Post on 21-Jul-2018
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
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
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