Genome Sequencer

FLX Titanium Research Applications Guide

October 2009

Genome Sequencer FLX System Research Applications Guide
October 2009 1




Table of Contents

1. Introduction to the Genome Sequencer FLX System...................................................... 2
1.1 The General Five-Step Workflow..................................................................................2
1.2 Overview of GS FLX Titanium System..........................................................................3
1.2.1 Library Preparation................................................................................................4
1.2.2 emPCR Amplification ............................................................................................5
1.2.3 Sequencing...........................................................................................................5
1.2.4 Data Processing....................................................................................................5
1.2.5 Data Analysis ........................................................................................................6
1.3 Use of Multiplex Identifiers ............................................................................................6
2. Ordering GS FLX Titanium Kits, Accessories, and Manuals .......................................... 7
2.1 Kits ................................................................................................................................7
2.2 Accessories...................................................................................................................8
2.3 Manuals and Guides .....................................................................................................9
3. Choosing the GS FLX Titanium Strategy For Your Experiment ................................... 10
3.1 Choose a Library Preparation Method........................................................................10
3.2 Calculate the Total Amount of Sequence Required and Choose the Regions Gasket
Size ....................................................................................................................................10
3.3 Choose an emPCR Amplification Method...................................................................11
3.4 Pick a Data Processing and Data Analysis Software..................................................11
4. Publications And Results................................................................................................. 12
5. Appendix............................................................................................................................ 13
5.1 Guidelines to cDNA Sequencing.................................................................................13
5.2 Guidelines to Amplicon Sequencing............................................................................13
5.3 NimbleGen Sequence Capture...................................................................................14
5.4 emPCR Shaker Adapters MV......................................................................................14

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1. INTRODUCTION TO THE GENOME SEQUENCER FLX SYSTEM
The Genome Sequencer FLX System is a DNA sequencing system capable of preparing,
amplifying, and sequencing a library of DNA fragments in a massively parallel fashion. The
system provides most of the components necessary for ultra-high throughput sequencing
experiments, including the Genome Sequencer FLX Instrument, kits, accessories, and software
to generate basecalls and interpret the raw reads.
1.1 The General Five-Step Workflow
The Genome Sequencer FLX System workflow can be divided in five general steps, from
sample input to analyzed output: Library Preparation, emPCR Amplification, Sequencing, Data
Processing, and Data Analysis.
The five general steps are briefly described as follows:
1. Library Preparation: The first step of the process is to prepare a DNA library from the sample
input. The DNA library must be composed of fragments appropriately modified for
amplification and sequencing in the Genome Sequencer FLX System. Several library
preparation methods are offered, depending on sample type and throughput required. All
include the modification of each DNA fragment by adding special sequences (Adaptors) that
will be recognized in later workflow steps.
2. emPCR Amplification: Once the library is constructed, the DNA fragments are immobilized
onto beads. Beads are amplified and enriched, resulting in the majority of the beads carrying
a single DNA fragment. Each bead is isolated in the aqueous phase of a water in oil micelle
(emulsification), along with the amplification reagents, for the clonal amplification of the DNA
fragments. Amplification is carried out in bulk, resulting in millions of individual beads that
are each coated with millions of clonal copies of different amplified DNA fragments.
3. Sequencing: After amplification of the library, the DNA-carrying beads are loaded into the
wells of a Pico Titer Plate (PTP) device such that each well contains only one DNA bead.
The loaded PTP device is then inserted into the Genome Sequencer FLX Instrument and
sequencing reagents, including each nucleotide, are sequentially flowed over the plate in
cycles. Each cycle generates light that is converted into digital images captured by the
camera.
4. Data Processing: These digital images, or raw data, are processed by the GS Run
Processor. This software application encompasses all the steps required to convert raw data
into basecalls and quality scores. The processing results in FASTA and Standard Flowgram
Format (SFF) files suitable for use in downstream analysis applications and upload to public
DNA sequence databases.
5. Data Analysis: Depending on sample type and experimental design, several data analysis
software have been tailored to generate the final output. These are the GS De Novo
Assembler for de novo sequencing and assembly, the GS Reference Mapper for
Resequencing and NimbleGen Sequence Capture, GS Amplicon Variant Analyzer (AVA) for
Amplicons. FASTA raw reads can be used for Metagenomics and 16S sequences.
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October 2009 3
1.2 Overview of GS FLX Titanium System
The 454 Sequencing workflow can be schematically represented in the diagram shown in Figure
1. The five steps of the workflow are represented with corresponding methods and kits for
different types of sequencing, from determining the sequence of unknown DNA or RNA, to ultra
deep sequencing of a small region of interest.
To reflect the flexibility of the GS FLX Titanium System, methods, kits and software are
displayed along the axis of the workflow. Below the axis are shown procedures involved in ultra
deep sequencing (also called amplicon or PCR product sequencing), and above the axis, the
procedures for other sequencing experiments.


Figure 1:The five steps of the sequencing workflow with methods, kits and software

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The five steps of a sequencing experiment are tabulated in Table 1.

Research
Application
Five Steps of Sequencing Experiment
Library Preparation
emPCR
Amp.
Sequencing
Data
Processing
Data Analysis
Contig Rapid
De Novo
Sequencing
Scaffold
PE 20-8 kb
Span
PE 3 kb
Span
Lib-L
Run
Processor
GS De Novo
Assembler
GS Reference
Mapper
Resequencing Rapid Lib-L
Run
Processor
GS Reference
Mapper

cDNA
/Transcriptome
sequencing
cDNA Rapid Lib-L
Run
Processor
GS De Novo
Assembler
GS Reference
Mapper

Targeted
Sequencing
(amplicon/PCR
products)
Amplicon Lib-A
Run
Processor
Amplicon
processing
scheme
Amplicon Variant
Analyzer (AVA)
Targeted
Sequencing
(NimbleGen
Sequence
Capture)
NimbleGen Arrays
Users Guide
(references the
General Lib Prep
Method Manual)
Lib-L






































M
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t
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o
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t
o

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s
e

w
i
t
h

t
h
e

k
i
t

X
L
R
7
0

Run
Processor
GS Reference
Mapper
Table 1: Methods corresponding to the five steps of a sequencing experiment (See Table 4 for the list of
method manuals)
Below is a brief introduction to the methods used in each step.
1.2.1 Library Preparation
There are 6 library preparation methods available, for shotgun Rapid and cDNA Rapid, three
Paired End (20 kb, 8 kb, and 3 kb Span), and amplicon (Amplicon) sequencing.
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For shotgun sequencing, the Rapid library preparation method should be used for DNA samples
and the cDNA Rapid should be employed for RNA samples. A shotgun sequencing experiment
will yield contigs.
When contig scaffolding is desired, preparing an additional library from the same sample as a
Paired End library will orient and order contigs into scaffolds. Three Paired End library
preparation methods are available, depending on the distance between the two paired
sequence tags (also known as the span). Paired End libraries also provide a certain proportion
of shotgun reads and in certain cases, these would suffice to provide all the sequencing
information required for the experiment (refer to TCB No. 008-2009). TCBs are viewable at
www.454.com/my454.
One can use either or both shotgun and Paired End sequencing for resequencing. Lists of
variations relative to a reference are produced in files containing local variations (SNPs and
short indels), as well as larger scale structural variations
The General library preparation is superseded by the Rapid library preparation, except for
NimbleGens Sequence capture experiments. Finally, Amplicon library preparation are used for
amplicon/PCR products in variant detection.
1.2.2 emPCR Amplification
Depending on the type of experiment performed, the GS FLX Titanium chemistry can be divided
in two main categories, amplicon and all other libraries.
Most experimental designs involving amplicons (PCR products) will use the emPCR Lib A kits
and the data analysis software Amplicon Variant Analyzer (AVA). Library preparation for
amplicons use custom fusion primers designed for the amplicon of interest that will also contain
additional sequences required with 454 Sequencing chemistry. This is why there is no library kit
for amplicon preparation.
All other experiments will use the Rapid, cDNA Rapid, or Paired End library preparation kits,
along with the emPCR Lib L kits, and the data analysis software GS De Novo Assembler
and/or GS Reference Mapper.
1.2.3 Sequencing
The sequencing workflow consists of three main parts, the Pre-Wash, the PicoTiterPlate (PTP)
device preparation, and the sequencing Run.
To begin a sequencing Run, the initial task is to close the previous Run, then to perform a pre-
wash of the fluidics system. Concurrently, the User prepares the PTP device and reagents.
Once the components are assembled, the User selects the desired Run script and specifies the
data processing scheme. Finally, the prepared PTP device is inserted into the PTP cartridge in
the instrument and the Run is launched.
1.2.4 Data Processing
The Data Processing software extracts data from the images taken during Sequencing, applies
correction algorithms, filters low quality data and generates files suitable for input to Data
Analysis applications.
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Data Processing can be broadly split into two phases, Image Processing and Signal Processing.
Image Processing is the extraction of uncorrected data from raw images and usually occurs
during the Sequencing Run. The second phase, Signal Processing, corrects for non-ideal
signals and performs the filtering and base calling. Signal Processing is significantly more
computationally intensive than Image Processing and is generally run on a separate computing
resource. Executing the Signal Processing on a separate computing resource frees up the
instrument for additional Sequencing run.
There are two main Signal Processing configurations, one for Shotgun/Paired End sequencing
and one for Amplicon sequencing. Performance of the Sequencing Runs can be evaluated by
viewing the results of the Data Processing with the 'gsRunBrowser' application.
1.2.5 Data Analysis
Three Data Analysis software applications are provided with the Genome Sequencer FLX
Titanium System. (1) Shotgun genomic data can be assembled into contigs, and the presence
of Paired End data allows contigs to be scaffolded, using the GS De Novo Assembler. With the
same software, shotgun transcriptome data can be assembled into isotigs, which are putative
transcripts (isoforms) constructed from the read data (2) When the application calls for
comparing the reads to one or more known references, the GS Reference Mapper can be used
(3) The AVA software is used to examine variations found in reads amplified from loci in multiple
individuals or populations.
1.3 Use of Multiplex Identifiers
It is possible to sequence several samples in the same emPCR amplification emulsion and to
separate them out after sequencing, using 454 Software. This is achieved with the use of
Multiplex Identifiers (MIDs), which are specific DNA sequences (sequence tags). MIDs allow for
an easier workflow when numerous samples are processed and for cost reduction by
multiplexing.
The MID kit to use with the Rapid library preparation contains a set of 12 MIDs and is
referenced in Table 2.
MID usage for amplicons is described in TCB No. 013-2009. A set of 14 MID adaptors has been
pre-loaded in the AVA software (TCB No. 013-2009). MID adaptors can also be designed by the
User (see TCB No. 004-2009 and 005-2009). TCBs are viewable at www.454.com/my454.
Please note that it is possible to sequence amplicons that do not have primers designed for 454
Sequencing Chemistry; these must have 454 Sequencing Chemistry specific sequences added
using the Rapid Library Preparation Kit.
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2. ORDERING GS FLX TITANIUM KITS, ACCESSORIES, AND MANUALS
2.1 Kits
Method Kit Name Kit Part Number
Rapid Library Preparation Kit 05 608 228 001 Rapid Library Preparation
Optional use of MID Adaptors:
Rapid Library MID Adaptors Kit
05 619 211 001
cDNA Synthesis System kit (Roche) 11 117 831 001
Primer random (Roche) 11 034 731 001
cDNA Rapid Library Preparation
Kits for Rapid Library Preparation (see above
entries)

GS FLX Titanium Paired End Adaptor Set 05 463 343 001 Paired End Library Preparation
20 kb, 8 kb, and 3 kb Span GS Nebulizer Kit 05 160 570 001
Amplicon Library Preparation None
emPCR - Lib-L:
GS FLX Titanium LV emPCR Kit (Lib-L) 05 618 428 001 LV
GS FLX Titanium emPCR Breaking Kits LV/MV
12 pcs
05 233 658 001
GS FLX Titanium MV emPCR Kit (Lib-L)
05 613 436 001
MV
GS FLX Titanium emPCR Breaking Kits LV/MV
12 pcs
05 233 658 001
GS FLX Titanium SV emPCR Kit (Lib-L) 05 618 444 001 SV
GS FLX Titanium emPCR Filters SV 64 pcs 05 233 674 001
emPCR Lib A:
GS FLX Titanium LV emPCR Kit (Lib-A) 05 619 114 001 LV
GS FLX Titanium emPCR Breaking Kits LV/MV
12 pcs
05 233 658 001
GS FLX Titanium MV emPCR Kit (Lib-A)
05 619 149 001
MV
GS FLX Titanium emPCR Breaking Kits LV/MV
12 pcs
05 233 658 001
GS FLX Titanium SV emPCR Kit (Lib-A) 05 619 165 001 SV
GS FLX Titanium emPCR Filters SV 64 pcs 05 233 674 001
GS FLX Titanium Sequencing Kit XLR70 05 233 526 001 Sequencing
GS FLX Titanium PicoTiterPlate Kit 70 x 75 05 233 682 001
GS FLX Titanium Sequencing Kit XLR70 05 233 526 001 Amplicon Sequencing
GS FLX Titanium PicoTiterPlate Kit 70 x 75 05 233 682 001
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Method Kit Name Kit Part Number
GS FLX Titanium Amplicon Control Beads
05 974 844 001
Maintenance wash GS FLX Maintenance Wash Kit 04 932 358 001
Table 2: List of the GS FLX Titanium System kits available
2.2 Accessories
Method Accessory Part Number
emPCR Lib-L and Lib-A:
MV GS FLX Titanium emPCR Shaker Adapter MV 05 618 487 001
LV GS FLX Titanium emPCR Shaker Adapters LV 05 233 887 001
70x75 Bead Deposition Device (2 large regions)
05 414 601 001
70x75 Bead Deposition Device (4 medium
regions)
05 414 610 001
70x75 Bead Deposition Device (8 M/S regions)
05 414 628 001
70x75 Bead Deposition Device (16 small
regions)
05 414 636 001
Bead Deposition Device
Counterweight for the Bead Deposition Device
05 414 644 001
Table 3: List of the GS FLX Titanium System accessories
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2.3 Manuals and Guides
October 2009 marks the release of the Amplicon library sequencing with the GS FLX Titanium
series chemistry for the Genome Sequencer FLX System. The collection of manuals and guides
for the GS FLX Titanium series is shown in Table 4.

Manual/Guide Title Release Date Part Number
Genome Sequencer FLX Manuals and Guides (binder):
Genome Sequencer FLX Instrument Owners Manual
Rapid Library Preparation Method Manual
Paired End Library Preparation Method Manual 20 kb and 8 kb
Span
Paired End Library Preparation Method Manual 3 kb Span
cDNA Rapid Library Preparation Method Manual
Amplicon Library Preparation Method Manual
emPCR Method Manual Lib-L LV
emPCR Method Manual Lib-L MV
emPCR Method Manual Lib-L SV
emPCR Method Manual Lib-A LV
emPCR Method Manual Lib-A MV
emPCR Method Manual Lib-A SV
Sequencing Method Manual
Oct 2009 05976855001
Genome Sequencer FLX System Software Manual Oct 2009 On-line
GS FLX Titanium Research Applications Guide Oct 2009 On-line
Tables of Material Required But Not Provided Oct 2009 On-line
Available by December 31
st
, 2009
Genome Sequencer FLX System Site Preparation Guide Oct 2009 On-line
Genome Sequencer FLX System Administrators Guide Oct 2009 On-line
Table 4: Manuals and guides of the GS FLX Titanium series
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3. CHOOSING THE GS FLX TITANIUM STRATEGY FOR YOUR EXPERIMENT
Choosing a sequencing strategy, and therefore the methods and kits to use for your experiment,
is a four step process:
1. Match your research application to the appropriate Library Preparation Method and choose
the method(s) that best fit(s) your needs.
2. Calculate the number of reads and / or the number of bases required for your experiment
and choose the appropriate regions gasket size.
3. Pick the type and amount of emPCR amplification reactions required.
4. Choose the appropriate data processing and analysis methods (which can be re-run if
incorrect).
3.1 Choose a Library Preparation Method
Use Table 1 for guidance in choosing the library preparation method that best fits your needs.
For each method there is a method manual.
3.2 Calculate the Total Amount of Sequence Required and Choose the Regions
Gasket Size
Sequencing on the PTP device can be performed with four bead loading gaskets that differ in
the size and number of regions available on the gasket. All regions of a specific multi-region
gasket are equal in size. The Large regions gasket is made of 2 regions, the Medium regions of
4, the M/S of 8, and the Small regions of 16.
The best gasket to use depends on the number of different samples that are to be loaded and
the number of reads/bases required. Use equation (1) to calculate the number of total bases
required for your experiment for Rapid and cDNA Rapid libraries, or the number of unique
Paired End reads for Paired End libraries, or the depth of coverage over the desired region for
Amplicon libraries, and equation (2) to choose the appropriate gasket. Table 5 will guide you to
choose the best gasket size for your experiment.
(1) For Shotgun sequencing (Rapid libraries): Size of the DNA sample to be sequenced x
desired coverage depth =Total number of bases required (Mb), where desired coverage depth
is 15x to 20x.
For Paired End libraries: see TCB No. 008-2009.
For cDNA Rapid libraries: see Section 5.1.
For Amplicon libraries: see Section 5.2.

(2) Total Mb required/ run capabilities =gasket size

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Number of Region / Size* Bases / Region
(Mbp)
Bases / full PTP Device
(Mbp)
Reads / Region
(x 10
3
)
2 / Large 180-280 360-560 450-650
4 / Medium 60-110 240-440 160-250
8 / M/S 30-55 240-440 80-120
16 / Small 10-20 160-320 25-40
Table 5: Determining the size and number of PTP regions to load with a DNA library sample
*for a PTP device size 70 x 75 mm.
3.3 Choose an emPCR Amplification Method
Once the Library Preparation Method picked and the regions gasket size established, it is time
to choose an emPCR amplification method. The emPCR amplification kits and methods come in
three sizes, large volume (LV), medium volume (MV), and small volume (SV).
Although there are several emPCR amplification and gasket size for sequencing a library on the
Genome Sequencer FLX Instrument, some permutations are more efficient than others as they
make better use of the PTP device. Table 6 shows the preferred emPCR amplification kit size to
use per amplification type and regions gasket size.

emPCR Amplification Type
Region Size
Lib - L Lib - A
16 (Small) SV SV
8 (M/S) MV SV / MV
4 (Medium) MV MV
2 (Large) LV MV / LV
Table 6: Preferred regions gasket size in emPCR amplification
3.4 Pick a Data Processing and Data Analysis Software
Use Table 1 to choose the appropriate software application for your experiment. Also refer to
Sections 1.2.4 and 1.2.5.
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4. PUBLICATIONS AND RESULTS
The Genome Sequencer FLX has enabled hundreds of peer-reviewed publications in a wide
range of research applications. The complete list of research publications is available on the
454 Life Sciences website at: http://www.454.com/publications-and-resources/publications.asp.
This page contains a publication search tool which allows researchers to search by Sequencing
Application, Field of Biology, Title, Author and Keyword (i.e. amplicons, 16S) and build
customized PDF lists.
Sequencing Applications
Ancient DNA
ChiP-seq/Metylation/Epigenetics
Eukaryote Whole Genome
Expression Tags
HIV Sequencing
Metagenomics & Microbial Diversity
Mitochondria/Viruses/Plastics/Plasmids
Prokaryote Whole Genome
Sequence Capture/Targeted Region
Small RNA
Somatic Variation Detection
Transcriptome Sequencing
Fields of Biology
Plants and Agricultural Biotechnology
Human Genetics & Genomics
Evolution & Ecology
Microbes, Viruses, Infectious Diseases
Metagenomics & Microbial Diversity
Model & Non-model organisms, Systems Biology
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October 2009 13
5. APPENDIX
5.1 Guidelines to cDNA Sequencing
The number of transcriptome sequencing runs required depends on several factors, including
the following:

1. The purpose of the experiment
2. The completeness of mRNA species in the sequenced sample
3. The estimated gene count
4. The sequence yield (total number of reads) per run
5. The average length of raw reads
6. The normalization of cDNA library
7. The quality of the mRNA sample

We recommend the following equation to estimate the required number of run:
Total number of bases required (Mb) =(Estimated gene count in the sample to be sequenced) x 40,000.
Or, for every 8,000 genes in a sequenced RNA sample, we recommend one full GS FLX
Titanium sequencing Run (approximately 1 million reads with an average length of 350 bases).
To obtain wider dynamic range in read count, add appropriate number of runs as desired.


Usage of pooled and normalized cDNA libraries is recommended for gene
discovery and genome annotation. Normalized samples are likely to produce higher
sensitivity and better gene coverage than non-normalized cDNA libraries.
Non-normalized cDNA libraries are used for measuring relative gene expression
using read counts.
5.2 Guidelines to Amplicon Sequencing
The processing and sequencing of amplicons is quite flexible and allows for a wide range of
experimental design. A researcher can choose a variety of options regarding design
parameters, such as the length of amplicons, the number of amplicons pooled together, the
number of reads desired for a given amplicon pool, and whether to read from the A end, the B
end, or both. Although the setup for a given experiment will depend on the specific project
goals, there are a number of general guidelines that will ensure the best possible result.
The highest confidence in low frequency variation will result from bi-directional reads.
A high-fidelity polymerase must be used in the amplicon generation step. Use of a low-
fidelity polymerase will result in many amplification induced variations in the sequence.
Although there are many choices of enzyme, Roches FastStart High Fidelity PCR
System has high fidelity coupled with robust amplification of a wide array of input
templates.
Greater confidence in results may be achieved by running replicates of the biological
material through the sequencing process and comparing the results.
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The level of multiplexing should be determined by:
o Number of amplicons of interest.
o Desired sensitivity/depth of coverage.
When sequencing mixtures of multiple amplicons, care must be taken in quantification
and pooling of amplicons.
o Equimolar mixtures will generate best results.
Forward and reverse reads will eliminate most systematic, context-dependent
sequencing errors.
o The ideal experiment has reads covering the amplicon forward and reverse.
Comparison of sample versus control will aid in identifying systematic errors.
o For example, a variation that shows at the same level in both the sample and the
control is not a biologically significant difference.
The number of amplicons that can be combined in an experiment, while theoretically unlimited,
is primarily determined by the desired sensitivity of detection.
These following guidelines are a general, though conservative, aid to help determine the level of
oversampling required for a desired level of detection. The following guidance accommodates
experimental realities such as variation in quantitation, pooling, amplification/sequencing
efficiencies of MID labeled amplicons, and amplification efficiencies of long versus short
amplicons.
Heterozygote detection.
o 40x
5% variation of single base changes and multibase deletions.
o 1000x coverage (good statistical chance for 50 variation reads)
1% variation of single base changes and multibase deletions.
o 5000x coverage (good statistical chance for 50 variation reads)
Single-base indels may require additional depth.
5.3 NimbleGen Sequence Capture
Targeted resequencing by sequence capture is a method of selecting a portion of the genome
of interest for sequencing. Sequence Capture works with custom designed genomic regions of
up to 5 Mb or on the whole human exome. The NimbleGen sequence capture method entails
preparing a GS FLX Titanium General library, hybridizing the library against an array that
selectively enriches for the desired genomic region(s), sequencing the enriched material, and
mapping the reads against the reference genome.
5.4 emPCR Shaker Adapters MV
The emPCR amplification, Medium Volume, both for Lib-L and Lib-A kits requires an emPCR
Shaker Adapters MV that comes pre-assembled to fit the Qiagen TissueLyser II. All prior
TissueLyser models require the following modifications to prevent the emulsion oil tube caps
Genome Sequencer FLX System Research Applications Guide
October 2009 15
from interfering with the safety shield: remove the four screws on the white back to expose the
two set screws, unscrew and adjust them into the off-set position. Screw in both set screws from
the accessory pack into the pre-tapped holes into the side of the cylinder and re-assemble the
adapter. Additionally, insert the two accessory screws into the pre-drilled holes on the edge.
Figure 2 shows a schematic representation of the emPCR Shaker Adapters MV assembly.

Genome Sequencer FLX System Research Applications Guide
October 2009 16

Figure 2: Assembling the emPCR Shaker Adapters MV

Published by
454 Life Sciences Corp.
A Roche Company
Brandford, CT 06405
USA
2009 454 Life Sciences Corp.
All rights reserved.
For Life Science Research Only.
Not for Use in Diagnostic Procedures
454, 454 LIFE SCIENCES, 454 SEQUENCING, FASTSTART, GS FLX TITANIUM,
emPCR, PICOTITERPLATE, and PTP are trademarks of Roche.
Other brands or product names are trademarks of their respective holders.
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