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Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
Allelic Imbalance for Pre-capture Whole Exome Sequencing
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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 …

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.

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  • Figure S5 Reference-allele biases at heterozygous SNP sites. Shown is box-plot of the percentage of reference allele depth. The percentage of reference allele depth at each heterozygous site was calculated for each replicate of the three platforms as well as for whole genome sequencing within the targeted and flanking regions of Agilent (AgiYH) and NimbleGen (NimYH).
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    • 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 | Denis.Bauer@CSIRO.au | Page 2
    • 3. My Background Brisbane Neustadt Berlin IMB Institute for Molecular Bioscience QBI Queensland Brain Institute Timothy Mikael Bailey Bodén Sumoylation Predictor Fabian Chikako NorahDesk Buske Raganhttp://meme.sdsc.edu/meme/intro.htmlExon Capture Comparison | Denis.Bauer@CSIRO.au | Page 3
    • 4. StreamQuantitative model of transcriptional regulation Bauer, D.C., Buske, F.A., Bailey, T.L., “Dual-functioning transcription factors in the developmental gene network of Drosophila melanogaster”; BMC Bioinformatics 11 (1), 366; PMID: 20594356. Cited: 4 Bauer, D.C., Bailey, T.L., “Optimizing static thermodynamic models of transcriptional regulation.”, Bioinformatics, 2009, 25, 1640-1646. PMID:19398449. Cited: 5 Bauer, D.C., Bailey, T.L., “STREAM: Static Thermodynamic REgulAtory Model of transcription.”, Bioinformatics 2008 24: 2544-2545. PMID:18776194. Cited: 1 Bauer, D.C., Bailey T.L., “Studying the functional conservation of cis-regulatory modules and their transcriptional output.”, BMC Bioinformatics, Apr 29;9(1):220. PMID: 18442418. Cited: 10http://www.bioinformatics.org.au/stream/Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 4
    • 5. Triplexator Sneak PreviewSearch/Design tool nucleic acid triple helices Fabian A. Buske et al., "Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data", Genome Research 2012, accepted Fabian A. Buske et al., "Potential in vivo ... roles of nucleic acid triple-helices", RNA biology, 2011, PMID: 21525785 Coming soon tohttp://www.bioinformatics.org.au/triplexator/Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 5
    • 6. NORAHDESKDetecting ncRNA in sequencing data Ragan, C., Mowry, B.J. and Bauer, D.C. “Hybridization based reconstruction of small non-coding RNA transcripts from deep sequencing data”, NAR, 2012, review received. Specifically useful for miRNA- and piRNA-clusters that are transcribed togetherhttp://www.bioinformatics.org.au/norahdesk/Exon Capture Comparison | Denis.Bauer@CSIRO.au | 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 | Denis.Bauer@CSIRO.au | 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 sample Sonicate Pull out fragments corresponding to the sequence of known “exons” However, with sequencing cost going down the capture reaction becomes the bottleneck. Solution: “Pre-capture pooling” Apply Bait Library to more than one sampleClark MJ, et al., Nat Biotechnol. 2011 PMID: 21947028.Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 8
    • 9. Pre-capture pooling for exome captureThe technical side Bait library design NG: empirically optimized AG: overlapping RNA-baites IL: 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 | Denis.Bauer@CSIRO.au | Page 9
    • 10. Oddities:targeted exons not follow the same length distribution as RefSeq exonsPresentation title | Presenter name | Page 10
    • 11. Oddities: cont’Theoretical vs actual capture efficiency of longest exonExon Capture Comparison | Denis.Bauer@CSIRO.au | Page 11
    • 12. Pre-capture pooling for exome captureThe potential problemPotential issue: “Allelic Bias”/ “Allelic imbalance” ? Bait Potentially underrepresented allele Reference conform + hom Het sample 4 bar-coded samples 1-3Sequence hapmap family (4 individuals) with• AG: Post capture• Ill: Precapture• NG: PrecaptureExon Capture Comparison | Denis.Bauer@CSIRO.au | 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 DBs NG: More Hets in post NG: slighly lower overlap Ill: More Hets in pre Ill: no difference Fraction of overlapHet/hom ratio known novel Hapmap 1000GExon Capture Comparison | Denis.Bauer@CSIRO.au | Page 13
    • 14. Allelic Bias ? SNPs Com Post Pre ... they would have lower coveragecoverage INDELS Com Post Pre Com Post Pre Asan, Xu Y et al. Genome Biol. 2011 PMID: 21955857 Illumina Nimblegen Exon Capture Comparison | Denis.Bauer@CSIRO.au | 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 | Denis.Bauer@CSIRO.au | Page 15
    • 16. Institute for Molecular Bioscience, UQ Timothy Bailey (MEME) School of Chemistry and Molecular Biosciences, UQ Mikael Bodén (Machine Learning) Queensland Brain Institute, UQ Vikki MarshallThank you Joon-Yong An Sam Lukowski Chikako Ragan (NorahDesk)CMISDenis C. BauerExon Capture Comparison Garvan Institute, UNSWt +61 2 9325 3174 John Matticke denis.bauer@csiro.au Fabian Buske (Triplexator)w www.csiro.au/cmisCMIS

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