1. GS-FLX Technology – How Does it Work?
Deborah J. Hollingshead, MS
Genomics Manager
Genomics & Proteomics Core Laboratories
University of Pittsburgh

2. FLX Sequencing Overview
Prepare library of single stranded DNA, 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)

3. 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 ( )

4. Depositing DNA Beads into the PicoTiter™Plate
Load Enzyme Beads
Load beads into PicoTiter™Plate
44 μm
Adapted from: Roche 454 James Grabeau 2007 ( )

5. 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 ( )

6. 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 ( )

7. FLX Sequencing Reaction

8. 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 ( )

9. 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

10. 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.

11. Shotgun Library Preparation
Create random DNA fragments, 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:

12. Shotgun Library Data Read Alignment

13. Shotgun Library Data Contig ID

14. Paired End Library Preparation
Image from:

15. Paired End Data
Image from

16. Amplicon Library Preparation
Target amplicon of 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

17. Variant Detection

18. 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

19. 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 ( )

20. 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

21. 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

22. 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

23. Acknowledgements GPCL Roche Bryan Thompson Janette Lamb Paul Wood
Janna Lanza