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Allelic Imbalance for Pre-capture Whole Exome Sequencing

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Exome sequencing has emerged as an economical way of focusing DNA sequencing efforts on the most functionally understood regions of the genome. Pre-capture pooling, where one bait library is used to pull down the exonic regions of several pooled samples simultaneously is a further financial improvement. However, rare alleles in the pool might not be able to attract baits at the same rate as reference conform sequences can, and may hence be underrepresented. We investigated this potential issue by sequencing a hapmap family (4 individuals) using the pre-capture protocol from Illumina and Nimblegen. We did not observe clear evidence that heterozygote variants are missed but noted a trend for indels to be imbalanced. Our findings do not provide clear evidence to rule out allelic imbalance or bias having an impact on research findings, this may be especially critical for low cellular cancer tissue where rare alleles are more ubiquitous.

Text of Allelic Imbalance for Pre-capture Whole Exome Sequencing

  • 1. Assessment of allelic bias in pre-capture platformsfor exome sequencingBack to the future?Denis Bauer | Research Scientist28 March 2012CMIS

2. Part 1:My Backgroundand a selection of bioinformatics tools developed inBrisbane in the Bailey Group and Boden GroupExon Capture Comparison | [email protected] | Page 2 3. My BackgroundBrisbane Neustadt BerlinIMB Institute for Molecular BioscienceQBI Queensland Brain InstituteTimothy Mikael Bailey BodnSumoylation PredictorFabianChikakoNorahDesk BuskeRagan Capture Comparison | [email protected] | Page 3 4. StreamQuantitative model of transcriptional regulationBauer, D.C., Buske, F.A., Bailey, T.L., Dual-functioningtranscription factors in the developmental gene network ofDrosophila melanogaster; BMC Bioinformatics 11 (1),366; PMID: 20594356. Cited: 4Bauer, D.C., Bailey, T.L., Optimizing static thermodynamicmodels of transcriptional regulation., Bioinformatics,2009, 25, 1640-1646. PMID:19398449. Cited: 5Bauer, D.C., Bailey, T.L., STREAM: Static ThermodynamicREgulAtory Model of transcription., Bioinformatics 200824: 2544-2545. PMID:18776194. Cited: 1Bauer, D.C., Bailey T.L., Studying the functionalconservation of cis-regulatory modules and theirtranscriptional output., BMC Bioinformatics, Apr29;9(1):220. PMID: 18442418. Cited: 10 Capture Comparison | [email protected] | Page 4 5. Triplexator Sneak PreviewSearch/Design tool nucleic acid triple helicesFabian A. Buske et al., "Triplexator:Detecting nucleic acid triple helices ingenomic and transcriptomic data",Genome Research 2012, acceptedFabian A. Buske et al., "Potential in vivo ...roles of nucleic acid triple-helices", RNAbiology, 2011, PMID: 21525785 Coming soon to Capture Comparison | [email protected] | Page 5 6. NORAHDESKDetecting ncRNA in sequencing dataRagan, C., Mowry, B.J. and Bauer, D.C. Hybridization basedreconstruction of small non-coding RNA transcripts fromdeep sequencing data, NAR, 2012, review received.Specifically useful for miRNA- and piRNA-clusters that are transcribed together Capture Comparison | [email protected] | Page 6 7. Part 2: Back to the future unbiasedExon capture is the economical way for angenome wide analysis.However, extensive sample manipulation can introduce biases thatwe might not be aware of. Is less sophistication saver?Exon Capture Comparison | [email protected] | Page 7 8. Pre-capture pooling for exome captureThe business side Economical way of focusing 2GS efforts on the most functionally understood regions.Whole DNA sampleSonicatePull out fragments corresponding to the sequence of known exons However, with sequencing cost going down the capture reaction becomes the bottleneck.Solution: Pre-capture poolingApply Bait Library to more than one sampleClark MJ, et al., Nat Biotechnol. 2011 PMID: 21947028.Exon Capture Comparison | [email protected] | Page 8 9. Pre-capture pooling for exome captureThe technical side Bait library designNG: empirically optimizedAG: overlapping RNA-baitesIL: Gapped tiles What is an exon ?Everything that is known to be transcribed/has function trust company Now AG: 72MbClark MJ, et al., Nat Biotechnol. 2011 PMID: 21947028.Exon Capture Comparison | [email protected] | Page 9 10. Oddities:targeted exons not follow the same length distribution as RefSeq exonsPresentation title | Presenter name | Page 10 11. Oddities: contTheoretical vs actual capture efficiency of longest exonExon Capture Comparison | [email protected] | Page 11 12. Pre-capture pooling for exome captureThe potential problemPotential issue: Allelic Bias/ Allelic imbalance ?Bait Potentially underrepresented alleleReference conform + homHet sample 4 bar-coded samples 1-3Sequence hapmap family (4 individuals) withAG: Post captureIll: PrecaptureNG: PrecaptureExon Capture Comparison | [email protected] | Page 12 13. Allelic Bias ? If Het-variances are not captured reliably in pre-capture thehet/hom ratio would be lower and they would not overlap with DBsNG: More Hets in postNG: slighly lower overlapIll: More Hets in preIll: no difference Fraction of overlapHet/hom ratio known novelHapmap1000GExon Capture Comparison | [email protected] | Page 13 14. Allelic Bias ? SNPs Com PostPre... they would have lower coveragecoverage INDELSComPostPreCom PostPreAsan, Xu Y et al. Genome Biol. 2011 PMID: 21955857 IlluminaNimblegen Exon Capture Comparison | [email protected] | Page 14 15. Conclusion1. We (and others) did not detect any obvious allelic imbalance, however no one tested samples with really rare alleles (e.g. Low cellularity in cancer)2. To be on the save side (BACK TO THE FUTURE): we go for post- capture whole-exom-sequencingExon Capture Comparison | [email protected] | Page 15 16. Institute for Molecular Bioscience, UQTimothy Bailey (MEME)School of Chemistry and Molecular Biosciences, UQMikael Bodn (Machine Learning)Queensland Brain Institute, UQVikki MarshallThank you Joon-Yong An Sam LukowskiChikako Ragan (NorahDesk)CMISDenis C. BauerExon Capture Comparison Garvan Institute, UNSWt +61 2 9325 3174 John Matticke [email protected] Buske (Triplexator)w

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