GS-FLX Technology – How Does it Work? Deborah J. Hollingshead, MS Genomics Manager Genomics &...

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

• Generation of millions of clonally amplified sequencing templates on each bead

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

BMicro-reactors

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

Load Enzyme BeadsLoad beads into

PicoTiter™Plate

Depositing DNA Beads into the PicoTiter™Plate

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

44 μm

3. Load Reagents in a single rack

454 Sequencing Instrument

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

Photons Generated

are Captured

by Camera

Reagent Flow

PicoTiterPlate Wells

SequencingBy 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 OrderTACG

Measures the presence or absence of each nucleotide at any given position

KEY (TCAG)

1-mer

2-mer

3-mer

4-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 DataContig 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 ThroughputMultiple 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