gs-flx technology – how does it work? deborah j. hollingshead, ms genomics manager genomics &...
TRANSCRIPT
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