Church dm grc_workshop
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Using the genome by Deanna Church
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Church dm grc_workshop
- 1. Real world challenges to using GRCh38 A view from the trenches Deanna M. Church Senior Director of Genomics and Content Pioneering Genome-Guided Medicine © 2014 Personalis, Inc. All rights reserved.
- 2. Acknowledgements Personalis Jason Harris Sarah Garcia Jeanie Tirch Gabor Bartha Mark Pratt Scott Kirk Michael Clark Rich Chen John West Genome Reference Consortium Personalis, Inc. | Confidential 2 and Proprietary NCBI Valerie Schneider Nathan Bouk Terence Murphy Alex Astashyn Donna Maglott Melissa Landrum Wendy Rubinstein Jennifer Lee
- 3. Who we are Inherited Disease Diagnostics Personalis, Inc. | Confidential 3 and Proprietary Cancer Services ACE Platform Research Services
- 4. Accuracy is key to what we do Novel 2bp deletion in GATAD2B Called by GATK as paternally inherited Personalis, Inc. | Confidential 4 and Proprietary • Both affected children – Macrocephaly – Low muscle tone, hypotonia – Delay in early milestones – Dysphagia – Esotropia • Affected Male (3 yr) – Intellectual disability – Mild hearing loss – High arched palate – Small cyst near eye • Affected Female (15 mo) – Sleep apnea – Failure to thrive – Laryngomalacia – Anisocoria – Small optic nerves Case courtesy of Geisinger Health System
- 5. Accuracy is key to what we do Sample GATK-determined Genotype Personalis, Inc. | Confidential 5 and Proprietary Ref Alt Depth Allele Freq. Father 0/1 108 11 121 0.09 Mother 0/0 111 0 112 0.00 Brother 0/1 63 44 109 0.40 Sister 0/1 64 52 119 0.44 Case courtesy of Geisinger Health System
- 6. Excitement about GRCh38 Personalis, Inc. | Confidential 6 and Proprietary DPYD GGAACGCAG GGAACACAG R->C Alt loci Model Centromere Sequences Miga et al., 2014
- 7. Medical content not on chromosome sequences Personalis, Inc. | Confidential 7 and Proprietary
- 8. Medical content not on chromosome sequences GRCh37 NT_113939: chr19 unlocalized contig GRCh38 Personalis, Inc. | Confidential 8 and Proprietary
- 9. Medical content not on chromosome sequences NT_167246.2: MHC alternate locus Sparse SNP No SNP annotation annotation Personalis, Inc. | Confidential 9 and Proprietary
- 10. By any other name chr19 vs 19 GenBank: CM00681.2 RefSeq: NC_000019.10 Personalis, Inc. | Confidential 10 and Proprietary
- 11. By any other name chr19_KI270938v1_alt CHR_HSCHR19KIR_G248_BA2_HAP_CTG3_1 GenBank: KI270886.1 RefSeq: NT_187640.1 Personalis, Inc. | Confidential 11 and Proprietary
- 12. Unflattening the data MICB Reporting formats (GFF, VCF, etc) don’t manage multiple locations easily Personalis, Inc. | Confidential 12 and Proprietary
- 13. NW_003871068.1 NC_000006.12 BestRefSeq gene 31494881 31511124 . + . ID=gene13336;Name=MICB;Dbxref=GeneID:4277 NT_167244.2 BestRefSeq gene 2827449 2843674 . + . ID=gene42005;Name=MICB;Dbxref=GeneID:4277 NT_113891.3 BestRefSeq gene 2972222 2988464 . + . ID=gene43669;Name=MICB;Dbxref=GeneID:4277 NT_167245.2 BestRefSeq gene 2742492 2758910 . + . ID=gene44377;Name=MICB;Dbxref=GeneID:4277 NT_167246.2 BestRefSeq gene 2810648 2816200 . + . ID=gene44827;Name=MICB;Dbxref=GeneID:4277 NT_167247.2 BestRefSeq gene 2836836 2853071 . + . ID=gene45127;Name=MICB;Dbxref=GeneID:4277 ID=gene13336;Name=MICB;Dbxref=GeneID:4277 ID=gene42005;Name=MICB;Dbxref=GeneID:4277 ID=gene43669;Name=MICB;Dbxref=GeneID:4277 ID=gene44377;Name=MICB;Dbxref=GeneID:4277 ID=gene44827;Name=MICB;Dbxref=GeneID:4277 ID=gene45127;Name=MICB;Dbxref=GeneID:4277 Building snpEFF Personalis, Inc. | Confidential 13 and Proprietary
- 14. Incremental steps: using fix patches Personalis, Inc. | Confidential 14 and Proprietary SHANK2
- 15. Personalis, Inc. | Confidential 15 and Proprietary Using Fix patches to improve alignments Incremental steps: using fix patches
- 16. Migrating to GRCh38: using Fix patches Personalis, Inc. | Confidential 16 and Proprietary Fix patch hs37d5
- 17. Migrating to GRCh38: using Fix patches Personalis, Inc. | Confidential 17 and Proprietary Fix patch hs37d5
- 18. Migrating to GRCh38: using Fix patches GRCh37 vs. Fix Patch GRCh38 Personalis, Inc. | Confidential 18 and Proprietary
- 19. GRCh37.p13 Improved alignments outside of fix patch regions Personalis, Inc. | Confidential 19 and Proprietary Jason Harris Regions outside of fix patches hs37d5 GRCh37.p13 hs37d5 GRCh37.p13 378 Ten kb windows that don’t overlap fix patches with >10 SNV call differences
- 20. GRCh37.p13 Improved alignments outside of fix patch regions Personalis, Inc. | Confidential 20 and Proprietary Jason Harris hs37d5 GRCh37.p13 hs37d5 GRCh37.p13 hs37d5 GRCh37.p13
- 21. Using Fix patches Personalis, Inc. | Confidential 21 and Proprietary
- 22. Aligning GRCh37 and GRCh38 A A B’ Seq in assembly 1 Personalis, Inc. | Confidential 22 and Proprietary Seq in assembly 2 B B Unique well aligned region in both assemblies. Second Pass (SP) alignments First Pass (FP) alignments SP only Expansion Assembly 1 SP + FP Collapse Assembly 2
- 23. Aligning GRCh37 and GRCh38 Personalis, Inc. | Confidential 23 and Proprietary
- 24. Mapping to GRCh38 Personalis, Inc. | Confidential 24 and Proprietary
- 25. Mapping to GRCh38 Dataset Starting loci Failure Unique to Personalis, Inc. | Confidential 25 and Proprietary Primary Unique to Alts Collapse in GRCh37 Collapse in GRCh38 GWAS catalog 7,991 0 7,827 0 14 0 ClinVar* 88,343 3 86,549 5 278 4 GO-ESP 6500 1,982,177 180 1,920,864 339 5,792 324 GIAB 2,915,713 274 2,874,786 47 1,662 4 NCBI assembly-assembly alignments from: Sept 20, 2014, software version 1.7 *clinvar_20140902.vcf
- 26. Remap vs. liftOver liftOver-dbSNP remap Personalis, Inc. | Confidential 26 and Proprietary rs141109950 chr7
- 27. Remap vs. liftOver Personalis, Inc. | Confidential 27 and Proprietary rs267602252 remap liftOver
- 28. Migrating to GRCh38 First Pass remap Second Pass remap Personalis, Inc. | Confidential 28 and Proprietary
- 29. Migrating to GRCh38 New PRODH paralog Sequence is unlocalized on chr22. Personalis, Inc. | Confidential 29 and Proprietary
- 30. Using GRCh38 to improve GRCh37 annotation Personalis, Inc. | Confidential 30 and Proprietary KCNE1 Alignment to new paralog added in GRCh38
- 31. Getting the most out of the reference Still challenging because tools and data structures expect a flat assembly Remap/liftOver not the final answer for moving variation Even modest changes (via fix patches) are promsing Personalis, Inc. | Confidential 31 and Proprietary
Editor's Notes
- Sarah mosacism/UDP slide
- Mutations in DPYD result in dihydropyrimidine dehydrogenase deficiency, an error in pyrimidine metabolism associated with thymine-uraciluria and an increased risk of toxicity in cancer patients receiving 5-flourouracil. Replace this with protein coding info and stats? And Valerie’s poster
- We can also see improvements outside of fix patch regions. Here we see another normalized read plot, with blue representing GRCh37 and green showing alignments to our fix patch version. Not only do we see alignment improvements, but this carries through to variant calling. We have identified 378 10 Kb windows that don’t overlap fix patches but have greater than 10 SNV call differences. Here is one such example, where a seeming SNP dense region, with lots of heterozygous SNPs now looks much cleaner- and has not heterozygous SNPs.
- The NCBI assembly-assembly alignment process uses a two step approach. In the first pass, a set of heuristics, including assembly structure are used to generate a set of essentially reciprocal best hits Then, the process does a second pass, looking for regions greater than 5kb in each assembly and tries to recover alignments in these regions. A report is produced that marks up whether a region is in a first pass or second pass alignment- analyzing this report can identify regions that are likely expanded in an assembly or collapsed in an assembly.
- We can use this data to plot the amount (expressed as an percentage of each chromosome) of collapse in each assembly. It is worth noting that this is some collapse in GRCh38, which is expected as several misassembled regions contained more than one haplotype and these haplotype expansions where removed in GRCh38. However, we can see the landscape is dominated by collapse in GRCh37. Variants called within these regions in GRCh37 are candidates for false positive variant calls.
- Susceptibility to thyrotoxic periodic paralysis