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 Mix DNA Library & capture beads (limited dilution) “Break micro-reactors” Isolate DNA containing beads Create “Water-in-oil” emulsion + PCR Reagents + Emulsion Oil Perform emulsion PCR Adapter carrying library DNA A B Micro-reactors 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 1. Genome is loaded into a PicoTiter™ plate 3. Load Reagents in a single rack 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 Photons Generated are Captured by Camera Reagent Flow PicoTiterPlate Wells Sequencing By Synthesis 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 Flow Order TACG Measures the presence or absence of each nucleotide at any given position KEY (TCAG) 4-mer 3-mer 2-mer 1-mer Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt )

GPCL Run Quality Metrics Region 1 2 3 4 Total Raw Wells 115,344 73,264 111,418 112,435 412,461 Keypass Wells 110,869 57,628 106,036 108,811 383,344 Passed Filter Wells 72,634 38,182 69,926 70,659 251,401 Total Bases 16,769,803 8,518,364 15,500,205 15,630,096 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 N2 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 Gaskets for existing 70 x 75 PTP: 2, 4, 16 lanes Gaskets for upcoming 20 x 75 PTP (under development): 1, 4, 12 lanes 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 Roche Bryan Thompson Janette Lamb Paul Wood Janna Lanza