Professional Documents
Culture Documents
0197
4J94-58
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
Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
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
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.
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.
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
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.
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.
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.
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.
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
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.
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.
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).
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.
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:
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
• 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.
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.
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.
4. Click OK.
5. In the Warning dialog box, click Yes to overwrite the previous settings.
3. General tab:
• Name: ViroSeq_Analysis_Protocol
• Description: optional
• Sequence File Formats: uncheck all check boxes.
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.
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.
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.
Do not use data in the process of collection if electrical power fails in the
middle of a run.
To Monitor a Run
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).
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).
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.
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.
1. From the Main menu, select Analysis > Analysis Defaults. The Analysis
Defaults window opens.
8. Click Done.
1. Click the Add Samples icon in the toolbar or select File > Add
Sample(s).
To add … Then …
All samples within a single folder Select the folder, 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.
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.
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.
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.
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.
1. To remove all files, select File > Remove All Samples from the Main menu.
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.
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
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.
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
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
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.
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.
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
Valine Val V
File Menu
IUB Codes
IUB Codes
Asparagine Asn N Save Ctrl+S Saves the open project with any edits that you
have made.
Glutamate or Glutamic acid Glu E Export Resistance Report Ctrl+X Allows you to export the Resistance Report.
Histidine His H
Isoleucine Ile I
Leucine Leu L
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
Window Menu Options
Adenine Green
Keyboard
Menu Item Description
Shortcut Cytosine Blue
Thymine Red
Help Menu
Mixed bases Pink
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.
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%.
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.
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.
Trimmed
sequence
Example of Trimming
Before Trimming
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.
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.
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.
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: 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.
<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.
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
ABC TDF
3TC & FTC
V118I 0.0625
M184I/V 0.2
NVP
Mutation Score Mutation Score
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
NFV LPV/r
APV/FOS
A71I/T/V 0.0625
V82A 0.0625
I50L/V 0.125
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.
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
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.
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
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.
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)
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.
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.
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.
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 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.
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
2. Run the Water Wash Wizard to wash the PDP chamber, lower polymer
block, channels, and tubing with distilled or deionized water.
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.
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.
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. 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.
2. In the Events Messages section of the window, view the status of each
capillary.
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:
Failed (tan)
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.
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.
Autosampler
To Calibrate
Calibrate the autosampler if you see:
• Poor injection for a small number of capillaries
• Low signal strength
• No evidence of sample
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.
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.
DRAFT
October 12, 2009 2:57 pm, ViroSeq IFU 3130 Back.fm
Celera Corporation
1401 Harbor Bay Parkway
Alameda, CA 94502 USA