = A, C, or T
M = A or C (aMino) V = A, C, or G Keyboard
Menu Item Description
Shortcut
R = A or G (puRine) N = aNy base
New Project Ctrl+N The Open dialog is displayed.
Amino Acid Codes By clicking the New button you can select and
open a Sequencing Analysis file in a folder and
the software assembles all sequences in that
folder into new projects.
Amino Acid Three-Letter Code One-Letter Code

Open Project Ctrl+O The Project Status window is displayed.


Alanine Ala A
Select a project from the list, or by clicking
the Find button, you can select and open a
Arginine Arg R ViroSeq project that has already been assembled.

Asparagine Asn N Save Ctrl+S Saves the open project with any edits that you
have made.

Aspartate or Aspartic acid Asp D


Save consensus [FASTA] Ctrl+F Stores the consensus sequence in an ASCII text
file with the extension .fasta.
Cysteine Cys C
Generate Resistance Report Ctrl+G Allows you to view and print the Resistance
Glutamine Gln Q Report.

Glutamate or Glutamic acid Glu E Export Resistance Report Ctrl+X Allows you to export the Resistance Report.

Glycine Gly G Quit Ctrl+Q Exits the ViroSeq software.

Histidine His H

Isoleucine Ile I

Leucine Leu L

ViroSeqTM HIV-1 Genotyping System v2.0 Page 31 of 68


Edit Menu • Reference sequence
ViroSeq software compares the consensus sequence to a reference sequence,
HXB-2.
• Consensus and reference translations
ViroSeq software shows the amino acid sequence in the reference virus strain
and all possible amino acid translations of the consensus sequence.
• Codon number
ViroSeq software numbers codons based on the reference sequence.

Edit Menu Options Review the Assembled Project

Keyboard
Menu Item Description
Shortcut
Once ViroSeq software has assembled a project, you must review and
verify basecalls at POIs in the consensus sequence. You can accept or change
Toggle Position Cursor Ctrl+T Turns the positioning line of the cursor on and
off. the basecalls where the software detects differences between the consensus
sequence and the reference sequence. You decide whether the suggested
Edit Report Information Ctrl+E Opens the Edit Report Information dialog basecall is justified by looking at the electropherograms. For example, dye
box, where you enter the patient, laboratory, blobs at the beginning of a sequence or several repeats of the same base can lead
and technician information for your final report the software to misjudge the sequence and to suggest a variation where there is
header.
none.
Change Passwords — When logged in as administrator, opens the
Administrator Access dialog box, where you
can change passwords and perform other
administrative functions.
POIs
When logged in as a user, opens the User A POI is a base in the consensus sequence that is one or more of the following:
Access dialog, in which you can change your
password. • Different from the HIV-1 reference strain
• Found in the internal table of known resistance positions
Login As New User — Opens the User Login window. • Identified as mixed base in at least one segment at that position
• An insertion relative to the reference sequence
• Different between segments
Window Menu Note: One or more POI criteria may apply to a given consensus position.

Base Color Codes

Base Color
Window Menu Options
Adenine Green
Keyboard
Menu Item Description
Shortcut Cytosine Blue

History — Opens the History log. Guanine Black

Thymine Red
Help Menu
Mixed bases Pink

To Set Navigation Options


Help Menu Options The Navigation Options window opens when you click the Navigation Options
button in the View*Edit window.
Keyboard
Menu Item Description
Shortcut

About ViroSeq — Displays product information.

Parts of an Assembled Project


The assembled project consists of:
• Electropherograms for the sequence segments
ViroSeq software represents bases as peaks with a different color for each of the
four bases.
• Consensus sequence of the sequence segments
With seven different primers, sequence segments overlap and cover each section
of the targeted gene at least twice and in both directions. (The exceptions are RT
codons at approximately 315-335.)

Page 32 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Navigation Options Explained Editing Sequence of Events
• Trim segment ends to remove poor quality sequence segments.
Option Description
• Verify and reconcile mixed basecalls and mismatched bases.
• Compare areas with mobility problems with other sequence segments and with
Global Settings the reference sequence to determine the correct peak position.

Additional variants from reference Locates mutations that are not used in the ViroSeq
software algorithm.
To Edit a Project

Multibase positions Allows you to go to the consensus sequence positions 1. Click Navigation Options to open the Navigation Options window.
that are derived from segments that have mixtures, or
more than one nucleotide, at a position.
2. Make sure all the check boxes for the options in the Navigation Options
Mismatches between segments Locates differences in a consensus sequence position
window are selected.
derived from forward or reverse primer segments that
do not agree at that position.
3. Select and trim areas of poor sequence quality to improve basecalling.
On the Navigation bar, dense groupings of vertical bars represent areas
Show saved edits Locates bases that were manually edited and saved in
the project. where poor sequence quality of one or more primers may exist.

Insertions Locates extra bases that are not part of the reference 4. Confirm or edit the basecalls.
sequence.
a. Begin at the leftmost POI in the protease gene by clicking on the
Note: For information on how the software handles button to edit or confirm the basecall.
insertions, please refer to Insertion Mutations.
b. Press the spacebar to confirm your selection.
c. Proceed to the next POI by pressing to edit or confirm the basecall.
Features from Gene Profile Repeat until you have edited or confirmed all the POIs in the consensus
sequence.
Show Resistance positions Makes every mutation used in the drug resistance Note: If overlapping sequences do not agree in the majority of positions,
algorithm a POI, whether or not the consensus investigate for possible sample mix-up or naming errors.
sequence differs from the reference sequence.
Note: This can only happen if you uncheck the Only
show if variant box. 5. In the Navigation Options window, clear all the check boxes except Show
Resistance positions and click OK.
Only show if variant Checking this box, in addition to the Show Resistance
positions box, selects only those drug resistance
codons in the consensus sequence for which mutations 6. Repeat the edit/confirm procedure starting at the far left and moving to the
are detected that have a role in determining drug right.
resistance.
This feature may be used when creating new projects. Note: You can use editing guideline 1 in the following summary to identify
Use with saved or edited projects is not recommended. mixtures that the software may have missed.

To Confirm or Edit the POI Reasons for Using Editing Guidelines


For each position of interest (POI) you select in the Navigation Options, you must IMPORTANT! Operator to operator consistency in the manner of basecalling is
manually confirm or edit the position. essential. The sequence editing guidelines provide you with a method for achieving
The cursor in the View*Edit window is a box around the letter code of a base or POI that consistency.
in the consensus sequence. You can either confirm or edit any mismatches between In most cases, the ViroSeq software accurately identifies mixtures. In those cases
overlapping segments or mutations that have been called by the software. where you need to make a determination, use these guidelines to distinguish real
mixtures.
IMPORTANT! The quality of the sequence data determines the accuracy of
To Distinguish Peaks from Noise basecalls. The lower the background or noise, the fewer the number of edits you will
have to make.
A peak is an electrophoretic signal that has a definable maximum and clearly
resolved slopes on either side. Peaks represent the signal strength of the fluorescent
dye bound to each DNA sequence fragment. Peaks are read and analyzed by the Summary of the Editing Guidelines for Identifying
Data Collection and Sequencing Analysis programs. Mixtures
You can call a mixture if:
1. Two opposite sense sequence segments contain secondary peaks clearly
above the local noise (approximately double the noise level).
2. One sequence segment contains a secondary peak at least 30% of the primary
peak and three times the local noise level.
Take note of resistance mutations in regions of single coverage – only editing
Well-Resolved Peaks Poorly-Resolved Peaks
guideline #2 can be used to identify a resistance mutation.
3. One sequence segment contains a secondary peak at least 30% of the primary
Note: Shoulders on peaks are defined as a peak only if they also have a definable peak and the segment in the opposite direction confirms the mixture.
maximum.
Noise is the background signal present in an electropherogram. It can be caused by
the instrument or by the quality of the capillary array, polymer, sample, buffer, or
Hi-Di formamide.
Mixture peaks should be distinct from noise peaks. However, when high levels of
continuous noise are present in a sequence, real data can be obscured and smaller
peaks cannot be distinguished from noise.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 33 of 68


Hierarchy for Project Editing Example of Diminished Peaks
1. For a first pass of the data, select all navigation features. This allows you to Diminished peaks, as seen in the example below, may appear across the entire
stop at all positions where a mixture has been called. This occurs because at sequence segment or may appear in limited regions within a sequence segment.
least one of the sequences shows a 30% mixture. These peaks are primarily caused by the presence of dye blobs at the beginning,
middle or end of a sequence segment. Less frequently, the diminished peaks are
2. Evaluate if the 30% mixture is real (that is, above the noise level). caused by low signal intensity.
3. If the 30% criteria are met, check if the opposite sequence shows a confirming If diminished peaks are evident, refresh the View*Edit window or trim the dye blobs
peak (Guideline 3). If the confirming peak is present, call the mixture. that appear at the beginning of the sequence segment using Sequencing Analysis
4. If the opposite strand does not show a confirming peak, determine if the 30% (refer to the Review Sequencing Analysis Data section). If the trimming does not
resolve this observation, then re-inject or repeat Cycle Sequencing.
peak (in the original strand) is 3 times the background. If the 3 times the
background criterion is met, call the mixture (Guideline 2). This is the rule for
a mixture in one direction only.
5. After you have made one pass through the data, the navigation options should
be changed to stop only at the resistance positions. Remember — you must
deselect the Only show if variant feature.
6. Make a second pass through all the data, looking for mixtures which meet
Guideline 1 (2  background in both directions). At this time, also review the
edits and automatic calls made in the other resistance positions.

Examples
Example of Good Sequence Data
The following example shows very low background and secondary peaks.

Examples of Guideline 1

Pass
Guideline 1: Two opposite sense sequence segments contain secondary peaks clearly
above the local noise (approximately double the noise level).
The ViroSeq® software automatically identifies mixtures at 30% of the primary
(larger) peak. In some situations, you see mixtures that are not called by the software
because they are below 30%.

Example of Poor Sequence Data


The following example shows very high background, and secondary peaks cannot be
confidently called. False mixtures can be called due to background noise. This
sample should be repeated.

To edit a basecall in this situation (second position of codon 20), follow Guideline 1
to accurately and consistently make the correct basecall. The correct basecall in this
example is W.

Page 34 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Fail Fail
Below is an example of a mixture peak that is present in both directions, but cannot Below is an example of a mixture (first position of codon 32) that cannot be called.
be called because it is the same size as the surrounding background noise peaks. The This is a false mixture that is caused by a dye blob at the start of primer B.
peaks themselves are noise.

Examples of Guideline 3

Pass
Examples of Guideline 2
Guideline 3: One sequence segment contains a secondary peak at least 30% of the
Pass primary peak and the segment in the opposite direction confirms the mixture.
The ViroSeq™ HIV-1 Genotyping System sequence primers provide double
Guideline 2: One sequence segment contains a secondary peak at least 30% of the
primary peak and three times the local noise level. coverage for the full length of the Protease and up to approximately codon 315 of the
RT gene, and single coverage from approximately codon 315 to 335. The mixture is
In rare situations a mixture sequence is seen in only one direction. To call this seen in both the forward and reverse directions, making mixture calls more reliable.
mixture, the single segment mixture peak should be well defined and unambiguous.
The ViroSeq software identifies this type of mixture as “. . .Mismatch,. . .Mixture”. In many cases, the mixture is greater than 30% of the primary peak in one direction
and less than 30% in the other. The lower sequence (the segment in the opposite
direction) is used to confirm the presence of the mixture.

The example above shows a mixture where one sequence segment contains a
secondary peak above 30%, and the other sequence segment shows no mixture (it
does not have a resolvable maximum). The mixture present in the top sequence is at
least 30% of the primary peak, and significantly greater than background.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 35 of 68


Fail
Editing Data
Below is an example of a false mixture (third position of codon 254) that is caused
by the high continuous background noise. The correct basecall is C.
About Trimming with ViroSeq® Software
Sequences in the beginning or the end of a segment are often of poor quality.
ViroSeq software automatically trims these sequences. The software also allows for
manual trimming.
The trimmed part of a sequence is still visible as part of an electropherogram but it
appears on a gray background on the screen. ViroSeq software ignores differences
between trimmed sequences and the reference sequence when determining the
consensus sequence. However, you can still use this information (in the grayed area)
when making your basecall.

Exceptions to the Guidelines


The examples below show anomalous positions at RT151 and RT215 that do not
follow the editing guidelines. When there are mixtures at these positions, they
frequently show a pattern like that illustrated below.
Compare these examples with your sample. If the patterns match then a mixture can
be called for these positions.

Trimmed
sequence

Example of Trimming

Before Trimming

Page 36 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


After Trimming To Edit a Basecall
To Edit a Mutation

1. Make the edit.


a. Click the button in the editing palette that corresponds to the type of
mutation you are editing or enter the appropriate base call letter (IUB
code) for the POI using the keyboard stroke.
b. Press the spacebar to confirm the edit.

2. Follow the editing guidelines for mixtures.


1. Two opposite sense sequence segments contain secondary peaks clearly
above the local noise (approximately double the noise level).
To Trim Segment Ends Manually 2. One sequence segment contains a secondary peak at 30% of the
primary peak and three times the local noise level.
Take note of resistance mutations in regions of single coverage – only
1. In the View*Edit window, select the region of poor quality by double- editing guideline #2 can be used to identify a resistance mutation.
clicking this portion in the electropherogram.
3. One sequence segment contains a secondary peak at 30% of the
The region from the point where you double-click to the closest end becomes primary peak and the segment in the opposite direction confirms the
highlighted in black. mixture.
IMPORTANT! Do not use sequences with high levels of continuous noise.
2. Click Trim.
The selected sequence is trimmed and now appears on a gray background.
Note: Trimming only excludes the use of trimmed data when making To Confirm a Basecall
consensus basecalls. The software does not exclude trimmed data from use
when it is assembling data. Therefore, if noisy data is causing poor assembly 1. Confirm a basecall by pressing the spacebar on the keyboard.
or alignment results, it must be trimmed using the Sequence Analysis
software.
2. To remove the confirmation, press the spacebar again.
3. To untrim a segment:
a. Select a smaller region of data and double-click to highlight it in black. To Revert to the Original Basecall
b. Click Trim.
1. Select the basecall you want to change.

Carefully review areas with insertions. These interpretations affect the 2. Click Revert.
results in the Antiretroviral Drug Resistance Report. The edit reverts to the original basecall made by the software.

To Find POIs
To Save Your Edits
There are four methods for moving the edit cursor to a new position:
To save your edits go to the File menu and select Save.
• Click the Forward ( ) or Reverse ( ) jump button to move to the next
POI as specified in the Navigation Options. The Progress window opens and indicates when the file has been saved. An entry is
made in the History window, and the file is saved in the Projects\Completed folder
• Use the scroll bar at the bottom of the View*Edit window to move through the within the ViroSeq software directory.
sequence. The edit cursor stays in the center of the window.
• Use the left or right arrow keys on the keyboard. • You can save your consensus sequence in an ASCII text format (FASTA format)
by selecting File > Save consensus [FASTA]
• Click once on the left or right side of the consensus sequence to move the Edit
window frame. The base where you click becomes the center POI. The file is saved in the directory C:\ViroSeq v2.8\Projects\Completed\Reports.
• The Antiretroviral Drug Resistance Report can be saved in XML format by
selecting File > Export Resistance Report.
The file is saved in the directory C:\ViroSeq v2.8\Projects\Completed\Export.
Note: Do not edit or modify the results after an XML version of the resistance
report is saved.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 37 of 68


To Review the Project in the History Window Example 1
The History window shows a permanent record of added, rejected, and trimmed
segments, as well as edited basecalls.
codon # 67 68 69 70 — — 71

To View the History Window


Reference D S T K W
Translation
1. From the Window menu, select History.
The History window opens.
Consensus N S S T S R W
Translation
2. Scroll through the log using the vertical scroll bar on the right to view your
activities.
Reference GAC AGT ACT AAA ––– ––– TGG
Sequence

Note: If you make edits to one base and save multiple times, the final edit is
recorded correctly in History. Consensus AAC AGT TCT ACA TCT AGA TGG
Sequence

Insertion Mutations
Example 1 shows a case where the placement of the insertion results in K70T.

In rare cases, insertions can result in an inaccurate Drug Resistance


Example 2
Report.
1. ViroSeq software is not designed to resolve cases for which there are two codon # 67 68 69 — — 70 71
dominant species of virus and one has no insertions while the other does.
This situation is easy to detect using ViroSeq and is covered below. D S T K W
Reference
2. Using gene sequencing to genotype viruses with insertions may yield non- Translation
unique solutions for the location of insertions and the existence of
mutations in the immediate vicinity of an insertion. This limitation is
illustrated and discussed below. Consensus N S S T S R W
Translation
3. In samples where an insertion is present, at least two primer sequences
are required for correct base calling of the insertion. Ensure that the area
with the insertion has at least two primer sequences that are untrimmed. If a Reference GAC AGT ACT ––– ––– AAA TGG
single primer sequence is available in the area of the insertion, inaccurate Sequence
results will be generated.
The ViroSeq software can detect the insertion mutations when the mutation is Consensus AAC AGT TCT ACA TCT AGA TGG
present in a homogeneous population. However, the ViroSeq software cannot Sequence
interpret a sample with a mixture of two or more virus populations where one
population contains an insertion and the other does not. This limitation exists with
all sequencing methodologies. Insertion mutations in a mixture result in sequence
shifts that are different for each virus population, causing the basecalls downstream Example 2 shows a case where the placement of the insertion results in K70R.
of the insertion to be seen as mixtures. Both placements are equally probable and valid because both have the same number
This phenomenon is plainly visible in the ViroSeq software as a series of strong of differences between the reference and consensus sequences.
mixtures beginning at the end of the insertion and continuing until the end of the The insertion at codon 69 occurs at a very low frequency, less than 1%, in patients
sample sequence for that primer. on prolonged therapy.25 A recent report indicates that the investigators were unable
In the example below, Virus 2 has a three-base insertion that offsets the alignment of to detect mixtures (wild type and mutant) in patients with virus containing
the two mixture sequences. the codon 69 insertion.26 This result suggests that the insertion mutation replaced the
wild type rapidly, and mixtures, if any, would be present at extremely low
frequencies.
Consensus: GAC AGT rsk Amw wrr wGr ArA

Virus 1: GAC AGT ACT AAA TGG AGA AAA Antiretroviral Drug Resistance Report
(no insertion)

Virus 2: GAC AGT GGG ACT AAA TGG AGA To Create the Antiretroviral Drug Resistance
(with insertion)
Report
You can include laboratory and sample-specific information into a project by using
The high incidence of mutations in the HIV-1 genome creates ambiguity with the Edit Report Information menu item. The laboratory-specific information can
determining the locations of insertions. This ambiguity can make it difficult to be saved as a default and printed in every Antiretroviral Drug Resistance Report. It
determine whether or not a certain resistance mutation exists. A prime example is is consistent with every user in your laboratory.
the insertion that occurs in the vicinity of codon 69. There are three important
resistance mutations in this vicinity: Technician, patient and sample information that is unique to each sample can be
entered for every project using the Edit Report Information menu item.
• D67N
• T69D • The special character, double quotation marks (“), is not supported for entry into
the resistance report header. Avoid using this in any of the Edit Report
• K70R Information fields.
Consider the following examples that show two possible interpretations of the • In the Comments field, you can enter a maximum of five lines and up to 256
insertion. characters, including spaces. If you enter text that exceeds this limitation, it may
not be displayed on the report.
• In the Comments field, do not use the Enter key, as this will result in loss of
information.

Page 38 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


• If you edit report fields in an existing project, it is recommended that you
complete generating and saving the resistance report before opening another
To Interpret the Antiretroviral Drug Resistance
project in the ViroSeq® software. Report
To Create a Default Header Laboratory and Sample Information
This portion of the report is user defined. In the Edit menu, the Edit Report
1. Select Edit > Edit Report Information.
Information option opens a dialog box for information entry.

2. In the Edit Report Information window, type in the information that you
want to appear as a default on all resistance reports. About Mutations and Resistance
It is well recognized that development of resistance to a drug is a continuing process
3. Click Set As Default, then click OK to close the dialog box. with some mutations conferring low-level resistance, others conferring higher-level
resistance, and combinations having additive or synergistic effects.27,29
Note: To edit your default information, type in your changes, then click
Set As Default and OK. Interaction between mutations conferring resistance to a drug is well defined for
some drugs, but not others. Additional studies are required to decipher the exact
contributions of each of these combinations of mutations to the development of
high-level resistance. In instances where the data do not clearly indicate evidence of
To Enter Sample-Specific Information a high level of resistance, the mutations detected may indicate impending resistance.
These are reported as markers of Possible Resistance.
1. From the Edit menu, select Edit Report Information. In some instances, mutations may cause hypersusceptibility to a particular drug.

2. In the Edit Report Information window, type in the assay operator, patient, HIV-1 Antiretroviral Resistance
and sample information and/or comments.
Antiretroviral resistance is reported for three classes of drugs:
Note: The default entries in the Edit Report Information window are a series
of five dashes. If the user does not customize the Edit Report Information • Nucleoside Reverse Transcriptase Inhibitors
window, then the series of dashes is automatically included in the drug • Non-Nucleoside Reverse Transcriptase Inhibitors
resistance report, the FASTA file, and the XML file. • Protease Inhibitors
Evidence of Resistance is reported in the far right column on page 1 of the report. It
3. Click OK to close the dialog box. is given for each drug within a class and is based on the mutations found in the
sample. The presence of a specific mutation or combination of mutations results in
4. Click Save to save changes. one of the following calls:
• Resistance indicates a high level of evidence of resistance.
• Possible Resistance indicates possible evidence of resistance. Mutations or
Note: When editing report information, the changes must be saved in order for edits combinations of mutations that do not meet the criteria required for Resistance,
to take effect. but are suggestive of resistance.
• None indicates insufficient evidence for resistance.
To View the Report
. Disclaimer Levels
1. Select File > Generate Resistance Report. The Evidence of Resistance levels are labeled with either none or up to three
Use the buttons at the top of the window to reduce or enlarge your view, or to asterisks. The meanings of the asterisks are described in the following table:
page through a multipage report.
Meaning in Terms of
2. Select the Save icon to save the generated report as a Portable Document Disclaimer Label Disclaimer Analytical and Clinical
Format (PDF) file. Studies

3. The Save Report dialog is displayed. Select the directory where the resulting (no label) none All mutations present have been
PDF report file will reside and click Save. verified in clinical and analytical
studies.

* NOTE: At least one mutation All mutations present have been


To Print used to determine Evidence of validated in clinical studies but
Resistance for this drug has not may not have been verified in
been fully validated. analytical studies.
1. Select File > Generate Resistance Report if the report is not already open.
At the top of the ViroSeq Report window, click the Print icon. ** NOTE: At least one mutation All mutations present have been
used to determine Evidence of verified in analytical studies but
Resistance for this drug has not may not have been verified in
2. When the Print dialog box opens, select your printer and click OK. been clinically verified. clinical studies.

*** NOTE: For at least one At least one mutation was not
mutation used to evaluate verified in both clinical and
Evidence of Resistance for this analytical studies.
drug, both notes above apply.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 39 of 68


The Protease Inhibitor drug class is labeled with a + indicating a disclaimer label. A low Total Score (below 0.5) is interpreted as insufficient for resistance. A medium
Total Score (between 0.5 and 1) is interpreted as possible resistance. A high Total
Score (1.0 or more) is interpreted as resistant. The table below shows how the Total
Score maps to the Level of Evidence for Resistance rating.
Disclaimer Label Interpretation
Relationship of Total Score to Level of Evidence for Resistance

The protease inhibitor (PI) evidence of resistance interpretations


were developed to estimate the expected virological response to Total Score Level of Evidence for Resistance
standard doses of protease inhibitors with pharmacokinetic
+ boosting by ritonavir. This has become the most common method
of administering each of the protease inhibitors, except nelfinavir,  1.0 Resistance
to ensure adequate drug levels in all patients. Boosted PIs are more
active in the presence of resistance than
non-boosted PIs. 0.5 to <1.0 Possible Resistance

<0.5 None
Mutation Notation at Resistance Positions
Mutations are represented using style, color, and punctuation conventions to indicate
We used the following criteria when assigning scores to specific mutations:
their relevance to the development of drug resistance.
• Mutations with the greatest contribution towards development of resistance,
• {Red Bold Curly Bracket} – mutations which by themselves confer viral
resistance and result in a Resistance call. usually those that confer resistance when present as a single mutation, were
assigned the highest score (1.0). Correspondingly, these mutations were also
• [Blue Bold-Italics Square Bracket] – mutations which by themselves confer the associated with the greatest increases in IC50 (50% Inhibitory Concentration)
possibility of viral resistance and result in a Possible Resistance call. when introduced into wildtype (WT) strains of HIV-1 and were clearly
• (Black Parenthesis) – mutations which contribute the least to the development associated with resistance in multiple clinical trials.
of resistance. More than one of these mutations is required to produce a Possible • Mutations with less pronounced effects and requiring accumulation of two or
Resistance call. This mutation must appear with at least one other mutation to more mutations for conferring resistance were assigned intermediate scores. For
confer the possibility of viral resistance. example, when considering resistance to AZT, we assigned scores of 0.625 for
• <Green Angle Bracket> – This mutation counters resistance. the T215F/Y mutation, 0.375 for the M41L mutation, and 0.25 for the K70R
mutation. The T215F/Y substitution results in a 12-16 fold increase in IC50 for
AZT whereas the M41L or K70R mutations cause a 3-5 fold increase. The
Additional Mutations combination of M41L and K70R results in a 9-fold increase in IC50.
The Additional Mutations section of the report identifies amino acids that differ • Mutations with the least impact on the development of resistance but observed in
from the reference sequence (HXB-2, accession number K03455) at the indicated patients treated with the drug and demonstrating small effects on the IC50 values
codon positions and may be useful as a baseline determination of virus genotype. in culture, were assigned low scores. Some of these mutations can enhance the
The performance characteristics of the additional mutations have not been effects of the major mutations. In some instances multiple mutations in this
established. Additional mutations are specified either for the Protease gene or for the category, in combination with mutations with intermediate scores, could result in
Reverse Transcriptase gene. resistance.
• Mutations causing an increase in susceptibility to drugs were assigned a negative
score based on the level of susceptibility.
Notation Key
For each drug, the tables on the following pages list the mutations that are relevant to
Pages three and four of the report contain the color key and the HIV-1 Resistance resistance to the drug and the assigned score for each mutation.
Mutation List that is used in the algorithm for determining drug resistance.

Algorithm
The interpretation is based on a proprietary algorithm that determines the relative
impact of mutations in the reverse transcriptase and protease open reading frames on
the development of resistance to antiretroviral drugs. Mutations included in the
algorithm were based on:
• Mutations defined by the FDA as having established clinical utility.28
• Mutations listed as primary and secondary in the recommendations by the
International AIDS Society-USA.13,30
– This list is periodically updated and can be found at the IAS-USA web site
( http://www.iasusa.org/).
• Mutations listed in GART14 and VIRADAPT15 studies as being predictive of
drug resistance.
• Recommendations by the Resistance Collaborative Group based on their
analysis of several prospective and retrospective studies.31
• Additional mutations were factored into the schema based on more recent reports
and consultations with experts in the field to provide an interpretation consistent
with current literature.8,16,32-50 The report has separate sections for Nucleoside
Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse
Transcriptase Inhibitors (NNRTIs) and Protease Inhibitors (PIs). The mutations
used in determining resistance are listed schematically along with the drug name
in the report.
An algorithm was developed to consider all possible combinations of mutations that
can confer resistance to a given drug. We used a numerical scoring system structured
so that the combinations of mutations known to generate resistance would be
assigned a level of evidence for resistance if detected in the assay. To determine the
level of evidence for resistance to a given drug, the scores of the mutations that are
present and pertinent to that drug are summed, giving a Total Score for that drug. For
each drug, a particular codon position is only counted once using the highest
mutation score among the mutations present at that codon position.

Page 40 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Mutations Tables A2. Stavudine, d4T, ZERIT® A3. Didanosine, ddI, VIDEX®

A. Resistance to NRTIs d4T ddl

A1. Zidovudine, AZT, RETROVIR® Mutation Score Mutation Score

T69ins 1.2 T69ins 1.0


AZT
Q151M 1.2 L74I/V 1.0
Mutation Score
V75A/M/S/T 1.0 Q151M 1.0
T69ins 1.2
Q151L 0.7 K65R 0.75

Q151M 1.2
T215F/Y 0.625 Q151L 0.5

Q151L 0.7
T215C/D/E/I/S/V 0.5 M41L 0.25
T215F/Y 0.625
M41L 0.375 K65N 0.25

T215C/D/E/I/S/V 0.5
K65R 0.25 T69D/N 0.25

M41L 0.375
D67E/G/N 0.25 V75A/M/T 0.25
D67E/G/N 0.25
K70R 0.25 V75S 0.25

K70R 0.25
L210W 0.25 L210W 0.25

L210W 0.25
K219E/N/Q/R 0.25 T215C/D/E/F/I/S/Y/V 0.25

K219E/N/Q/R 0.25
V75I 0.125 D67E/G/N 0.125

V75I 0.125
F77L 0.125 K70E 0.125
F77L 0.125
F116Y 0.125 V75I 0.125

F116Y 0.125
E44D 0.0625 F77L 0.125

E44D 0.0625
A62V 0.0625 F116Y 0.125
A62V 0.0625
V118I 0.0625 K219E/N/Q/R 0.125

V118I 0.0625
M184I/V 0.2 E44D 0.0625

K65N 0.125

A62V 0.0625
M184I/V 0.2

V118I 0.0625
K65R 0.25

ViroSeqTM HIV-1 Genotyping System v2.0 Page 41 of 68


A4. Lamivudine, 3TC, EPIVIR®, A5. Abacavir, ABC, ZIAGEN® A6. Tenofovir, TDF, VIREAD®
Emtricitabine, FTC, EMTRIVA®

ABC TDF
3TC & FTC

Mutation Score Mutation Score


Mutation Score
T69ins 1.0 T69ins 1.2
M184I/V 1.0
Q151M 1.0 K65R 1.0
K65R 0.5
K65R 0.75 Q151M 0.375
T69ins 0.5
L74I/V 0.75 M41L 0.35
K65N 0.25
Y115F 0.5 T215Y 0.35
Q151M 0.25
Q151L 0.5 L210W 0.3
T215F/Y 0.25
T215F/Y 0.3 K65N 0.25
E44D 0.125
M41L 0.25 K70E 0.25
K70E 0.125
K65N 0.25 Y115F 0.2
V75I 0.125
M184I/V 0.25 T215C/D/E/I/S/V 0.15
F77L 0.125
T215C/D/E/I/S/V 0.25 D67E/G/N 0.125
F116Y 0.125
L210W 0.2 K70R 0.125
V118I 0.125
K70E 0.125 V75I 0.125
Q151L 0.125
V75I 0.125 F77L 0.125
A62V 0.0625
F77L 0.125 F116Y 0.125
M41L 0.05
F116Y 0.125 Q151L 0.125
D67E/G/N 0.05
D67E/G/N 0.1 T215F 0.125
K70R 0.05
E44D 0.0625 K219E/N/Q/R 0.125
L210W 0.05
A62V 0.0625 E44D 0.0625
K219E/N/Q/R 0.05
V118I 0.0625 A62V 0.0625

V118I 0.0625

M184I/V 0.2

Page 42 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


B. Resistance to NNRTIs B2. Delavirdine, DLV, RESCRIPTOR® B3. Efavirenz, EFV, SUSTIVA®

B1. Nevirapine, NVP, VIRAMUNE®


DLV EFV

NVP
Mutation Score Mutation Score

Mutation Score K103H/N/S/T 1.0 K103H/N/S/T 1.0

K103H/N/S/T 1.0 V106M 1.0 V106M 1.0

V106A/M 1.0 Y181C/I/V 1.0 Y188L 1.0

Y181C/I/V 1.0 Y188L 1.0 G190C/E/Q/S/T/V 1.0

Y188C/H/L 1.0 P236L 1.0 L100I 0.5

G190A/C/E/Q/S/T/V 1.0 Y318F 1.0 K101P/E 0.5

A98G 0.5 A98G 0.5 V106A 0.5

L100I 0.5 L100I 0.5 Y181C/I/V 0.5

K101E/P 0.5 K101E/P 0.5 Y188C/H 0.5

V108I 0.5 V106A 0.5 G190A 0.5

E138K 0.5 V108I 0.5 P225H 0.5

V179D/E/F 0.5 E138K 0.5 M230L 0.5

P225H 0.5 V179D/E/F 0.5 A98G 0.25

F227C/L 0.5 Y188C/H 0.5 K101Q 0.25

M230L 0.5 G190E/Q 0.5 K103R 0.25

K238T 0.5 P225H 0.5 V108I 0.25

Y318F 0.5 F227C 0.5 E138K 0.25

K101Q 0.25 M230L 0.5 V179D/E/F 0.25

K103R 0.25 K238T 0.5 F227C 0.25

K101Q 0.25 K238T 0.25

K103R 0.25 Y318F 0.25

ViroSeqTM HIV-1 Genotyping System v2.0 Page 43 of 68


B4. Etravirine, ETR, INTELENCE™ C. Resistance to Protease Inhibitors C2. Saquinavir, SQV, FORTOVASE®, INVIRASE®
®
C1. Indinavir, IDV, CRIXIVAN
ETR SQV
IDV
Mutation Score Mutation Score
Mutation Score
V179F 0.5 G48V 1.0

V82A/F/M/S/T 0.5
Y181C/I/V 0.5 I84A/C/V 0.5

I84A/C/V 0.5
L100I 0.25 L90M 0.5

L90M 0.5
K101E/P 0.25 I54A/L/M/S/T/V 0.25

V32I 0.25
E138K 0.25 G73A/C/S/T 0.25

M46I/L 0.25
Y188L 0.25 V82A/F/S/T 0.25

I47A 0.25
G190C/E/Q/S/T/V 0.25 L10F/I/R/V 0.125

G48V 0.25
M230L 0.25 L24I 0.125

I54A/L/M/S/T/V 0.25
A98G 0.125 M46I/L/V 0.125

N88S 0.25
K103H/N/S/T 0.125 F53L 0.125

L24I 0.125
V106A/M 0.125 A71I/T/V 0.125

M46V 0.125
V179D/E 0.125 I50L 0.125

I47V 0.125
Y188C/H 0.125 L76V 0.125

F53L 0.125
G190A 0.125

G73A/C/S/T 0.125
P225H 0.125

L76V 0.125
F227C/L 0.125

N88D/T 0.125

L10F/I/R/V 0.0625

A71I/T/V 0.0625

I50L 0.125

Page 44 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


C3. Nelfinavir, NFV, VIRACEPT® C4. Amprenavir, APV, AGENERASE®, C5. Lopinavir/Ritonavir, LPV/r, KALETRA®
Fosamprenavir, FOS, LEXIVA®

NFV LPV/r
APV/FOS

Mutation Score Mutation Score


Mutation Score
D30N 1.0 I47A 1.0
I50V 1.0
N88D/S/T 1.0 I50V 0.5
I47A 0.5
L90M 1.0 V32I 0.25
I54L/M 0.5
L23I 0.5 M46I/L 0.25
I84A/C/V 0.5
M46I/L/V 0.5 I54A/L/M/S/T/V 0.25
V32I 0.25
G48V 0.5 V82A/F/S/T 0.25
L33F 0.25
I54A/L/M/S/T/V 0.5 I84A/C/V 0.25
M46I/L 0.25
V82A/F/S/T 0.5 L24I 0.125
I47V 0.25
I84A/C/V 0.5 L33F 0.125
L76V 0.25
I47A 0.25 M46V 0.125
V82A/F/S/T 0.25
L10F/I/R/V 0.125 I47V 0.125
L90M 0.25
L24I 0.125 G48V 0.125
L10F/I/R/V 0.125
A71I/T/V 0.125 F53L 0.125
M46V 0.125
G73A/C/S/T 0.125 G73A/C/S/T 0.125
I54A/S/T/V 0.125
I50L 0.125 L76V 0.125
A71I/T/V 0.125
L90M 0.125
G73A/C/S/T 0.125
L10F/I/R/V 0.0625
I50L 0.125
A71I/T/V 0.0625
N88S 0.125
I50L 0.125

ViroSeqTM HIV-1 Genotyping System v2.0 Page 45 of 68


C6. Atazanavir, ATV, REYATAZ® C7. Tipranavir, TPV, APTIVUS® C8. Darunavir, DRV, PREZISTA®

ATV TPV DRV

Mutation Score Mutation Score Mutation Score

I50L 1.0 V82L 0.5 I50V 0.375

N88S 1.0 V82T 0.375 V32I 0.25

I84A/C/V 0.5 I84A/C/V 0.375 I47A/V 0.25

V32I 0.25 L33F 0.25 I54L/M 0.25

M46I/L 0.25 M46I/L 0.25 L76V 0.25

I47A 0.25 I47A/V 0.25 I84A/V 0.25

I54A/L/M/S/T/V 0.25 I54A/V 0.25 V11I 0.125

V82A/F/S/T 0.25 V82F/S 0.25 L33F 0.125

N88D/T 0.25 V32I 0.125 G73A/C/S/T 0.125

L90M 0.25 E35G 0.125 I84C 0.125

L10F/I/R/V 0.125 K43T 0.125 L89V 0.125

M46V 0.125 M46V 0.125 M46I/L/V 0.0625

G48V 0.125 I54L/M/S/T 0.125 I54A/S/T/V 0.0625

A71I/T/V 0.125 Q58E 0.125 V82A/F/L/M/S/T 0.0625

G73A/C/S/T 0.125 G73A/C/S/T 0.125 L90M 0.0625

L24I 0.0625 T74P 0.125 I50L -0.125

L33F 0.0625 N83D 0.125

F53L 0.0625 L90M 0.125

L76V 0.125 L10F/I/R/V 0.0625

A71I/T/V 0.0625

V82A 0.0625

I50L/V 0.125

Page 46 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Software Error Messages
Message Explanation Message Explanation

A unique non-blank user name Add User button is clicked in the Administrator Access window Unable to assemble files in: An attempt was made to create a project with fewer than two
must be used. Please select a when trying to create the user that already exists. <folder> segment files with the same sample ID.
different user name and try Select OK and type a unique User Name. Or
again.
Corrupted sample files were selected for assembly in the Open
dialog box.
Current Password incorrect. An incorrect or blank Current Password was entered in the User Select OK and check that two or more, non-corrupted ab1 files
Check ‘Caps Lock’ and try Access window. with same sample ID’s are presented in the working folder.
again. Select OK and type correct Current Password.
Unable to load project: <file The platform Operating System fails in opening Project file (vpr),
Default Password incorrect. There is an incorrect Default Password in Change Administrator name> due to low memory or corrupted disk.
Check ‘Caps Lock’ and try Password dialog, while launching the software for the first time. Or
again. Select “OK” and type correct Default Password (hiv1). An attempt was made to open a corrupt or incomplete project file.
Select OK and check that the vpr file is not corrupted or read-only.
New passwords must be the A valid new password was given in the Change Administrator
same. Try entering the Password dialog or User Access dialog, but the confirmatory
passwords again. password does not match. ViroSeq detected a deletion, An attempt was made to generate a drug report, FASTA file, and/or
frame shifting insertion, and/or XML report when deletion/frame shifting insertion/multiple
Select OK and type same password in confirmatory password as multiple insertions. The insertions are present in consensus sequence.
given in New password field. software has not been validated The drug resistance report, FASTA file, and/or XML report cannot
for such mutations. Output will be generated if deletion/frame shifting insertion/multiple insertions
not be created. are detected in the sequence.
Password length should be 6 The new password given in the Change Administrator Password
to 14 characters. Please choose dialog, Administrator Access dialog or User Access dialog is of
a longer password and try insufficient length.
again. ViroSeq detected that an Wrong version of JRE is installed
Select OK and type a password with length of 6 to 14 characters. incorrect version of Java Select OK and copy the correct JRE folder from the CD. Launch
Runtime Environment [JRE] is the ViroSeq software.
being used. Please refer to
The passwords entered were A valid new password was given in the Administrator Access installation instructions for Windows Platform /JRE version
not the same. Please enter the dialog, but the confirmatory password does not match. installing the appropriate JRE. Windows XP & 2000/ JRE 1.4.2_10
exact same password twice. Or
Add User button is clicked in the Administrator Access dialog ViroSeq software is not An attempt was made to install ViroSeq v2.8 on a Windows NT
with a valid new User Name and mismatched confirmatory supported on Microsoft NT operating system.
password. operating system.
Select OK and type same password in confirmatory password as
given in New password field.

User Name or password


incorrect. Check ‘Caps Lock’
Incorrect User Name or Password is entered in the User Login
dialog. ViroSeq® Software and Results
and try again. Select OK and type correct user name and password. • Only trained users are allowed to use the ViroSeq software.
• Accurate and reliable results are dependent on good sequence quality. High
Launch failed. A file is missing A ViroSeq system file or folder is missing or corrupt. background and noisy data can interfere with precise basecalling.
or damaged. Please re-install
the software. If the problem
Select OK and install the ViroSeq software again. • Understanding and using the editing guidelines found in this document will
persists, please contact assist in making more accurate basecalls, and may help identify mutations that
technical support. otherwise may be missed.
• Multiple nucleotide changes within the wild type codon are necessary to
One or more “q” or “?”s were An attempt was made to generate a drug report, FASTA file, and/or generate some mutations such as: V82T, V82S, T215Y, etc. In these cases, when
detected in the consensus XML report after editing base with “?” (with Keyboard) or “q” (by mixtures of the wild type and mutant are present, the software report indicates
sequence. ViroSeq will not clicking on the Edit Palette). the presence of two amino acids, but also reports amino acids corresponding to
generate output. The drug resistance report, FASTA file, and/or XML report cannot other possible codon combinations. For example, a mixture of Val (GTC) and
be generated if one or more “q” or “?” is detected in the sequence. Ser (TCC) at codon 82 results in a report indicating the presence of Val, Ser, Ala
(GCC) and Phe (TTC) – V, S, A, and F. This is a limitation of all population
Selected file is invalid. A sample file (ab1) is selected when the software was expecting a sequencing methodologies in that they do not define linkage of the nucleotides
project file (vpr). [File > Open Project + Find] from a single template.
Or
In accordance with the FDA Guidance document for HIV-1 genotyping, analytical
A project file (vpr) is selected when the software was expecting a studies using the ViroSeq HIV-1 Genotyping System have not been completed at
sample file (ab1). [File > New Project/File > Open Project
+ New] the limit of detection for the following mutations:
Or
Selected file type is wrong in the Open dialog box. Gene Mutation
Click OK and select correct file as expected.
RT E44D, K65N, D67E/G, D67N, T69N, K70E, L74I, V75A/I/M/S/T, A98G, L100I,
K101E/P/Q, K103H/S/T/R, V106M, V108I, Y115F, E138K, Q151L, V179D/E/F,
Sequence data does not cover An attempt was made to generate a drug report, FASTA file, and/or Y181I/V, Y188H, G190A/C/E/Q/S/T/V, T215C/D/E/I/S/V, K219N/R, P225H, F227L/C,
critical portions of the reference XML report when reference sequence does not cover between M230L, P236L, K238T, Y318F
sequence. ViroSeq will not protease codon 9 through RT codon 320. Report cannot be generated
generate output. if there is no coverage between protease codon 9 through RT codon Protease L10F/V, V11I, L23I, L24I, V32I, L33F, E35G, K43T, M46L/V, I47A/V, I50L, F53L,
320. I54A/L/M/S/T, Q58E, A71I/T, G73A/C/S/T, T74P, L76V, V82L, N83D, I84A/C,
N88D/S/T, L89V
The file is in use by another An attempt was made to save new report with the existing file
application. You must close name which is opened with MS word (or any other program which
the file in that application locks file).
before ViroSeq can output into Select OK, close the file and then save it again.
that file.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 47 of 68


Genotyping Success
Genotyping success is determined qualitatively during ViroSeq software analysis. In
order to successfully genotype a sample, six of the seven ViroSeq system primers
must assemble into a project to generate the consensus sequence (A or D, B, C, F, G,
H). In addition, a qualitative assessment of the sequence quality should be made to
ensure the reliability and accuracy of results. High background noise can interfere
with the determination of viral mixtures. The ViroSeq Software v2.8 recognizes a
viral mixture when a secondary peak, which is at least 30% of the primary peak, is
present.
The expected genotyping results for the ViroSeq controls are:

Positive Control Negative Control

Expected Value • All seven primers Do not use a negative control to monitor genotyping
assemble success or failure. Negative controls, which contain
• No drug resistance no DNA, can actually contribute to sample failure due
mutations to the adverse electrical effects that empty capillaries
• Two additional have on capillary-based sequencing instruments.
mutations
– Protease V3I
– RT L214F
• No mixed bases

Perform a qualitative visual inspection of the


electropherograms to assess the sequence
quality and degree of background in the
control.
Examine the run data for accurate instrument
calibration. If the run shows consistent and
proportional pull-up peaks (that is, smaller
peaks of another color) then perform a new
spectral calibration (refer to To Perform A
Spectral Calibration.).

The HIV-1 8E5 Positive Control is a non-infectious HIV-1 virus and should contain
little or no background. Any mutations reported, other than those given above, may
signify an editing mistake or problems with sequence quality. A comparison with the
other sample results is necessary to determine the cause of failure. For example:
• If background peaks are responsible for the failed control, and if these peaks are
seen in every sample, and if all samples have the same color noise for a given
base, then perform a new spectral calibration (refer to To Perform A Spectral
Calibration); all samples and controls should be repeated from “Reverse
Transcription” through “ViroSeq Software Analysis”.
• If the background peak colors are random, then all samples and controls should
be repeated from “Reverse Transcription” through “ViroSeq Software Analysis”.
If repeated failure occurs, please contact Abbott Molecular customer support.
Note: The HIV-1 8E5 Positive Control has a single-base insertion at codon 219 of
the RT gene that renders the virus non-infectious. The 219 mutation does not appear
during editing or on the antiretroviral drug resistance report because it is a frame-
shifting mutation that is not found in clinical viral samples.

Page 48 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Section 5: Performance Table 1. Fully Verified Performance List at a Limit of Detection (LOD) of 2,000
copies/mL

Characteristics M41L A62V K65R


In Reverse Transcriptase

T69D K70R L74V F77L K103N


F116Y V118I Q151M Y181C M184V L210W T215Y T215F
K219E K219Q

In Protease
Performance L10I K20R D30N M36I M46I G48V I54V L63P

The performance characteristics of the ViroSeq HIV-1 Genotyping System were A71V V82T V82A I84V L90M
evaluated using viral and clinical samples containing defined mutations that confer
viral resistance to antiretroviral therapy. The clinical samples used in the analysis
were obtained both prospectively and retrospectively from adults and infants and Table 2. Fully Verified Performance List at an LOD of 1,000 copies/mL
pediatric patients (from 4 months to 17 years of age) on antiretroviral therapy. The
presented results define assay sensitivity, specificity, reproducibility, and precision In Reverse Transcriptase
with reference to accurate detection of specific mutations in the RT and protease
genes of HIV-1 that confer resistance to approved medications. Clinical specimens M41L A62V K65R L74V F77L K103N F116Y V118I
were also used in studies to determine sequencing success rates and the ability to
detect mutations in mixtures, and to define clinical utility. The ViroSeq HIV-1 Y181C M184V L210W T215F K219Q
Genotyping System’s ability to detect specific mutations that confer viral resistance
in a patient sample, to evaluate drug resistance assessments, and to generate a final In Protease
Antiretroviral Drug Resistance Report that accurately follows a rules-based L10I D30N M36I M46I G48V I54V L63P A71V
interpretation algorithm have been evaluated. The use of the ViroSeq HIV-1
Genotyping System will enhance physicians’ capability to assess drug efficacy and V82T I84V L90M
design individualized therapeutic strategies in conjunction with other clinical results
such as clinical presentation, laboratory markers, and antiretroviral history.
Table 1 is a list of mutations that have been fully verified at 2,000 RNA copies/mL Table 3. Mutations used in the rules-based interpretation algorithm to evaluate viral
in accordance with the FDA “Guidance for Industry: Class II Special Controls resistance include:
Guidance Document: In Vitro HIV Drug Resistance Genotype Assay.”
Table 2 is a list of mutations that have been fully verified at 1,000 RNA copies/mL In Reverse Transcriptase
in accordance with the FDA “Guidance for Industry: Class II Special Controls
Guidance Document: In Vitro HIV Drug Resistance Genotype Assay.” M41L* E44D A62V* K65N K65R* D67E D67G D67N

Table 3 is a list of mutations that are used in the ViroSeq System rules-based T69D* T69N K70E K70R* L74I L74V* V75A V75I
algorithm. V75M V75S V75T F77L* A98G L100I K101E K101P
Table 4 is a list of drugs for which drug resistance is evaluated in the ViroSeq
K101Q K103N* K103H K103R K103S K103T V106A V106M
System Antiretroviral Drug Resistance Report.
V108I Y115F F116Y* V118I* E138K Q151L Q151M* V179D
V179E V179F Y181C* Y181I Y181V M184I M184V* Y188C
Fully Verified Performance List
Y188H Y188L G190A G190C G190E G190Q G190S G190T
As required by the FDA in the “Guidance for Industry: Class II Special Controls G190V L210W* T215C T215D T215E T215F* T215I T215S
Guidance Document: In Vitro HIV Drug Resistance Genotype Assay”, mutations are
listed below that met the greater than 90% sensitivity specification in both: T215V T215Y* K219E* K219N K219R K219Q* P225H F227C
• Analytical Performance Studies at the limit of detection (2,000 copies/mL) with F227L M230L P236L K238T Y318F Insertion mutation at codon 69
40% mutant samples (69 insertion)
• Clinical Validity studies with a panel of 50 clinical isolates (VL = 1,800 to In Protease
10,500)
Mutations M184I, V82F, and V82S were detected in clinical samples that were part L10F L10I* L10R L10V V11I L23I L24I D30N*
of the high viral load (VL greater than 10,500) Clinical Validity Study. They are not V32I L33F E35G K43T M46I* M46L M46V I47A
included in the Verified List given in Table 1.
I47V G48V* I50L I50V F53L I54A I54L I54M
I54S I54T I54V* Q58E A71I A71T A71V* G73A
G73C G73S G73T T74P L76V V82A* V82F V82L
V82M V82S V82T* N83D I84A I84C 184V* N88D
N88S N88T L89V L90M*
* Fully verified mutations

ViroSeqTM HIV-1 Genotyping System v2.0 Page 49 of 68


Table 4. The ViroSeq Antiretroviral Drug Resistance Report reports viral Table 5. Mutations Detected With 100% Sensitivity at 100% Mutant Samples at
resistance in the following drugs: 1,000 copies/mL

Nucleoside RT Inhibitors a Non-Nucleoside RT Inhibitors Protease Inhibitors In Reverse Transcriptase


EPIVIR®/lamivudine/3TC INTELENCE™/etravirine/ETR AGENERASE®/amprenavir/ M41L A62V K65R D67N T69D K70R
APV
® ® ® L74V V75I F77L K103N V106A Y115F
EMTRIVA /emtricitabine/FTC RESCRIPTOR /delavirdine/ APTIVUS /tipranavir/TPV
DLV F116Y V118I Q151M Y181C M184I/V Y188C/L
RETROVIR®/zidovudine/AZT SUSTIVA®/efavirenz/EFV CRIXIVAN®/indinavir/IDV L210W T215F/Y K219E/Q T69S + SS
[Insertion
VIDEX®/didanosine/ddI VIRAMUNE®/nevirapine/NVP FORTOVASE®/INVIRASE®/ mutation at
saquinavir/SQV codon 69 (69
insertion)]
VIREAD®/tenofovir/TDF KALETRA®/lopinavir/ritonavir/
LPV/r
In Protease
ZERIT®/stavudine/d4T LEXIVA®/fosamprenavir/FOS
L10I/R K20R D30N M36I M46I G48V
ZIAGEN®/abacavir/ABC PREZISTA®/darunavir/DRV
I50V I54V L63P A71V V82A/F/S/T I84V
REYATAZ®/atazanavir/ATV
L90M
VIRACEPT®/nelfinavir/NFV

a
The report also evaluates multinucleoside resistance due to the presence of the Q151M complex
• When a mutation was present in a 40/60 mutant/wild type mixture, the aggregate
or the 69 insertion.13 Resistance to COMBIVIR® and TRIZIVIR® can be deduced from the sensitivity was greater than 97% and the aggregate specificity of detection was
report. greater than 99.9% for all mutations at all viral loads greater than 1,000
copies/mL.
• At a viral load of 2,000 copies/mL only, and at a 40/60 mutant/wild type mixture,
Analytical Sensitivity the aggregate sensitivity was 98.1% (4 false negatives [FN]/206) and the
aggregate specificity was 99.9% (1 false positive [FP]/1422).
Two parameters were used to define analytical sensitivity. • At a viral load of 1,000 copies/mL only, and at a 40/60 mutant/wild type mixture,
the aggregate sensitivity was 92.7% (15 FN/206) and the aggregate specificity
• Determination of the minimum HIV-1 viral load for reliable mutation detection. was 99.9% (1 FP/1422).
• Determination of the lowest amount of mutant virus in a mixture that allowed • Table 6 (reverse transcriptase) and Table 7 (protease) list the sensitivities of the
reliable mutation detection. individual mutations in a 40% mutant mixture at viral loads of 1,000 copies/mL
and greater.
Limit of Detection In addition, each mutation has been assigned a Limit of Detection value.

Detection of Mutations Table 6. Sensitivity of Detection of Reverse Transcriptase Mutations at 40%


Mixture Level
The ViroSeq System was able to accurately detect the specific drug resistance
mutations listed below at 1,000 copies/mL in 100% mutant samples and at 2,000
copies/mL in 40% mutant samples. Reverse Transcriptase
Sensitivity at Sensitivity at
LOD Claim
>1,000 copies/mL >2,000 copies/mL
Description of the Study. For these studies, the Limit of Detection is defined as the Mutation (comments)
(No. of True Positives) (No. of True Positives)
lowest viral load with a sensitivity of 100%. This study utilized a single lot of
reagents. Twelve recombinant viruses were formulated in negative plasma as 100% M41L 98.8 100 2,000
pure and 40/60 mutant/wild type. (82) (71)
A62V 100 100 1,000
Results (41) (35)

K65R 100 100 1,000


• When a mutation was present in 100% of the viral population, the limit of (13) (11)
detection for all mutations was 1,000 copies/mL – that is, a 100% sensitivity and
100% specificity was obtained at 1,000 copies/mL for all of the following D67N 91.6 93 Not determined (1)
mutations. (76) (66)
In a subsequent three-
• In subsequent studies at 2,000 copies/mL in a 40/60 mixture, the sensitivity for lot study, a different lot
D67N and T69D was 83% and 50% respectively for one of three reagent lots. gave 83% sensitivity at
2,000 copies/mL; the
other two lots gave
100% sensitivity.
T69D 92.9 100 2,000
(13) (12)
In a subsequent three-
lot study, a different lot
gave 50% sensitivity at
2,000 copies/mL; the
other two lots gave
100% sensitivity.
K70R 98.2 100 2,000
(54) (47)
L74V 100 100 1,000
(14) (12)
V75I 97.6 97.1 6,000 (2)
(40) (34)
F77L 100 100 1,000
(41) (35)
K103N 98.6 100 2,000
(68) (59)

Page 50 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Table 6. Sensitivity of Detection of Reverse Transcriptase Mutations at 40% Table 7. Sensitivity of Detection of Protease Mutations at 40% Mixture Level
Mixture Level (continued) (continued)

Sensitivity at Sensitivity at
Reverse Transcriptase LOD Claim Sensitivity at Sensitivity at
>1,000 copies/mL >2,000 copies/mL
Mutation (comments) Protease >1,000 copies/mL >2,000 copies/mL LOD Claim
(No. of True Positives) (No. of True Positives)
Mutation (No. of True (No. of True (comments)
V106A 100 100 1,000 Positives) Positives)
(25) (21)
Y115F 85.2 95.7 6,000 (3) L63P 100 100 1,000
(23) (22) (26) (22)

F116Y 100 100 1,000


(41) (35) A71V 91.7 90.0 1,000 (8)
(11) (9)
V118I 100 100 1,000
(28) (24)
V82A 96.4 100 2,000
Q151M 97.6 100 2,000 (27) (24)
(40) (35)
Y181C 100 100 1,000 V82F 100 100 1,000
(54) (46) (13) (11)

M184I 100 100 1,000


(14) (12) V82S 85.7 83.3 1,000 (9)
(12) (10)
M184V 98.9 98.7 1,000 (4)
(92) (78)
V82T 100 100 1,000
Y188C 100 100 1,000 (27) (23)
(14) (12)
Y188L 100 100 1,000 I84V 100 100 1,000
(13) (11) (26) (22)

L210W 100 100 1,000


(53) (45) L90M 98.8 98.5 1,000 (10)
(79) (67)
T215F 98.8 100 2,000
(82) (71)
T215Y 95.8 100 2,000 Comments:
(23) (20) 1. RT mutation D67N is a mutation that the assay detects with a lower
K219E 78.6 83.3 2,000 (5) sensitivity. There were seven FN results out of 83 detections at viral loads
(11) (10) >1,000 copies/mL. D67N was detected with 100% sensitivity in five of the
viral panels (MC1, MC2, MC3, MC4, and MC5). However, with one of the
K219Q 97.6 97.1 1,000 (6)
(40) (34) panel members (GT1a) there were seven FN results.
2. The claimed LOD is 6,000 copies/mL for RT mutation V75I (6 true positive
determinations). At 2,000 copies/mL and above, there was one FN out of 42
Table 7. Sensitivity of Detection of Protease Mutations at 40% Mixture Level expected detections. At 1,000 copies/mL only, the sensitivity was 100%.
3. The claimed LOD is 6,000 copies/mL for RT mutation Y115F (4 true positive
determinations). There was 100% sensitivity at viral loads of 6,000
copies/mL and higher; at 2,000 copies/mL, there was one FN out of four
Sensitivity at Sensitivity at expected detections.
Protease >1,000 copies/mL >2,000 copies/mL LOD Claim
Mutation (No. of True (No. of True (comments) 4. RT mutation M184V was detected with 100% sensitivity (28 detections) at
Positives) Positives) 1,000 and 2,000 copies/mL. However, there was one FN at 6,000 copies/mL
(out of 84 detections). Therefore, the LOD for this mutation is claimed at
L10I 100 100 1,000 1,000 copies/mL.
(12) (10)
5. RT mutation K219E has a total of three FN results, with one FN at
1,000 copies/mL and the other two FNs at 60,000 copies/mL. This was an
L10R 100 100 1,000 anomalous result since the mutation was detected with 100% sensitivity at
(14) (12) other viral loads (6,000, 20,000, 200,000, and 750,000 copies/mL). Therefore
the LOD is claimed at 2,000 copies/mL.
K20R 91.7 100 2,000
(11) (10) 6. RT mutation K219Q is associated with one FN result at 6,000 copies/mL (out
of a total of 40 detections) and is detected with 100% sensitivity at 1,000 and
2,000 copies/mL viral load (12/12). Therefore, the LOD for this mutation is
D30N 100 100 1,000
(14) (12) claimed at 1,000 copies/mL.
7. Protease mutation M46I is associated with one FN result at 2,000 copies/mL
M36I 100 100 1,000 (out of 40 detections) and is detected with 100% sensitivity at 1,000
(12) (10) copies/mL and all viral loads greater than 2,000 copies/mL. Therefore, the
LOD for this mutation in a 40% mixture is 1,000 copies/mL.
M46I 97.6 97.1 1,000 (7) 8. Protease mutation A71V is associated with one FN result at 6,000 copies/mL
(40) (34) (out of a total of 12 detections) and is detected with 100% sensitivity at 1,000
and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is
G48V 100 100 1,000 claimed at 1,000 copies/mL.
(14) (12)
9. Protease mutation V82S is associated with two FN results at 6,000 copies/mL
(out of a total of 12 detections) and is detected with 100% sensitivity at 1,000
I50V 100 100 1,000
(14) (12) and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is
claimed at 1,000 copies/mL.
I54V 100 100 1,000
(12) (10)

ViroSeqTM HIV-1 Genotyping System v2.0 Page 51 of 68


10. Protease mutation L90M is associated with one FP result at 6,000 copies/mL Results indicate that commonly occurring biological substances and anti-reverse
(out of a total of 80 detections) and is detected with 100% sensitivity at 1,000 transcriptase drugs or inhibitors found in HIV-1 infected individuals did not interfere
and 2,000 copies/mL viral load. Therefore, the LOD for this mutation is with the assay. These include:
claimed at 1,000 copies/mL. • Antiretroviral drugs AZT, ddI, 3TC, d4T, abacavir, nevirapine, efavirenz.
Note: Reverse transcriptase mutations V118I and Q151M were detected in 14
Detection of Mixtures of 16 samples (88% sensitivity). All other mutations had greater than 94%
sensitivity.
The ViroSeq System can accurately detect:
Note: The anti-retroviral drugs listed above have not been fully validated with
• 24 mutations when present as a 40% mixture level at 2,000 copies/mL regards to actual plasma drug levels. Instead, the plasma samples used were from
• 11 mutations when present at a 30% mixture level at 2,000 copies/mL patients currently taking approved reverse transcriptase inhibitors and had viral
• 3 mutations when present at a 20% mixture level at 2,000 copies/mL loads below the limit of detection of the Roche Ultrasensitive AMPLICOR
Description of the Study. In this study, eleven recombinant clones were formulated HIV-1 MONITOR® test (50 copies/mL), thus suggesting that these drugs were
at three viral loads (2,000, 50,000, and 750,000 copies/mL) at six viral mixtures fully active.
(100, 80, 60, 40, 30, and 20% mutant). • Lipid (30 mg/mL), bilirubin (0.6 mg/mL), and hemoglobin (5 mg/mL).
Results.
• At the viral load of 2,000 copies/mL, mutations were detected with >95%
sensitivity at the mixture levels given below. Precision and Reproducibility
The ViroSeq System is able to reproducibly detect specific drug resistance
Table 8. Detection at 2,000 copies/mL mutations between different sites and/or operators, different manufactured
lots, different replicates within the assay, and different runs of the assay.
Mixture Level of Mutant
Reverse Transcriptase Gene
Protease This claim is supported by both the Analytical Imprecision Study and the
(%) Gene Clinical Reproducibility Study. The Imprecision Study shows that the range of
20 — L10R
run-to-run variability (%CV) was 0.1 to 1.4% and that the range for the intra-
D30N assay variability (%CV) was 0.1 to 5.3%. The Clinical Reproducibility Study
I50V shows that the ViroSeq System can reproducibly detect specific mutations that
confer drug resistance in HIV-1 infected patient samples with low viral loads
30 K65R V82F (1,800 to 10,500 copies/mL) when the mutation comprises 40% or greater of the
K70R V82S
Y181C V82T quasispecies with a %CV of <0.1%, well below the level of significance (>5%).
M184I
Y188C
T215F Imprecision Study
K219E/Q
The Imprecision Study tested six mutant/WT viral mixtures at a viral load of 2,000
40 M41L L10I copies/mL and 40% mixture level in duplicate in ten assay runs to evaluate the run-
T69D K20R
L74V M36I to-run variability of the ViroSeq HIV-1 Genotyping System (refer to results in
V75I M46I Table 9).
F77L G48V
K103N I54V Table 9. Estimates of Coefficient of Variation by Recombinant Virus and Gene
V106A L63P
F116Y A71V
V118I V82A %CV
Y188L I84V Mutant Virus Gene
M184V L90M Run-to-Run Intra-Assay
L210W RT <0.1 2.6
T215Y GT-4 Protease <0.1 <0.1
RT <0.1 3.7
MC-1
Analytical Specificity Protease
RT
<0.1
<0.1
1.8
2.3
MC-2 Protease <0.1 2.2
The ViroSeq System is able to accurately genotype mixtures of viral samples in the
presence of infectious agents found in HIV-1 infected patients. This Analytical RT 1.4 1.6
Specificity Study demonstrates that the presence of HIV-1 related or non-related MC-5 Protease <0.1 <0.1
viruses, at approximately five times the HIV-1 viral load, did not affect the RT <0.1 10.9 a
performance of the assay. The presence of these infectious agents did not result in a MC-6 Protease <0.1 3.3
concordance value below 90% for the 72 samples tested. This proves that none of
the infectious viruses decrease the specificity of the ViroSeq System to accurately RT <0.1 5.3
MC-7
detect specific mutations (previously listed) in HIV-1 that confer viral resistance to Protease <0.1 2.2
drugs.
a
Results indicate that none of the organisms listed below interfered with or were This value is the result of one sample in which only 3 of 13 mutations were detected. For this
detected by the ViroSeq assay. sample, the RT-PCR yield did not meet specifications – it was approximately 10 ng, which is
less than the required 20 ng minimum RT-PCR yield needed for sequencing reactions.
Related Viruses Coinfecting Organisms
The data in Table 9 show that the run-to-run %CV is in the range <0.1 to 1.4. Five of
HIV-2 Cytomegalovirus the 6% CV values are <0.1 and one is 1.4. The intra-assay %CV is in the range <0.1
to 5.3.
HTLV-I Epstein-Barr Virus
Results from this study support the Celera claim that the ViroSeq HIV-1
HTLV-II Hepatitis B Genotyping System has a run-to-run variability which is not significant ( 5%).
Hepatitis C

Clinical Reproducibility Study


Interfering Substances The clinical reproducibility of the ViroSeq HIV-1 Genotyping System was
measured through a multi-center study to determine the site-to-site and lot-to-lot
Commonly occurring biological substances and anti-reverse transcriptase (anti-RT) variation of the assay. Three sites tested 11 patient samples ranging in viral load
drugs or inhibitors that are present in patient plasma were tested for their potential to from 1,800 to 10,500 copies/mL. Each of these 11 samples was tested in duplicate
interfere with the detection of mutations that confer viral resistance by the ViroSeq
HIV-1 Genotyping System. In this study any substance that caused less than 90%
concordance between the genotype of the test sample and the expected genotype was
considered to be interfering with assay performance.

Page 52 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


with three different lots, generating 18 data points per specimen (3 sites x 3 lots x 2 Data quality control for this clinical validity study consisted of a phylogenetic
replicates). The detection of all mutations present in a specimen was measured and analysis on 300 samples (50 specimens x 6 replicates each). The phylogenetic tree
compared to establish the variability (%CV). The results in Table 10 show that the that was derived showed no evidence of PCR amplicon carryover contamination.
site-to-site and lot-to-lot variation of the ViroSeq System are negligible. This study shows that the AmpErase® Uracil N-glycosylase and recommended
Table 10. Clinical Reproducibility Results containment procedures effectively control PCR contamination.
Data were also analyzed for all-base accuracy.
%CV Range

Source of Variation RT Protease Clinical Utility Study


Site-to-site <0.1 <0.1
The ViroSeq® System Software uses drug resistance mutations to generate drug
Lot-to-lot <0.1 <0.1 resistance assessments with proven clinical utility.
Intra-assay <0.1 <0.1 to 4.7 a This claim is supported by resequencing samples from the GART study using the
Total <0.1 <0.1 ViroSeq System. The GART study had previously demonstrated the clinical utility
of several mutations in RT and protease listed in Table 12.14 Additional mutations
a
For most of the samples analyzed, the intra-assay %CV was <0.1. Two samples had values of were factored into the rules-based interpretation algorithm based on recent literature
1.3 and 4.7. reports.
Results from this study support the Celera claim that the ViroSeq HIV-1 Table 12. Additional Mutations Detected in the GART Study, But Not in the
Genotyping System demonstrates site-to site, lot-to-lot, and assay variability Original Study List of Analyzed Mutations
that is not significant (<5%).
In Reverse Transcriptase
RT-PCR and Sequencing Success Rates M41L A62V K65R D67N T69D K70R L74V V75I
V75T F77L K103N V106A F116Y Q151M Y181C Y181I
The ViroSeq System is able to successfully genotype a significant portion
( 98%) of the targeted population samples when sufficient RT-PCR product is M184V Y188C L210W T215Y T215F K219Q K219E
available. In Protease
The 100 adult HIV-1 infected patient specimens were screened to obtain samples
representing a geographical cross section consisting of varying viral loads and L10I L10R L10V K20M K20R L24I D30N M36I
resistance mutations. 50 of these samples were grouped into a low viral load M36L M46I M46L G48V I54V L63P A71T A71V
category (1,800 to 10,500 copies/mL) and 50 were grouped into a high viral load V77I V82A V82F V82T I84V N88D L90M
category (above 10,500 copies/mL).
For the PACTG 377 study, three laboratories (Johns Hopkins, UCLA, and the
University of Medicine and Dentistry of New Jersey) and three operators tested the The Genotyping Antiretroviral Resistance Testing (GART) study was one of the
196 samples that had viral loads >1084 copies/mL. pivotal clinical utility trials. It proved that if physicians had access to genotyping
data before altering therapy, they made better treatment decisions and improved the
short term patient response to therapy.14 There were two arms to this study – one
Mixture Detection in Patient Samples (GART) allowed treating physicians access to genotyping results and expert panel
treatment recommendations based on those results, while the other (nonGART) did
The ViroSeq System is able to accurately detect specific drug resistance not allow access to this information before treatment. These two arms were
mutations in clinical EDTA plasma samples in a low viral load range (1,800 to compared with regards to average viral load decreases at 4, 8, and 12 weeks. The
10,500 copies/mL) and viral mixture limit ( 40% mutant). GART arm showed a significant decrease in viral load compared to the nonGART
arm, especially when the treating physicians showed a high adherence to expert
This claim is supported by the Clinical Validity Study, which compared the treatment advice. Because of this protocol and others like it, resistance testing has
percentage of mutations found in each sample when PCR product was cloned versus become the standard of medical care when designing therapeutic strategies for
the mutations detected by population sequencing using the ViroSeq System. This patients experiencing treatment failure. To demonstrate that our assay detects the
study shows that mutations representing 40% or more of the quasispecies in low same resistance mutations detected by the homebrew assay used in the GART study,
viral load range samples (1,800 to 10,500 copies/mL) can be detected by three we have obtained all of the original 153 patient samples from the GART study's
clinical sites reproducibly with a concordance value of >98% to the lower limit of original investigators, as well as the original sequencing results from the GART trial.
the 95% confidence level. Table 13 shows that the comparison of the genotyping data demonstrates high
Table 11. The mutations detected in this study include: concordance.
Table 13. Sample Statistics for the GART Concordance by Gene
In Reverse Transcriptase
M41L E44D A62V K65R D67N S68G T69D K70R Gene
L74V V75I F77L L100I K101E K103N V108I Y115F
RT Protease
F116Y V118I Q151M Y181C Y181I M184V G190A G190S
L210W T215Y T215F K219Q K219E Mean 0.969 0.954
In Protease Median 1.000 1.000
L10I L10F L10V K20M K20R D30N V32I L33F
Standard Deviation 0.066 0.086
M36I M46I M46L I47V G48V F53L I54V L63P
A71I A71T A71V G73S V77I V82A V82T I84V Standard Error of Mean 0.005 0.007

N88D L90M Minimum 0.652 0.538

For this study low viral load samples were chosen randomly with respect to age Maximum 1.000 1.000
(above 18), sex, and clinical and treatment history. Samples were collected
prospectively from HIV-1 infected patients from multiple geographic locations in the
targeted population. The detected mutations and their frequency are considered The concordance between the GART homebrew assay and the ViroSeq HIV-1
representative of the targeted HIV-1 infected population. Within this population, the Genotyping System is quite good (>95%), achieving the level needed to claim the
ability of the ViroSeq System to detect the selected list of drug resistance mutations clinical utility of the ViroSeq System. Also, the majority of the samples were
is consistently high across all mutations detected. In some cases the assay was able successfully analyzed (149/153, or 97%), which allows the original conclusions
to detect mutations present below the 40% limit, although this was not consistent from the analysis of the data for clinical utility to apply without reanalysis.
across all of the sites or with all of mutations. Therefore, the above study supports the Celera claim that clinical utility is
proven for the mutations detected and included in the GART study analysis
when detected by the ViroSeq HIV-1 Genotyping System.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 53 of 68


Section 6: Maintenance Item

Hi-Di™ Formamide, 25 mL 4311320

and Calibration of the Contains Formamide.

Applied Biosystems® R 61 May cause harm to the unborn child.


R 36/38 Irritating to eyes and skin.
S 53 Avoid exposure - Obtain special instructions before use.
S 24/25 Avoid contact with skin and eyes.

3130 and 3130xl Genetic S 35 This material and its container must be disposed of in a safe
way.
S 36/37/39 Wear suitable protective clothing, gloves and eye/face
protection.

Analyzers S 45 In case of accident or if you feel unwell, seek medical advice


immediately (show the label where possible).

Care of the Applied Biosystems®


Required Materials 3130 and 3130xl Genetic Analyzers
For illustrations and diagrams that explain how to conduct a specific task on the
Applied Biosystems ® 3130/3130xl Genetic Analyzer, refer to the Applied Maintenance
Biosystems ® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and
Reference Guide (PN 4352716). Follow the maintenance schedules below for optimal instrument performance.
IMPORTANT! The Applied Biosystems® 3130 and 3130xl Genetic Analyzers
should be installed by a qualified Applied Biosystems field service engineer. Keep
Item the instruments in a room with an ambient temperature of 15 to 30°C (59 to 85°F).
Temperatures outside of this range, or high humidity, can cause condensation,
decreased performance, and damage to the instrument.
Applied Biosystems ® 3130xl Genetic Analyzer with: 3130XL
• Data Collection Software v.3.0 IMPORTANT! Do not Note: After opening the door of the Applied Biosystems ® 3130 or 3130xl Genetic
• Firmware version 6286200-02 or 6286250-02 download software from the Analyzer, you must allow the autosampler homing sequence to complete before
• PC computer with: internet for this intended use. issuing further commands.
– Microsoft® Windows® XP Professional edition,
Service Pack 2
– 2 GHz Intel® Pentium® 4 processor Tasks To Perform Before Each Run
– 1 GB RAM
– Dual 36 GB hard drives Ensure that the plate septa are firmly seated and flat.
Applied Biosystems ®3130 Genetic Analyzer with: 3130
• Data Collection Software v.3.0 IMPORTANT! No software Ensure that the plate assembly was put together properly.
• Firmware version 6278250-02 should be downloaded from
• PC computer with: the internet for this intended IMPORTANT! Make sure the plate septa lies flat on the plate. The holes in the
– Microsoft® Windows® XP Professional edition, use. plate retainer must align with the holes in the septa or the capillary tips will be
Service Pack 2 damaged.
– 2 GHz Intel® Pentium® IV processor
– 1 GB RAM
– Dual 36 GB hard drives Ensure that the plate assembly is positioned on the plate deck properly. Plate
should sit snug on the deck.
ABI PRISM ® 3130 Capillary Array, 50 cm 4315930
IMPORTANT! Never use warped plates.
®
ABI PRISM 3130xl Capillary Array, 50 cm 4333466
Lower Polymer Block Cleaning Kit 4359572 Replenish the deionized water and 1X Running Buffer reservoirs on the
MicroAmp® 96-Well Support Base N801-0531 instrument.
Reservoir Septa 4315932
Check for bubbles in the polymer block and polymer block channels. Remove all
96-Well Plate Base 4317237 bubbles with the Bubble Remove Wizard.
96-Well Plate Retainer 4317241
96-Well Plate Septa 4315933
Check that the capillary tips are not crushed or damaged.

MicroAmp® Optical 96-Well Reaction Plate N801-0560


Check the level of polymer in the polymer bottle to ensure there is enough for all
Applied Biosystems ® Sequencing Analysis Software v5.3.1 with 4360967 your runs.
KB™ Basecaller v1.4 To obtain additional licenses,
please contact your Applied
Biosystems representative. Check the lower polymer block to ensure it fits securely on the instrument.
IMPORTANT! Do not
download software from the Clean the instrument surfaces.
internet for this intended use.
POP-6™ Polymer for 3130/3130xl Genetic Analyzers 4363783 (3500 µL) or Check for dried polymer around the lower polymer block and clean as necessary.
4352757 (7000 µL)
®
BigDye Terminator Sequencing Standard v1.1 4336791
Check for leaks around the lower polymer block, array knob and interconnecting
Running Buffer, 10X 402824 tube nuts.

Page 54 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Tasks to Perform Once Per Week 2. Using a long narrow implement, such as a straightened paper clip, press the
Reset button on the front of the instrument.
Flush the water trap.

Clean the lower polymer block. To Reset by Powering Down

Replace the polymer using the Replenish Polymer Wizard. 1. Close the instrument doors.

Check the storage conditions of the used arrays. 2. Turn off the instrument by pressing the on/off button on the front of the
instrument.
Check data base space. Delete plate records from the instrument database and
archive sample files when the database is getting full (approximately 70 to 75%). 3. Restart the computer.
Refer to the Applied Biosystems ® 3130/3130xl Genetic Analyzers Maintenance, a. Select Start > Turn Off Computer.
Troubleshooting, and Reference Guide (PN 4352716). b. In the dialog box, select Restart and click OK.
IMPORTANT! Wait until the computer has completely restarted before
Restart the computer and instrument. proceeding.

4. Turn on the instrument and wait for the solid green light.
Tasks To Do As Needed
5. Launch the Data Collection software. (Service console application start
Maintenance Task Frequency automatically.)

Run the Water Wash Wizard. Flush the array Every month or as needed
port during this wizard, whether or not there are
bubbles present in the array port. Shutting Down the Instrument
Defragment the hard drive. Every month
If the instrument
will be unattended Perform this shutdown procedure. . .
Clean the drip trays. As needed for. . .

Change the array. After 100 runs No more than 1 week Short-term.
with a full buffer IMPORTANT! The key to a successful short-term
Remove dried polymer from the capillary tips. As needed reservoir shutdown is keeping the capillary array in 1X Running
Buffer. This prevents the polymer from drying in the
Calibrate the autosampler. As needed capillaries.

For more than 1 week Long-term.


To Clean the Instrument
To Perform a Short-Term Shutdown
1. Press the Tray button on the front of the instrument to move the autosampler
to the forward position.
1. Ensure that the oven and instrument doors are closed.
2. Wipe off any liquid on or around the autosampler using a lint-free tissue.
2. Collect polymer waste:
3. Clean out the drip trays with deionized water and lint-free tissue. a. Select GA Instruments > ga3130 or ga3130xl > instrument name >
Manual Control.
b. In the Send Defined Command drop-down menu, select Autosampler.
4. Clean off any polymer build-up (crystals) on the instrument and the stripper c. In the Command Name drop-down menu, select Move autosampler to
plate with deionized water and lint-free tissue. site.
IMPORTANT! Never use organic solvents to clean the instrument. d. In the Value menu, select Waste.
e. Click Send Command. Wait for the autosampler to stop moving and
Send Command becomes active, before proceeding.
5. Clean the array port knob, plug, or opening threads of these parts with
moistened lab wipes.
3. Fill the capillary array with fresh polymer using manual control:
a. In the Send Defined Command for drop-down menu, select Polymer
Delivery Pump.
Resetting the Instrument b. In the Command Name, select the appropriate Fill <length> cm
capillary array length.
Reset the instrument when: c. Click Send Command.The array fill is finished when Send Command
• There is a fatal error as indicated by the red status light becomes active.
• The instrument does not respond to the Applied Biosystems ® 3130 or 3130xl d. Return the buffer reservoir to the capillaries.
Data Collection software
There are two ways to reset the instrument.

To Reset With the Reset Button

1. Close the instrument doors.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 55 of 68


4. Cleaning the Reservoirs
To Replace or Replenish the Polymer
a. Press the Tray button to move the autosampler forward. CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and
b. Open the doors, then remove the: respiratory tract irritation. Wear appropriate protective eyewear, clothing, and
– Plates gloves.
– Cathode buffer reservoir and water reservoirs
c. Dispose of remaining fluids and rinse out the reservoirs with deionized
water. 1. Select Wizard > Replenish Polymer Wizard.
Note: Follow your company’s waste disposal practices for appropriate
disposal procedures. 2. Follow the directions given in the wizard to put fresh polymer on the
d. Rinse the cathode reservoir with 1X Running Buffer, and then fill to the instrument.
line with 1X Running Buffer (about 16 mL).
e. Fill the three water reservoirs to the line with quality deionized water
(about 16 mL). Ensure that the septa fits snug and flush on the tops of the
reservoirs in order to prevent damage to the capillary tips.
f. Place a clean reservoir septa on each reservoir, and dry the outside of the
Capillary Array
reservoirs using a lint-free wipe.
g. Place the reservoirs into position on the autosampler as shown below.
When to Change a Capillary Array
Water reservoir Water reservoir Expect your capillary array to last approximately 100 runs.
(waste) (rinse)
A new capillary array may be needed if there is:
• Poor sizing precision
Cathode reservoir Water reservoir • Poor resolution and/or decreased signal intensity
(1X Running Buffer) (spare) • Blocked or broken array

Home position Before You Install a Previously Used Capillary


h. Close the instrument doors.
Array
Note: Closing the doors returns the autosampler to the home position, • Clean the front of the detection cell
placing the tips of the capillaries in buffer. • Check that the cathode bar is dry
i. Shut down the computer and turn off the instrument.
To Clean the Detection Cell
To Perform a Long-Term Shutdown Note: This procedure is unnecessary for new arrays unless you have accidently
touched the detection cell.
Select the Instrument Shutdown Wizard and follow the prompts.
IMPORTANT! Make sure all parts are dry before long-term storage.
CHEMICAL HAZARD. Methanol is a flammable liquid and vapor.
Exposure may cause eye, skin, and respiratory tract irritation, and central
nervous system depression and blindness. Wear appropriate protective
When to Replenish the Polymer eyewear, clothing, and gloves. Refer to the manufacturer’s Safety Data Sheet
for additional information.
IMPORTANT! Always replace polymer that has been on the instrument more than
one week. The polymer is good at 25°C for 7 days. 1. Put one drop of methanol on the front surface of the detection cell.

If polymer on the instrument. . . Then. . . 2. Allow the cell to air dry.


Note: Do not use pressurized air to dry the detection cell.
has been on less than one week and is Remove all bubbles, and proceed with
sufficient in quantity to complete your instrument preparation.
runs a
To Check the Cathode Bar
has been on less than one week, and is Add fresh polymer to the polymer When putting a used array back on the instrument, be sure that the cathode bar is dry.
insufficient in quantity to complete supply by following the Replenish A wet bar could lead to arcing.
your runs Polymer Wizard.
has been on longer than one week Replace with fresh polymer by ELECTRICAL SHOCK/FIRE HAZARD. Do not leave liquid in the
following the Replenish Polymer cathode bar. This can lead to electric shock or even fire if not properly
Wizard. maintained.
is the wrong type (a change between Replace the installed polymer type
POP-4, POP-6, and/or POP-7 with a different type by following the To Install or Remove the Capillary Array Using the
polymers is required) Change Polymer Type Wizard.
Wizard
a
A 3130xl Genetic Analyzer run uses 50 to 80 µL of polymer and a 3130 Genetic Analyzer run
uses ~25 to 40 µL of polymer. IMPORTANT! Wear gloves while performing the following procedure, and any
other time you handle the capillary array, septa, or buffer reservoirs.

CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and


respiratory tract irritation. Wear appropriate protective eyewear, clothing, and
gloves.

Page 56 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


To Fill the Capillary Array with Polymer Using Manual
1. Close the oven and instrument doors, and then press the Tray button. Control
2. Select Wizards > Install Array Wizard.
1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Manual
Control.
3. Open the oven and instrument doors.
2. In the Send Defined Command drop-down list, select Polymer Delivery
4. Follow the directions given in the wizard to replace or install an array. Pump.
IMPORTANT! For the ViroSeq assay, use a 50 cm capillary array. The
capillary array length that you are using must match the capillary array 3. In the Command Name drop-down list, select Fill 50 cm capillary array.
length in the instrument database.
Note: Proper capillary array information (e.g., capillary array length and lot 4. In the Value drop-down list, select 50 cm for array length and POP-6 for
number) must be entered. polymer.

5. Check that the capillary tips are not crushed or damaged. 5. Click Send Command. The array fill is finished when Send Command
becomes active.
6. Click Finish.
6. Return the buffer reservoir to the capillaries.
7. Close and lock the oven door, then close the instrument door.

To Store a Capillary Array On the Instrument


To Update the Capillary Array Information Store the capillary array on the instrument only when the capillary array will be
unused for less than 1 week.
Use the Update Cap Array Info Wizard to: To store the capillary array on the instrument, perform a short-term shutdown.
• Update the capillary array length and serial number information into the database
• Correct an entry error after using another wizard To Store a Capillary Array Off the Instrument
IMPORTANT! The capillary array length defined in the wizard must match the
array length you are using. Store the capillary array off of the instrument when the capillary array will be
unused for longer than 1 week. Store at 15 to 25°C.
IMPORTANT! Before storing the capillary array for long periods, we recommend
Maintenance filling the capillaries with fresh polymer.

To Care for the Capillary Array and Work Area CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and
respiratory tract irritation. Wear appropriate protective eyewear, clothing, and
• Wear gloves and handle the capillary array gently. gloves.
• Do not touch the detection cell. If it is dirty, refer to To Clean the Detection
Cell. IMPORTANT! Wear gloves while performing the following procedure, and any
other time you handle the capillary array, septa, or buffer reservoirs.
• Keep the ends of the capillary array wet at all times.
• Do not overtighten the capillary array knob.
• Clean off any polymer buildup (crystals) on the instrument, including the 1. Remove the capillary array from the instrument by selecting Install Array
capillary electrodes and the stripper plate, with deionized water and lint-free Wizard.
tissue.
2. Select Store Array and follow the prompts.
To Clean the Capillary Array
3. Replace the cover over the detection cell.

CHEMICAL HAZARD. POP-6 polymer causes eye, skin, and 4. Fill a buffer reservoir with fresh 1X Running Buffer and cover with a septa
respiratory tract irritation. Wear appropriate protective eyewear, clothing, and strip. Insert the capillary tips into the buffer.
gloves.

5. Fill the shipping vial with fresh 1X Running Buffer and insert the detection
1. Flush the capillary array with fresh polymer as instructed in the section To end of the capillary array.
Fill the Capillary Array with Polymer Using Manual Control below.
6. Store the capillary array upright.
2. Clean off any polymer buildup (crystals) on the instrument, including the
capillary electrodes and the stripper plate, with deionized water and lint-free 7. Check the 1X Running Buffer level in the reservoir and tube weekly and
tissue. replenish the buffer as needed.
Note: When cleaning the capillary electrodes, be careful not to bend them
out of position.
IMPORTANT! Never use organic solvents to clean the instrument.

3. Clean the detection cell.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 57 of 68


Flushing and Filling the Water Trap Polymer Block
Overview
Polymer Block Care
The polymer delivery pump (PDP) water trap should
be flushed with either distilled or deionized water at
least once per week to wash out any diluted polymer Avoiding Damage to the Blocks
and to clear bubbles. Leave the trap filled with either The pump and lower polymer blocks can be irreversibly damaged if:
distilled or deionized water.
• Polymer dries in the polymer channels of the pump or lower polymer block
• The inside surface of the channel is scratched
• The block is exposed to organic solvent
• The block is exposed to temperatures greater than 40 °C
IMPORTANT! Use clean, powder-free, silicone-free latex gloves whenever you
To flush the water seal trap: handle the polymer blocks or any item in the polymer path to minimize background
fluorescence.
1. Fill the 20 mL, all-plastic Luer lock syringe (supplied in the Lower Polymer
Block Cleaning Kit, PN 4359572) with distilled or deionized water. Expel
any bubbles from the syringe. When to Clean the Polymer Block Using the Lower
Note: Do not use a syringe smaller than 20 mL. Doing so may generate Polymer Block Cleaning Kit
excessive pressure within the trap.
In nearly all circumstances, the Water Wash Wizard is very effective in cleaning
2. Attach the syringe to the forward-facing Luer fitting at the top of the pump the polymer delivery pump (PDP), including the lower polymer block. Occasionally,
block. Hold the fitting with one hand while threading the syringe onto the the Water Wash Wizard may not sufficiently clean the polymer block, and the
fitting with the other hand. Lower Polymer Block Cleaning Kit (Applied Biosystems PN 4359572) should be
used. Refer to the Applied Biosystems Lower Polymer Block Cleaning Kit Protocol
3. Open the Luer fitting by grasping the body of the fitting and turning it and (PN 4363356 Rev. A).
the attached syringe approximately one-half turn counterclockwise. Note:
• Polymer has dried in the channels of the lower block due to maintenance
4. Open the exit fitting at the top left side of the pump block by turning it oversight.
approximately one-half turn counterclockwise. Problems such as electrical arcing or mechanical malfunctions may cause dried
polymer to appear in the lower polymer block. Washing with either the Water
5. Hold an empty tube or beaker under the exit fitting to receive approximately Wash Wizard or this kit may not correct these problems- replacing the lower
polymer block may be necessary.
5 mL of waste. Flush the trap by pushing steadily on the syringe plunger.
• Some contaminant in the lower polymer block is suspected of causing problems.
IMPORTANT! DO NOT USE EXCESSIVE FORCE when pushing the • A replacement lower polymer block is to be installed. In this case, follow the
syringe plunger as this may damage the trap seals. It should take protocol for removing the installed lower polymer block, then clean and install
approximately 30 seconds to flush 5 mL of either distilled or deionized water the replacement lower polymer block.
through the trap.
IMPORTANT! Do not expose the polymer blocks to any organic solvents.
Note: Because the water trap volume is approximately 325 L, a relatively
small volume of water is required to completely flush the water trap. IMPORTANT! Use clean, powder-free, silicone-free latex gloves whenever you
However, a larger volume improves flushing as long as force and flow rate handle the polymer blocks or any item in the polymer path to minimize background
are within the limits given above. fluorescence.

6. Close the fittings in this order by turning each clockwise until the fittings Removing the Lower Block
seal against the block:
a. Luer fitting
CHEMICAL HAZARD. POP-6™ polymer cause eye, skin, and
b. Exit fitting respiratory tract irritation. Read the MSDS, and follow the handling
IMPORTANT! Do not over-tighten the fittings. Very little pressure instructions. Wear appropriate protective eyewear, clothing, and gloves.
develops within the trap during pump operation, so the fittings require only To remove the lower polymer block if the channel is not completely blocked with
enough tightening to prevent water leaks. Excessive tightening can damage
the fittings. dry polymer:
c. Remove the syringe from the Luer fitting. Hold the fitting with one hand 1. Run the Water Wash procedure:
while turning the syringe counterclockwise with the other hand. a. Select Water Wash Wizard:
b. Follow the instructions in panels 1 and 2.
c. Exit the wizard after executing the water wash procedure at step 2.
Water Wash d. Click Cancel and then Yes in the dialog box to leave the wizard

When to Use the Water Wash Wizard


When dried polymer is observed in the lower polymer block, use the following
procedure.
1. Select Wizards > Water Wash Wizard.

2. Run the Water Wash Wizard to wash the PDP chamber, lower polymer
block, channels, and tubing with distilled or deionized water.

Page 58 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


2. Open the buffer valve from the Manual Control panel of Data Collection 3. Fill the 20-mL silicone-free plastic syringe (P/N 4324463) with warm water
by selecting: (40°C or below).

4. Fit the 6-mm plastic syringe adaptor (P/N 4322928) onto the 20-mL silicone-
Send Defined Command for: Buffer Valve free plastic syringe (P/N 4324463).
Command Name: Close/Open buffer valve
5. Thread the 6-mm plastic syringe adaptor into the polymer block where the
Value: Open interconnect tube was originally attached.

Click Send Command 6. Force several plastic syringe volumes of warm water (40 °C or below)
through the channel.

7. Inspect the channels for dried polymer, which appears as white residue. Wash
3. Verify that the buffer valve is open (valve lever in the up position). partially obstructed channels with warm water (40°C or below) until the
dried polymer is removed.Verify that all polymer is removed before
4. Open the oven and detection block doors. proceeding.
IMPORTANT! Some time may be required for the warm water to clear the
5. If you have not already removed the buffer jar during the wizard, do so now obstruction. Do not use any pointed or sharp objects to clear the channel,
and set it aside to avoid spilling the contents. even if the channel is completely obstructed with dried polymer.

6. Remove the array comb nearest to the detection cell. 8. Remove the syringe and syringe adapter.

7. Loosen the array knob about halfway by turning it counterclockwise. 9. Rinse the lower polymer block and all the fittings a final time with water.

8. Pull the pump and lower polymer blocks forward to the stop position on the 10. Dry the fittings and exterior surfaces of the lower polymer block with lab
alignment pins. The detection cell comes out of the holder wipes.
IMPORTANT! Do not use compressed gas to blow water from the
9. Attach the detection cell cover to the detection cell to protect the window. channels, valve, or any part of the lower polymer block.

10. Completely loosen the connecting nut for the interconnect tube at the lower
polymer block. Reinstalling the Lower Polymer Block
11. Pull the lower polymer block off its mounting pins and away from the 1. With the pump block in the forward stop position, slip the interconnect tube
interconnect tube. This operation may be slightly awkward because of from the pump block loosely into the socket of the lower polymer block
resistance from the stiff interconnect tube. while sliding the block into the mounting pins. This operation may be
slightly awkward because of resistance from the stiff interconnect tube.
Dried Polymer Blocking Channel
2. Verify that the end of the interconnect tube is flush with the bottom of the
To remove the lower polymer block if the channel is completely blocked with dry connector port. Tighten the interconnect tube connection to the lower
polymer. polymer block. Check again to make sure that the end of the interconnect
tube and the bottom of the lower polymer block connector port are in contact.
1. Follow the steps under Removing the Lower Polymer Block starting with
step 2, omitting the Water Wash as described in step 1.
3. Remove the detection cell cover from the detection cell window.
2. Immerse the removed block in warm (40°C or below) water.
4. IMPORTANT! Push the pump and lower polymers blocks back
against the rear the pump panel. Verify that the buffer valve pin
3. Inspect the block and replace the warm water as necessary until the block can engages the valve lever.
be cleaned using the procedure that follows (refer to Cleaning the Lower
Polymer Block.). If the block cannot be cleared sufficiently for cleaning, a 5. Insert the array comb nearest the detection cell into its holder in the oven.
new lower polymer block should be installed.
6. Carefully insert the array detection cell into the detection cell holder, making
certain that it is mounted flat and secure in the mounting.
Cleaning the Lower Polymer Block
7. Close and secure the detection block door.
1. Rinse all the fittings with warm (40 °C or below) purified (distilled or
8. Tighten the array knob into the pump block.
deionized) water to remove dried polymer.
IMPORTANT! Do not use water above 40°C to rinse the fittings or the 9. Close and secure oven and instrument door.
polymer block.
10. Run the Water Wash Wizard to flush the system with water and then to
2. With the lower polymer block in warm water (40 °C or below), move the replace the water in the PDP with polymer. Since the water bottle is already
buffer valve in and out to ensure any encrusted polymer is removed from the at the polymer supply position, verify that you have enough water volume
guide channel. when starting the wizard.
IMPORTANT! Do not remove any of the components from the lower
polymer block.
IMPORTANT! Do not twist the buffer valve against the seat.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 59 of 68


To Remove Air Bubbles from the Polymer Block 2. Accept or reject a spatial calibration as follows:

1. Select Wizards > Bubble Remove Wizard to clear bubbles. If the Spatial Calibration
Then. . .
Profile. . .
IMPORTANT! Remove bubbles from the interconnect tubing and the
channel of the lower polymer block. These areas are part of the Passed Click Accept to write the calibration data to the database
electrophoresis current path. Absence of bubbles in the current path is and .ini file.
important for problem-free electrophoresis.
Failed Click Reject, then refer to If the Spatial Calibration
Fails.
2. Replace the buffer if excess polymer is expelled into the anode buffer jar.

Spatial and Spectral Calibration If the Spatial Calibration Fails


• Repeat the spatial calibration.
• Fill the capillaries with polymer, and then repeat the spatial calibration.
To Perform A Spatial Calibration • Clean the detection cell, and then repeat the spatial calibration.
• Reposition the array window in the detection cell, and then repeat the spatial
When to Perform calibration.
• Try another capillary array.
You must perform a spatial calibration whenever a new or different capillary array is • Contact an Applied Biosystems field service engineer to check laser alignment.
installed on the instrument, the array is temporarily removed from the detection Refer to the Applied Biosystems® 3130/3130xl Genetic Analyzers Maintenance,
block, or the instrument is moved. Troubleshooting, and Reference Guide.
Note: The Applied Biosystems® 3130 and 3130xl Genetic Analyzers should be
moved by a qualified Applied Biosystems field service engineer.
To Display a Spatial Calibration Profile
A spatial calibration maps the position of each capillary detected on the CCD
camera. A spatial calibration with filling of the capillaries takes approximately six
minutes. During this time, multiple frames of data are collected and summed. The 1. To view a current spatial calibration profile, select GA Instruments >
collected data is analyzed and saved as a spatial map. ga3130 or ga3130xl > instrument name > Spatial Run Scheduler.

How To Perform 2. To view a previous spatial calibration profile:


a. In the Data Collection Viewer window, click GA Instruments >
1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Spatial ga3130 or ga3130xl > Run History.
Run Scheduler. b. In the Run History, search for the run with the associated spatial
calibration profile you want to view or select Find all.
2. In the Spatial Protocols section, select one of the following: c. Select Spatial Calibration Viewer (under the Run History icon).
• If the capillaries contain fresh polymer, select d. Select the run with the associated spatial calibration profile you want to
Protocol > 3130SpatialNoFill_1 view from the Run to view pull-down list.
• Otherwise, select Protocol > 3130SpatialFill_1
The capillaries do not have to be filled each time a spatial calibration is
performed. To Perform A Spectral Calibration
3. Click Start.
When to Perform
Note: The spatial profile window will be displayed as black when the spatial
calibration is started. You must perform a spectral calibration:
• Whenever you use a new dye set on the instrument
• When you change capillary array length
To Evaluate a Spatial Calibration File and Save the Data • When you change polymer type
• Whenever a service engineer realigns/replaces the laser, optics, or CCD camera
• If you begin to see a decrease in spectral separation (pull-up and/or pull-down
1. Evaluate the spatial calibration profile. peaks)
While viewing the calibration profile, use the following criteria to evaluate
the data: For spectral calibration of the ViroSeq System, use BigDye® Terminator Sequencing
Standard v1.1 (Applied Biosystems PN 4336791).

Peak Attribute Criteria What Happens?


Height Similar heights for all peaks Run the spectral standards in all 16 or 4 capillaries. The Data Collection software
Orange crosses One orange cross marking the top of every peak. No
then:
misplaced crosses. • Collects the data and stores it in 16 or 4 separate temporary files
To move a cross: • Analyzes the data and generates a matrix for each capillary
a. Change the value in the Positions (pixels) box.
b. Click outside of that box. • Stores the spectral calibration data for the dye set run
c. Click Enter using the keyboard.
Shape • Single sharp peak for each capillary. How To Perform
• Small shoulders are acceptable.
A spectral calibration is a run that produces a mathematical description of the
Spacing The difference between adjacent positions is 13 to 16
pixels. Theoretical spacing between capillaries is 15. overlap in the emission spectra of a given dye set. This mathematical description is
called a matrix, and is required for each dye set. The process of applying a matrix to
sample data is called multicomponenting.

Page 60 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


To Prepare and Load the BigDye® Terminator 3. Complete the New Plate dialog box:
Sequencing Standard onto the 3130 or 3130xl Genetic
a. Name: enter a name for the plate.
Analyzers b. Description: enter a description for the plate record (optional).
c. Application: Spectral Calibration.
1. Resuspend a tube of the BigDye Terminator Sequencing Standard v1.1 with d. Plate Type: 96-Well.
170 L of Hi-Di formamide. e. Owner Name: enter a name for the owner.
f. Operator Name: enter a name for the operator.
g. Click OK.

Contains Formamide. 4. Complete the Spectral Calibration Plate Editor dialog box:
R 61 May cause harm to the unborn child.
a. Sample Name: enter the name for the samples.
R 36/38 Irritating to eyes and skin. b. Comment: enter any additional comments or notations (optional).
S 53 Avoid exposure - Obtain special instructions before use. c. Priority: the value 100 automatically displays in the column.
S 24/25 Avoid contact with skin and eyes. d. Instrument Protocol 1: select the
ViroSeq_spectral_instrument_protocol. Refer to To Create a ViroSeq
S 35 This material and its container must be disposed of in a safe way. Spectral Instrument Protocol.
S 36/37/39 Wear suitable protective clothing, gloves and eye/face e. Click OK.
protection. IMPORTANT! Verify that you have selected the correct spectral
S 45 In case of accident or if you feel unwell, seek medical advice instrument protocol file. Selecting the incorrect instrument protocol file will
immediately (show the label where possible). cause the spectral calibration to fail.

2. Vortex the standard 10 to 15 seconds to mix well.


To Start the Calibration
3. Spin the mixture for 10 to 15 seconds at 2,000  g in a microcentrifuge.
1. Select GA instruments > ga3130 or ga3130xl > instrument name > Run
4. Dispense 10 µL of the standard into the MicroAmp Optical 96-Well Scheduler > Plate View.
Reaction Plate, loading wells A1 through H2 for the 3130xl Genetic
Analyzer or wells A1 through D1 for the 3130 Genetic Analyzer. 2. Search for the spectral plate record. There are two search options:
• Find All
5. Cover the plate and centrifuge it for 5 to 10 seconds at 2,000  g. • Advanced
Under Find All:
6. Heat the plate at 95°C for 2 minutes to denature the standards. • Select Barcode in the Type of Search drop-down list.
• If you have a limited number of plates in the database, click Find All. All
7. Quick chill the plate on ice for 2 minutes. plates in the database will display in the plate record section.
Under Advanced:
8. Immediately load the plate onto the 3130 or 3130xl Genetic Analyzer. a. Select Advanced in the Type of Search drop-down list.
IMPORTANT! Do not store the plate. b. Use the drop-down list to define search conditions for a category or
multiple categories (Run Name, Results Group Name, Plate Name,
etc.)
c. Use the Plate Name for the Plate ID category.
To Create a ViroSeq Spectral Instrument Protocol d. For each category with a condition selected, type a value (primary search
string) in the Value 1 column.
1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager to e. Click Search. All plates in the database that match the search criteria
open the Protocol Manager window. display in the plate record section.

2. In the Instrument Protocols pane, click New. The Protocol Editor dialog 3. Select the plate record, then click the plate position indicator that
box opens. corresponds to the plate being linked. The plate position indicator changes
from yellow to green when linked and the green Run Instrument button
3. Complete the Protocol Editor dialog box by entering information or making is active.
selections from drop-down lists:
a. Name: ViroSeq_spectral_instrument_protocol. 4. Click the Run Instrument button to start the run. The run time is
b. Description: enter a description for the protocol (optional). approximately 95 minutes.
c. Type: Spectral
d. Dye Set: E-BigDyeV1 5. At the end of the run, the calibration results are generated. The spectral
e. Polymer: POP6 calibration profile is updated and saved to the instrument database unless all
f. Array Length: 50 capillaries fail.
g. Chemistry: Sequencing Standard
h. Run Module: Spect50_POP6_1

4. Click OK.

To Create a Spectral Plate Record

1. Select GA Instruments > ga3130 or ga3130xl > Plate Manager.

2. Click New to open the New Plate Dialog box.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 61 of 68


Evaluate a Spectral Calibration Profile 4. Evaluate the spectral profile for the selected capillary.
Note: To magnify an area of interest in the spectral profile or raw data,
To View the Pass/Fail Status After the Spectral click-drag the cursor to create a box. Release the mouse button to display
Calibration Run the selected region. Press the r key to reset the view.
a. Verify that the order of the peaks in the spectral profile from left to right
are blue-green-yellow-red.
1. Select GA Instruments > ga3130 or ga3130xl > instrument name >
Instrument Status > Event Log. Blue Green Yellow Red

2. In the Events Messages section of the window, view the status of each
capillary.

To Evaluate the Spectral Profile and Raw Data


Note: Refer to the Applied Biosystems® 3130/3130xl Genetic Analyzers
Maintenance, Troubleshooting, and Reference Guide if:
• the spectral calibration fails (no spectral profiles are created),
• the peaks are not in the correct order or extraneous peaks are adversely
affecting the spectral profile, or
• the peaks are not separate and distinct.

1. Select GA Instruments > ga3130 or ga3130xl > instrument name > Example of a 4-dye spectral profile
Spectral Viewer.

2. In the Dye Set drop-down list, select E-BigDyeV1. b. Verify that the peaks in the spectral profile do not contain gross
overlaps, dips, or other irregularities (e.g., wide peaks that overarch
other peaks; peaks that are not distinct).
3. In the plate diagram, select a well on the plate diagram to view the capillary
spectral results. Peaks are distinct, regular and in the proper order – pass

Capillary status:

Passed (dark green)

Selected (light green)


Red peak is not distinct, regular or in the proper order – fail

Failed, Not Selected (light tan)

Failed (tan)

Note: A failing capillary is automatically assigned the spectral profile of its


nearest passing capillary.
IMPORTANT! Evaluate the spectral calibration profile for each capillary, 5. Click each well to review the data for each capillary. For a good-quality
even if the Spectral Calibration Results box indicates that they passed. calibration, each capillary must have a:
– Q-value above 0.95
– Condition number within the range 3.0 to 5.0

If a Capillary Fails
A failed capillary is automatically assigned the spectral profile of its nearest passing
capillary to the left. If there are no passing capillaries to the left, it will be assigned
the profile of the nearest passing capillary to the right. These capillaries are marked
in tan instead of green in the Array Viewer.
IMPORTANT! For ViroSeq analysis, where pull-up and pull-down peaks will
cause critical errors, repeat the spectral calibration and use a unique spectral for each
capillary.

Page 62 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


If the Calibration Fails
Try one or more of the following:
• Verify that the correct parameter file and run module were selected. If not, make
the correction and repeat the run.
• Verify that fresh reagents were used. If the reagents used were not fresh, stored
improperly, or expired, prepare fresh reagents and repeat the run.

To Activate a Spectral Calibration


IMPORTANT! A run cannot start unless a calibration file that matches the dye set
and capillary array length combination to be used for the run, is active.
IMPORTANT! Every new spectral calibration is automatically the active one for
that dye set.

Setting an Active Spectral Calibration

1. Select GA Instruments > ga3130 or ga3130xl >instrument name > Spectral


Viewer.
IMPORTANT! If the Spectral Viewer window is blank and deactivated,
then either:
– The spectral calibration for that dye set is not in the database, or,
– You changed the array length and you do not have a spectral
calibration file activated for that dye set and array length
combination.

2. In the Dye Set drop-down list, select E-BigDyeV1.

3. In the List of Calibrations for Dye Set drop-down list, select a spectral
calibration. The spectral profile and raw data is displayed.

4. If the spectral calibration is acceptable, then click Set. Otherwise, run a new
spectral calibration.

To Display a Spectral Calibration Profile

1. To view a current spectral calibration profile, select GA Instruments >


ga3130 or ga3130xl > instrument name > Spectral Viewer.

2. To view a previous spectral calibration profile:


a. Select GA Instruments > ga3130 or ga3130xl > Run History.
b. In the Run History, search for the run with the associated spectral
calibration profile you want to view or select Find all.
c. Select Spectral Calibration Viewer (under the Run History icon).
d. Select the run with the associated spectral calibration profile you want to
view from the Run to view pull-down list.

Autosampler

To Calibrate
Calibrate the autosampler if you see:
• Poor injection for a small number of capillaries
• Low signal strength
• No evidence of sample

1. Select Wizards > Autosampler Calibration Wizard.

2. Follow the directions in the wizard.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 63 of 68


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ViroSeqTM HIV-1 Genotyping System v2.0 Page 65 of 68


Appendix A: Auditing and To Enable Features of the Audit Map Configuration
1. In the AB Navigator left -pane, double-click Audit Map Configuration.
Software Access Control 7
2. Select the Enabled check box for each audit map to activate it.
• DC Plate Record
• DC Run Module
• DC Results Group
This appendix is written for the Administrator who implements auditing and access Note: An audit record is generated in Data Collection software when you:
control to the Data Collection software on the Applied Biosystems 3130/3130xl
• Create or edit a Plate Record
Genetic Analyzer instruments. For more information, refer to the Applied
• Create a run module or edit the parameters of a Run Module
Biosystems AB Navigator Software Administrator Guide (PN 4359472).
• Create or edit a Results Group

3. Exit Audit Map Configuration.


AB Navigator
AB Navigator is the access point for these applications:
• Access Control Administration
• Audit History Viewer
Audit Map Configuration Tool
• Audit Map Configuration The Audit Map Configuration Tool is used to manage audit maps. Audit maps
are used to control how auditing is done for a given data type.
To Start the AB Navigator Some features of the Audit Map Configuration Tool:
• Audit states of an audit map can be set to On, Off, or Silent.
IMPORTANT! At a minimum, you must start the first two Data Collection • There is no Save command. All changes to audit maps are saved
software services in order for AB Navigator to function properly. automatically.
Note: Access to the Audit Map Configuration is restricted to those with an
1. Start the Data Collection Software v3.0: Start > All Programs > Applied Administrator Profile.
Biosystems > Data Collection > Run Data Collection 3130 or Run Data When auditing is ON and a change occurs, the Reason(s) for Change dialog
Collection 3130xl. displays and contains:
• The attribute that was changed, created, or deleted
2. Start the AB Navigator application: Start > All Programs > Applied • The old and new values, if applicable, in the top half of the dialog box
Biosystems > AB Navigator. • A text box to enter the reason for the change:
– When you click OK, changes to the attribute and the audit data are
3. In the AB Navigator dialog box, type “Administrator” (default login name saved
and password) for the Login Name and Password or type your login name – When you click Cancel, no changes are saved and you return to the
and password if you have changed them and click OK. previous window.
IMPORTANT! Record your login name and password. Failure to When auditing is Silent, auditing still occurs in the database but the Reason(s) for
remember the login and password may require uninstalling and reinstalling Change dialog does not display.
the Data Collection software!
IMPORTANT! Always run with Auditing enabled.
4. In the AB Navigator window, click Administration to expand the Audit
1. Double-click the Audit Map Configuration icon in the left pane tree.
Map Configuration, Audit History Viewer, and Access Control
Administration options. The System Authentication dialog box displays.

2. Type your password in the System Authentication dialog box

Enabling Applications 3. Click OK.


The Audit Map Configuration window displays.

To Enable Access Control Administration


1. In the AB Navigator window, double-click Access Control
Administration.

2. Select Applications > FoundationViewerApp.

3. Select the Challenge check box to activate it. The Login and Password
dialog box is now enabled.
4. Individually select a cell in the Enabled column in the Audit Map Objects
4. Select File > Save. pane for DC Run Module, DC Results Group, and DC Plate Record. After
each selection, enter the reason(s) in the Reason(s) for Change dialog box
5. Exit Access Control Administration. and click OK.

Page 66 of 68 ViroSeqTM HIV-1 Genotyping System v2.0


Toolbar Menu Command Function
5. Individually select the object (e.g., DC Run Module) in the Audit Map
Objects pane then select an attribute in the Attributes pane. From the State Go To Displays a list of applications that are currently running.
Select an application to go to that application.
drop-down menu, select ON for each attribute. Enter text in the Reason(s)
for Change dialog box and click OK. Repeat this step until all of the Exit Application Exits the Audit Map Configuration application.
respective attributes are in the ON state. Exit AB Exits the AB Navigator application.
Navigator
View Filter Displays the filter pane on the top of the frame when
State Change Description Parameter selected. It allows the user to specify criteria that limits
the amount of audit records in the Audit Record table.
ON Reason for change required Records old and new values
OFF Reason for Change dialog box does not Does not record old or new value To Use Filter Command in Audit History
display changes
The filter allows you to limit the audit history records that are displayed.
SILENT Reason for Change dialog box does not Records old and new values
display 1. Click Filter. The Filter Audit Records pane displays.
2. Enter search criteria in the applicable text boxes.
3. Click Find Now. You can filter audit records by:
6. Exit Audit Map Configuration. • Name
• Date (includes before or after a date or between two dates)
• User name
7. Restart the software. • Matching whole words
IMPORTANT! Any changes to an audit map do not take effect until you • Case-sensitivity
restart the software

Access Control Administration


Audit History Viewer The Access Control Administration tool allows an administrator to manage the
creation, deletion, and modifications of users and profiles. It also allows an
The Audit History Viewer is used to view historical audit data. This tool is used as administrator to restrict or grant users access to features and functions of the
a read-only viewer for audit records. The tool provides data filtering so that Audit software.
Records can be viewed in different formats.
IMPORTANT! Do not misplace the administrator password! If the administrator
Note: Access to the Audit History Viewer is restricted to those with an password is lost, access control must be reset, causing the loss of all users and
Administrator Profile. profiles.
Audit Records that you can view with the Audit History Viewer are: The default administration user is always associated with the Administration User
• Date and time the Audit Record was created Group and cannot be deleted. In addition, only one administrator is allowed to
• The user who triggered the Audit Event modify access control data at one time.
• The Attribute that was changed
Default Profiles:
• The old and the new values
• The reason for the change Complete access to Instrument Protocols, Instrument
Administrator
Operation, and Instrument Maintenance
To Start the Audit History Viewer Scientist Complete access to Instrument Protocols, Instrument
Operation, and Instrument Maintenance
1. Start the AB Navigator. Refer to To Start the AB Navigator section. Technician Access to Instrument Operation and Maintenance

Access Rights:
2. In the AB Navigator left Viewer window, double-click Audit History
Viewer. Includes Run Module Operations, Results Group
Instrument Protocols Operations, Analysis Protocol Operations, Instrument
Protocol Operations and Re-extraction.
3. In the Audit History Viewer dialog box, type Administrator for the Login
Name and Password or type your login name and password if you have Instrument Operation
Includes Plate Operations, Event Log, and Instrument
changed them and click OK. The Audit History Viewer displays. Control Operations.
Includes Spatial Calibration Operations, Manual
Instrument Maintenance
Instrument Control, and Miscellaneous Operations.
To View an Audit History
To Create a New User
1. In the Audit Objects pane, navigate to the object of interest
IMPORTANT! You must set a default password for each new user.
2. Highlight an object and then select View > Details Panel to display audit
record details. 1. Click the New User icon. The New User dialog displays.
Note: Click the column headers to sort the read-only columns.
2. Click Next. The Configure pane displays.

Filter Commands in Audit History 3. Complete the information in the Create User window, click Set Password.
enter the new password and click OK.
Toolbar Menu Command Function
File Reload Refreshes the Audit History Viewer with the latest 4. Click Finish to complete the creation of a new user.
changes.
Report Customize and then print a report of the selected Audit
History Record. 5. Click Save.
Print Preview Customize and then preview a report of the selected
Audit History Record.
Page Setup Customize the page setup of the Report printout.

ViroSeqTM HIV-1 Genotyping System v2.0 Page 67 of 68


Page 68 of 68 ViroSeqTM HIV-1 Genotyping System v2.0
© 2001-2009 Celera Corporation. All rights reserved.
NOTICE TO PURCHASER: LIMITED LICENSE
This product is sold under licensing arrangements between Celera Corporation and Life Technologies Corporation.
The purchase price of this product includes limited, nontransferable rights under US Patents 5,035,996; 5,683,896;
5,945,313 and 6,287,823 and foreign equivalents owned by Life Technologies Corporation to use only this amount of
the product to practice the claims in said patents solely for activities of the purchaser within the field of human
diagnostics. Further information on purchasing licenses under the above patents may be obtained by contacting the
Licensing Department, Life Technologies Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008.
U.S. Patent No. 6,806,046
U.S. Patent No. 6,531,588
U.S. Patent No. 6,379,957
TRADEMARKS
Celera and its Spirit Design and ViroSeq are trademarks and registered trademarks of Celera Corporation or its
subsidiaries in the U.S. and/or certain other countries.
ABI PRISM, BigDye, Hi-Di, MicroAmp, and POP-6 are trademarks and registered trademarks of Life Technologies
Corporation or its subsidiaries in the U.S. and/or certain other countries.
Amplicor HIV-1 Monitor, AmpErase, AmpliTaq, AmpliTaq Gold, and GeneAmp are registered trademarks of Roche
Molecular Systems, Inc.
CENTRISEP 96 is a trademark of Princeton Separations, Inc.
Intel, Pentium, and Core are trademarks and registered trademarks of Intel Corporation.
Java ia a trademark of Sun Microsystems, Inc.
Microsoft, Windows, and Windows NT are registered trademarks of Microsoft Corporation.
All other trademarks are the sole property of their respective owners.

DRAFT
October 12, 2009 2:57 pm, ViroSeq IFU 3130 Back.fm
Celera Corporation
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Alameda, CA 94502 USA

Worldwide Sales and Support


For sales office locations and technical support,
please call your local Abbott Molecular office.

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