GS-FLX Technology – How Does it Work?

Deborah J. Hollingshead, MS Genomics Manager Genomics & Proteomics Core Laboratories University of Pittsburgh

FLX Sequencing Overview

• Prepare library of single stranded DNA, 200-500 bp long and ligate adapters • Perform emulsion PCR, amplifying a single DNA template molecule in each microreactor (bead).

• Sequence all clonally amplified sample fragments in parallel using pyrosequencing technology • Analyze sequence results – Align overlapping sequence of individual reads to define contigs (Shotgun) – Order and orient contigs, create scaffolds (Paired End) – Identify variants (Amplicon) – Determine gene expression patterns (Transcriptome)

Emulsion Based Clonal Amplification

A B Adapter carrying library DNA Mix DNA Library & capture beads (limited dilution) + PCR Reagents + Emulsion Oil Create “Water-in-oil” emulsion Micro-reactors “Break micro-reactors” Isolate DNA containing beads Perform emulsion PCR

• Generation of millions of clonally amplified sequencing templates on each bead From: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

Depositing DNA Beads into the PicoTiter™Plate Load Enzyme Beads Load beads into PicoTiter™Plate 44 μm Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

454 Sequencing Instrument 3. Load Reagents in a single rack 1. Genome is loaded into a PicoTiter™ plate 2. Load PicoTiter plate into instrument Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

Reagent flow and image capture

PicoTiterPlate Wells Reagent Flow Sequencing By Synthesis Photons Generated are Captured by Camera Sequencing Image Created

Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

FLX Sequencing Reaction

https://www.roche-applied-science.com/servlet/RCConfigureUser?URL=StoreFramesetView&storeId=10357&catalogId=10356&langId=-1&countryId=jp

454 Sequencing: BaseCalling • Count the photons generated for each “flow” • Base call using signal thresholds • Delivery of one nucleotide per flow ensures accurate base calling

4-mer Flow Order T A C G 3-mer KEY (TCAG) 2-mer Measures the presence or absence of each nucleotide at any given position 1-mer

Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

GPCL Run Quality Metrics

Raw Wells Keypass Wells Passed Filter Wells Total Bases Region 1

115,344 110,869 72,634 16,769,803

2

73,264 57,628 38,182 8,518,364

3

111,418 106,036 69,926 15,500,205

4 Total

112,435 412,461 108,811 383,344 70,659 15,630,096 251,401 56,418,468

Different Library Preparation Methods for Different Project Aims

• • •

Shotgun Library Preparation

for

de novo

or resequencing of genomic DNA or long PCR product. Align overlapping reads to define contigs

Paired End Library Preparation

provides regions of sequence a known distance apart, allowing for ordering of contigs and analysis of genetic rearrangement.

Amplicon Library Preparation

for detection of rare variants.

Shotgun Library Preparation

Create random DNA fragments, 300-800 bp, by nebulization with compressed N 2 Ligate universal adpaters “A” and “B”. Select for “A” – “B” fragments. Remove second strand Attach to library beads via “B” adapter at calculated concentration to yield a single template molecule per library bead Proceed to emPCR Images from: https://www.roche-applied-science.com/

Shotgun Library Data Read Alignment

Shotgun Library Data Contig ID

Paired End Library Preparation

Image from: http://www.nature.com/nmeth/journal/v5/n5/images/nmeth.f.212-F1.jpg

Paired End Data

Image from http://www.nature.com/nmeth/journal/v5/n5/images/nmeth.f.212-F2.jpg

Amplicon Library Preparation

• Target amplicon of 200-500 bp – 200 bp for uni-direction reads – 500 bp requires bi-directional reads • Amplify using fusion primers that include template specific primer and primers A and B •Purify and quantify •Proceed to emPCR

Variant Detection

Transcriptome Analysis

• Technology under development • rRNA reduction prior to labeling essential • Different RT priming strategies are under investigation in several labs – Oligo(dT) – Random primers – Nugen RNA amplification system • ds-cDNA is processed as shotgun library • cDNA input requirement (3-5 ug) is challenging

GS FLX Throughput Multiple Gasket Formats and Plate Sizes Provide Flexibility in Sample Loading and Throughput Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

Sample Multiplexing

• Use of MID (multiplex identifier) tags allows multiple samples to be run in a single region – 12 different 10 base MID sequences supported by Roche – Software update due out by December will support 14 MID tags for amplicon sequencing – Can be included in PCR primer design or kit with MID adapters is available for shotgun library prep – Can design your own, but the ones from Roche have certain quality check characteristics in design

Sample Enrichment Techniques

• 15-20X coverage needed • Some samples include a lot of off target sequence • How to target the area of interest?

– Long range PCR followed by shotgun prep – Array or solution based sequence capture • Roche/Nimblegen service • Agilent do it yourself products – arrays and solution based • Both fully configurable custom content

Coming Soon – Titanium Assay

• Longer reads – 500 bp • More reads per PTP – 1M per full 70 x 75 • Training due and full roll out expected by Jan. 2009

Acknowledgements

• GPCL – Bryan Thompson – Janette Lamb – Paul Wood • Roche – Janna Lanza