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Genome Sequencer FLX Titanium Research Applications Guide October 2009
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GS FLX System Research Aplications Guide

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Page 1: GS FLX System Research Aplications Guide

Genome Sequencer FLX Titanium Research Applications Guide

October 2009

Page 2: GS FLX System Research Aplications Guide
Page 3: GS FLX System Research Aplications Guide

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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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 User’s Guide (references the General Lib Prep Method Manual)

Lib-L

Met

hod

to u

se w

ith th

e ki

t XLR

70

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 NimbleGen’s 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 Owner’s 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 31st, 2009

Genome Sequencer FLX System Site Preparation Guide Oct 2009 On-line

Genome Sequencer FLX System Administrator’s 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 103)

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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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, Roche’s 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

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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.

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Figure 2: Assembling the emPCR Shaker Adapters MV

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Published by

454 Life Sciences Corp. A Roche CompanyBrandford, CT 06405USA

© 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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