Grc ashg2015 workshop_mudge
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150224 grc kms
- 1. Characterizing extreme diversity in the human genome using a single haplotype genomic resource Karyn Meltz Steinberg, Ph.D. AGBT 2015 GRC Workshop @KMS_Meltzy
- 2. 1 bp 1 chr Frequency SNP Trisomy monosomy Copy number variants Size of variant 1 kb 1 Mb Types of genetic variants Slide courtesy of S. Girirajan Human Genetic Variation
- 3. 1 bp 1 chr Frequency SNP Trisomy monosomy Copy number variants Size of variant 1 kb 1 Mb Types of genetic variants 1 bp 1 chr Throughput 1 kb 1 Mb Size of variant How do we assay them? Slide courtesy of S. Girirajan Human Genetic Variation
- 4. 1 bp 1 chr Frequency SNP Trisomy monosomy Copy number variants Size of variant 1 kb 1 Mb Types of genetic variants SNP genotyping 1 bp 1 chr Throughput 1 kb 1 Mb Size of variant How do we assay them? Slide courtesy of S. Girirajan Human Genetic Variation
- 5. 1 bp 1 chr Frequency SNP Trisomy monosomy Copy number variants Size of variant 1 kb 1 Mb Types of genetic variants Array-CGH Karyotyping SNP genotyping 1 bp 1 chr Throughput 1 kb 1 Mb Size of variant How do we assay them? Slide courtesy of S. Girirajan Human Genetic Variation
- 6. 1 bp 1 chr Frequency SNP Trisomy monosomy Copy number variants Size of variant 1 kb 1 Mb Types of genetic variants Array-CGH Karyotyping Sequencing SNP genotyping 1 bp 1 chr Throughput 1 kb 1 Mb Size of variant How do we assay them? Slide courtesy of S. Girirajan Human Genetic Variation
- 7. Extreme diversity in the human genome • <99.5% identity to the reference • Refractory to traditional sequencing efforts • Loci often contain gene families associated with immune response and xenobiotic metabolism
- 8. HLA is a classic example of an extremely diverse locus • Critical to immune response • Characterized by overdominant selection • Alleles are linked and segregate as distinct haplotypes • Shaped by gene duplication and diversification
- 9. Segmental duplications can predispose loci to further rearrangement via NAHR
- 10. Segmental duplications can predispose loci to further rearrangement via NAHR
- 11. A A C T C G C C Repeat Copies (noted by color difference) Allelic Copies Diploid Genome With a diploid genome, there is significant ambiguity sorting allelic copies from repeat copies A C C C Haploid Genome Repeat Copies (ONLY but noted by color differences) With a haploid genome, allelic differences are eliminated, and base differences are likely indicative of repeat copies
- 12. Hydatidiform mole
- 13. SRGAP2 Homology between genes Shows nearly identical segments between SRGAP2A and SRGAP2 paralogs Shows homology between SRGAP2B and SRGAP2C Dennis, et.al. 2012 SRGAP2A SRGAP2B SRGAP2C
- 14. 1q21 1q21 patch alignment to chromosome 1 1q32 1q21 1p21
- 15. Hydatidiform mole Let’s sequence and assemble the whole genome!
- 16. CHM1_1.1 Assembly • Reference-guided assembly • SRPRISM v2.3, R. Agarwala • Alignment of Illumina reads to GRCh37 primary assembly • CHORI-17 BAC clone tilepaths were then incorporated • 428 total clones • 324 clones in 45 tilepaths • 104 clones as singletons http://www.ncbi.nlm.nih.gov/assembly/GCF_000306695. 2 Total Sequence Length 3,037,866,619 bp Total Assembly Gap Length 210,229,812 bp Number of Scaffolds 163 Scaffold N50 50,362,920 bp CHM1 Assembly Paper - Genome Research Steinberg et al. 2014
- 17. CHM1_1.1 assembly is highly contiguous compared to other WGS based assemblies
- 18. Integrating BAC tiling paths improved assembly
- 19. Integrating BAC tiling paths improved assembly
- 20. Alignment of CHM1 Illumina data to assembly revealed regions of extreme heterogeneity Heterozygous Homozygous Total Variants 64033 22513 86546 In RepeatMasked (RM) sequence 37060 14833 51893 In Segmental duplication (SD) 30670 4843 35513 In RM and SD 51466 17174 68640 Ts:Tv 1.5 0.7 1.2 Mean SNV density/kb 0.02 0.008 0.03 There are significantly more heterozygous variants in repetitive sequence than expected (p<1x10-16). BAC ends mapping discordantly and in multiple loci are significantly enriched for segmental duplications (p<1x10-5).
- 21. Identified 549 novel protein coding genes not annotated in GRCh37
- 22. CHM1 BioNano Genome Map Aligned to GRCh38 GRCh38 CHM1 BioNano Map ~15kb additional data
- 23. BioNano SV Calls Identified a Assembly Problems Collapse Expansion inAssembly Gap in SequenceCHM1_1.1 Assembly CHM1 BioNano Map
- 24. Conclusion • Extremely diverse regions of the genome are difficult to characterize due to issues distinguishing allelic from paralogous duplications • CHM1_1.1 highly contiguous single haplotype representation of the genome • Identified regions of misassembly or reference-ized regions • Utilize long read technology and nanopore technology to attempt to fix these regions
- 25. Need to add more diversity to reference • Finish another hydatidiform mole to platinum status • Finish 5 genomes to gold status • NA19240 (Yoruban) • NA12878 (European) • HG00513 (Han Chinese) • 2 “wildcards” • Looking for underrepresented minority population • Add high quality alternative sequences to reference to create a population reference graph or “pan genome”
- 26. Use colored de Bruijn graph structure to represent population reference graph
- 27. Bioinformatic tool development in the future • Alignment of short reads to population reference graph • Variant calling • Variant reporting/Haplotype resolution
- 28. Adapted from Weinstein et al, 2009
- 29. The GRCh37 reference sequence was assembled from three lymphoblastoid cell lines Not a true haplotype Incomplete
- 30. The CH17 haplotype is quite different from the reference
- 31. Novel insertion The CH17 haplotype is quite different from the reference
- 32. Complex Indel The CH17 haplotype is quite different from the reference
- 33. Hotspot/Recurrent Mutation The CH17 haplotype is quite different from the reference
- 34. 60 kbp Insertion (Hotspot) African Asian European
- 35. Duplication (influenza) The CH17 haplotype is quite different from the reference
- 36. 44 kbp Duplication (influenza) African Asian European
- 37. Summary of hydatidiform mole sequence • 47 functional V genes • 24 total variants (SNV and CNV) involving 29 IGHV genes • 5 structural variants • 19 single nucleotide variants • 15 non-synonymous mutations • 20 out of 24 variants represent differences in amino acid sequence or gene copy number
- 38. Summary of hydatidiform mole sequence • 47 functional V genes • 24 total variants (SNV and CNV) involving 29 IGHV genes • 5 structural variants • 19 single nucleotide variants • 15 non-synonymous mutations • 20 out of 24 variants represent differences in amino acid sequence or gene copy number 100 kbp of novel sequence
- 39. Current status of CHM1 resources • CHORI-17 BAC Library (created from CHM1 cell line) • CHORI-17 BAC end sequences (n=325,659) • CHORI-17 multiple enzyme fingerprint map (1560 fpc contigs) • CHORI-17 BACs (>750 have been sequenced, with 592 of them in Genbank as phase 3) • Active cell line • >100X coverage Illumina 100bp reads • 300, 500bp, 3kb inserts • Reference assisted assembly CHM1_1.1 • BioNano genome map • >50X coverage of PacBio long read data
- 40. CHM1_1.1 Assembly • Reference-guided assembly – SRPRISM v2.3, R. Agarwala • Alignment of Illumina reads to GRCh37 primary assembly • CHORI-17 BAC clone tilepaths were then incorporated • 428 total clones • 324 clones in 45 tilepaths • 104 clones as singletons • Comparison back to GRCh37 reference to provide appropriate gaps sizes • Assembly submitted to Genbank • http://www.ncbi.nlm.nih.gov/assembly/GCF_000306695.2 • Steinberg et al, 2014 • Genome Research (Dec;24(12):2066-76)
- 41. LILR (leukocyte immunoglobulin-like receptor)/KIR (killer immunoglobulin receptor) Immunoglobulin Kappa chain Immunoglobulin Lambda chain TCRA/B 17q21.31 inversion polymorphism Immunoglobulin heavy chain locus CYP2D6 SRGAP2 15q13.3 inversion polymorphism
