Victor Guryev. European Research Institute for the Biology of Ageing

Transcription

1 Victor Guryev European Research Institute for the Biology of Ageing September 29, 2014 Genomic resequencing in Medical diagnostics course Erasmus MC, Rotterdam

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4 Low coverage whole genome and deep exome sequencing of 2,500 individuals to discover 95% of variants at 1% frequency

5 [Boomsma et al, 2013] [Francioli et al, 2014] Median base coverage: 12x bp 500 bp

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8 1) Twice as many bases per slide! 2) Structural information!!! SINE SINE CONTIG 1 CONTIG 2 G A Molecular haplotyping (phasing) A T Genome assembly Structural variants Better repeat coverage Profiling of transcript isoforms

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10 [van Heesch et al, 2013] + Fosmid libraries: 40kb tags Protocol for Illumina [Williams et al 2012]

11 Average coverage: 5 WGS /site 5 WGS/site 10 WGS/site 5 WGS/s

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17 Per individual genome (compared to reference genome) 3.7M SNPs 360k short indels (1-20bp) 5.2k medium deletions ( bp) 3.3k large deletions ( 100+ bp)

18 Q: Medium coverage de novo variants validation? A: Possible, but a lot of validation will be required because of FP due to undercovered positions in parents: 917 candidates -> 284 de novo indels; 601 candidates -> 41 de novo SVs.

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22 Repeat content in human genome L1 Simple repeats AluYa5 AluYb8 SVA

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24 Mechanism: polymerase errors Tool example: GATK Haplotype Caller Prevalence: up to 10% of all indels are non-simple

25 Father Mother Child Mechanism: gene conversion Tool example: assembly, discordant pairs Prevalence: currently only several cases

26 Mechanism: (retro)transposition Prevalence: very high (>13,000 MEIs in GoNL) Tool example: Mobster [Thung et al, Genome Biology 15:488]

27 Chr15: 40.85Mb Chr7: Mb to chr deletion Chr15: 40.85Mb 210 Chr7:26.24Mb 534 to chr15 Mechanism: (retro)transposition Prevalence: GoNL about 40 cases Tool example: Discordant pairs (123SV)

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29 37kb, AF=28% ZF724 ZF257 ZF431 2 genes ZF253 ZF737 ZF100 New ZF expressed => 13 genes ZF93 ZF430 Including: FGRF1, EVC, FKBP10, PLOD2 ZF85 ZF681 ZF708 ZF675 ZF730 ZF724 ZF492 New ZF gene ZF98 ZF726 ZF728 ZF92 GO-term analysis: anomality of the limb diaphyses and bowing of the long bones ZF734 ZF429 ZF273

30 WGS Father WGS Mother WGS Child WES Father WES Mother WES Child Same methodologies are applicable for WES RD analysis: need additional correction to account for variation in enrichment Very limited sensitivity if SV breakpoint is outside of enriched area Tool examplea: GATK HaplotypeCaller, CONIFER, ExomeCNV

31 Father, WGS Mother, WGS Child, WGS Father, WES Mother, WES Child, WES Gene annotation

32 Father, WGS Mother, WGS Child, WGS ather, WES other, WES hild, WES Gene annotation

33 1. Next to the SNP world, there is a whole new world of structural alterations in our genomes. 2. Study design: # of libraries, type (PE, MP), insert sizes, read length 3. Combine methods for SV discovery: read depth, readpair, split-reads, de novo assembly 4. Do verifications ( PCR/Sanger-Seq, acgh, Enrichment -> NGS)

34 GoNL SV Team Victor Guryev UMCG Wigard Kloosterman UMCU Laurent C. Francioli UMCU Jayne Y. Hehir-Kwa UMCN Tobias Marschall CWI/MPI Alexander Schoenhuth CWI Matthijs Moed LUMC Eric-Wubbo Lameijer LUMC Abdel Abdellaoui VU Slavik Koval EMC/LUMC Joep de Ligt UMCN Najaf Amin EMC Freerk van Dijk UMCG Lennart Karssen EM/Polyomica Leon Mei LUMC Kai Ye LUMC/WASHU GoNL steering committee Paul de Bakker UMCU Dorret Boomsma VU Cornelia van Duin EMC Gert-Jan van Ommen LUMC Eline Slagboom LUMC Morris Swertz UMCG Cisca Wimenga UMCG University of Washington Fereydoun Hormozdiari Evan E. Eichler BGI Shenzen Jun Wang ERIBA, RuG, UMC Groningen Diana Spierings Marianna Bevova Rene Wardenaar Tristan de Jong Peter Lansdorp