Agenda Item0102 Agenda Item

03 Agenda ItemThe right answer the first time - why accuracy mattersSOLiD Sequencing

Roland Wicki, Dir Mkt Dev Sequencing, Life TechnologiesREADNA Workshop – Berlin - 14 Jan 2010

SOLiD Development Road Map

12 January 2010

Chairman and CEO Greg Lucier told investors at the JP Morgan Health Care conference held here that the firm plans to present more information on the single-molecule sequencer, as well as make a major announcement regarding updates to SOLiD at the Advances in Genome Biology and Technology meeting in February.

SOLiD™ 3 Plus SystemLaunched 1st October, 2009

• Walk away operation, remote monitoring• Throughput Specs

— 60Gb Mate Pair, 1.2B reads• 2 Flow Cells, independent• Sample multiplexing

— RNA and DNA, mix-and-match• 10x faster mapping• Color and/or Base Call Output

(Nucleotide Sequence)— Preserving industry-highest

two-base encoding accuracy• Additional Application Kits

2 color change = SNP

Differentiate between misreads and real SNPAccuracy advantage of 2-Base Encoding – every base read twice

Reference sequence

Sequence reads

1 color change = misread

TACGTCGATATGCATGCTAGCGTACGGATCA

2 color change = SNP

Differentiate between misreads and real SNPAccuracy advantage of 2-Base Encoding – every base read twice

Reference sequence

Sequence reads

1 color change = mis-read

TACGTCGATATGCATGCTAGCGTACGGATCACT

AC TG A TCGCC ACC TG A TCGAA T

Other platformsNo two-base encoding

SOLiD™ System Enables Mutation Detection at Low Coverage due to Two-Base Encoding

AB SOLiD™ 3 Plus SystemWith two-base encoding

C C G A T C A G A C T

?

C C G A T A G A C TAC

SNP??

*schematic not to scale for typically SNP detection coverage

SOLiD Alignment BrowserMis-read vs. real SNP – toggel between color-data and bases

Two valid adjacentcolor changes suggest nucleotide change (SNP)

Single color change suggests instrument mis-call

Three valid adjacent color changes suggest two adjacent nucleotide changes (SNPs)

Accuracy SOLiD vs. SBS Platform

• Same DNA sample sequenced on both platforms

• SOLiD System’s Accuracy requires less sequence reads to accurately call SNPs and mutations

• Particularly useful in low coverage regions or when funding limited

SOLiD needs 2 readsper allele to call SNPs

SBS platform needs 3-4 readsper allele to call SNPs

< 5 6-10 11-150%

10%

20%

30%

40%

50%

60%

70%

0

0.005

0.01

0.015

0.02

0.025% of bases Error Rate

16-20 21-25 26-30 31-35 36-40Base QV Score

% B

ases

SOLiD 3 Plus System Read AccuracyGreatest number of bases > QV30

System Accuracy DifferencesPublic 1000 Genomes Data in Broad Integrative Genomics Viewer

SOLiD 3 System

Non-SOLiD

http://www.broadinstitute.org/igv-beta/index.html

SOLiD Whole Genome Cancer SequencingSmoking 15 cigarettes introduces one mutation

Nature 463, 184-190 (14 January 2010)

SOLiD Whole Genome Cancer Sequencing

• Validated Insertions• Validated deletions• Substitutions• Copy Number

Variations• Validated Intra-

Chromosomal and Inter-Chromosomal Rearrangements

Nature 463, 184-190 (14 January 2010)

SOLiD Whole Genome Cancer SequencingValidated Complex Rearrangements and Fusion Transcripts

Nature 463, 184-190 (14 January 2010)

SOLiD Structural Variation AnalysisComprehensive characterization of all variants in the genome

SOLiD Sequencing, 10kb mate pairs

aCGH

Slide courtesy of Yjjun Ruan, GIS

Chromosomal rearrangementsMasked as CNV on CGH Arrays

CGH ArrayDetect copy number?

SOLiD Distinguish translocations!

SOLiD Structural Variation AnalysisComprehensive characterization of all variants in the genome

Slide courtesy of Yjjun Ruan, GIS

SOLiD 3 Plus - Data Analysis Made SimpleBioScope™ 1.0

RNA-Seq and DNA-Seq in single SOLiD runMatched Normal/Tumor Samples

Deletions

Known Tumor Suppressor Gene

Candidate Tumor Suppressor Gene

SOLi

D R

NA

-Seq

0.5x1x 1x

Normal (+)

Normal (-)

Tumor (+)

Tumor (-)

aCGHSOLiD Seq

Chr1 - NM_. . . .02Chr2 - NM_. . . .97

158 readsFusion Candidate 1

Fusion transcript detected using BioScopeTaqMan® validation of putative fusion transcripts

Normal* Tumor*Position Strand p-value* Coverage

NormalCoverage

TumorReference Nucleotide

Observed Nucleotides A C G T A C G T

1,973,235 - 1.4E-81 323 194 T C, T 0 319 0 1 0 36 0 156

1,973,238 - 9.7E-77 318 197 T C, T 0 307 0 1 0 35 0 144

1,973,500 - 3.7E-20 60 35 A A, G 0 0 59 0 33 0 1 0

1,973,686 - 3.2E-14 52 26 C C, G 0 0 41 0 0 21 0 0

1,974,744 - 8.1E-30 94 63 C C, T 0 0 0 85 0 51 0 1

1,975,429 - 2.5E-30 126 43 G A, G 118 0 0 0 3 0 32 0

2,108,628 - 1.4E-05 24 24 G A, G 0 0 21 0 16 0 8 0

SOLiD Allele-specific Expression AnalysisTwo-Base Encoding highly beneficial at low expression

* Matched normal and tumor tissues** P-value from a χ2 test (2x4 contingency table)

Variant Detection by Microbial Sequencing

• Yersinia pestis strain sequenced had lost virulencfactor due to large deletion

Virulence positive

Virulence negative

SOLiD 2.0 data

SOLiD™ Targeted Resequencing Workflow

Shotgun Sequencing for Quantitative Analysis

• SOLiD 3 Plus System

• 5ml maternal plasma input

• 12M uniquely mapped reads per sample, random distribution

Chiu et alClinical Chemistry 56:3 (2010)

Family of Solutions for RNA Analysis

Whole Transcriptome

SOLiD™System

WT Analysis Pipeline

Taqman®Gene

Expression Assays

SAGE

RecoverAll

SAGE Analysis Tool

Small RNA

mirVana™miRNA

Isolation Kit

Small RNA Analysis Pipeline

RiboMinus™Transcriptome

Isolation Kit

SOLiD™ Whole Transcriptome

Analysis Kit

SOLiD™ Small RNA Expression Kit

SOLiD™ SAGE

SOLiD Single Cell Expression AnalysisGetting the most out of n=1

• 61.4% of RefSeq genes expressed

• Many genes expressed more than one splicing isoform concurrently in the same cell

• >1,700 novel splice junctions were identified

• 5,270 more genes detected than microarray, incl. 1,027 that the array doesn't test

Tang et al, Nature Methods, 6, 377-382 (2009)

Greg Lucier, CEO Life TechnologiesJP Morgan Healthcare Conference; 12 January, 2010

… expect our sequencing technologies to migrate into clinical diagnostic

applications…

The single molecule sequencing …ultra-long reads of DNA within hours. … will further the movement of this

science into the clinical realm

A lot of short-run sequencing will go on and that will be ever more used in a

diagnostic setting

3500DX (CE-IVD) SOLiD System Single Molecule SequencingSystem

3500 DX Series Genetic Analyzers

• 1st capillary electrophoresis system to meet the requirements of the In Vitro Diagnostic Medical Devices Directive (98/79/EC)

• Launched in Autumn 2009 in Europe

• RUO version available