Assessment of allelic bias in pre-capture platforms for exome sequencing

CMIS

Back to the future?Denis Bauer | Research Scientist 28 March 2012

Part 1:

My Backgroundand a selection of bioinformatics tools developed in Brisbane in the

Bailey Group and Boden Group

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 2

My Background

Berlin

Neusta

dt Brisb

ane

IMB Institute for Molecular Bioscience

QBI Queensland Brain Institute

NorahDesk

Sumoylation Predictor

Timothy Bailey

Mikael Bodén

Fabian Buske

Chikako Ragan

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 3

http://meme.sdsc.edu/meme/intro.html

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: 10

StreamQuantitative model of transcriptional regulation

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 4

http://www.bioinformatics.org.au/stream/

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

TriplexatorSearch/Design tool nucleic acid triple helices

Sneak P

review ... C

oming soon to

http://www.bioinformatics.org.au/triplexator/

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 5

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.

http://www.bioinformatics.org.au/norahdesk/

Specifically useful for miRNA- and piRNA-clusters that are transcribed together

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 6

Part 2: Back to the future

Exon capture is the economical way for an unbiased genome wide analysis.

However, extensive sample manipulation can introduce biases that we might not be aware of.

Is less sophistication saver? Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 7

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 sample

Pre-capture pooling for exome captureThe business side

Clark MJ, et al., Nat Biotechnol. 2011 PMID: 21947028.

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 8

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 companyNow AG: 72Mb

Pre-capture pooling for exome captureThe technical side

Clark MJ, et al., Nat Biotechnol. 2011 PMID: 21947028.

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 9

Oddities: targeted exons not follow the same length distribution as RefSeq exons

Presentation title | Presenter name | Page 10

Oddities: cont’ Theoretical vs actual capture efficiency of longest exon

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 11

Potential issue: “Allelic Bias”/ “Allelic imbalance” ?

Sequence hapmap family (4 individuals) with• AG: Post capture

• Ill: Precapture

• NG: Precapture

Pre-capture pooling for exome captureThe potential problem

Bait

Het sample 4

Potentially underrepresentedallele

Reference conform + hom bar-coded samples 1-3

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 12

• If Het-variances are not captured reliably in pre-capture the

het/hom ratio would be lower and they would not overlap with DBs

Allelic Bias ?H

et/h

om r

atio

known novel

NG: More Hets in postIll: More Hets in pre

Hapmap 1000G

Fra

ctio

n of

ove

rlap

NG: slighly lower overlap Ill: no difference

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 13

... they would have lower coverage

Allelic Bias ?

Asan, Xu Y et al. Genome Biol. 2011 PMID: 21955857

cove

rage

Illumina Nimblegen

INDELS

SNPs Com Post Pre

Com Post Pre

Com Post Pre

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 14

1. 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-sequencing

Conclusion

Exon Capture Comparison | Denis.Bauer@CSIRO.au | Page 15

Thank youCMISDenis C. BauerExon Capture Comparisont +61 2 9325 3174e denis.bauer@csiro.auw www.csiro.au/cmis

CMIS

Institute for Molecular Bioscience, UQTimothy Bailey (MEME)

School of Chemistry and Molecular Biosciences, UQ

Mikael Bodén (Machine Learning)

Queensland Brain Institute, UQVikki MarshallJoon-Yong An Sam LukowskiChikako Ragan (NorahDesk)

Garvan Institute, UNSWJohn MattickFabian Buske (Triplexator)