Transitioning to gr_ch38
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GRC Workshop: Advancing the Human Reference Assembly Speaker: Deanna Church
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Transitioning to gr_ch38
- 1. © 2014 Personalis, Inc. All rights reserved. Pioneering Genome-Guided Medicine Deanna M. Church Senior Directory of Genomics and Content Transitioning to GRCh38
- 2. Personalis, Inc.2 Who we are Inherited Disease Diagnostics Cancer Services ACE Platform Research Services
- 3. Personalis, Inc.3 Reference assembly influence Gene1 Gene2 Gene1 Sample Ref Assembly
- 4. Personalis, Inc.4 Excitement about GRCh38 GGAACGCAG GGAACACAG DPYD R->C Alt loci Model Centromere Sequences Miga et al., 2014
- 5. Personalis, Inc.5 CCL3: region: GRCh37 NC_000017.10 (chr17): 34,442,621-35,005,379
- 6. Personalis, Inc.6 CCL5-TBC1D3 region: GRCh38 NC_000017.11 (chr17): 36,032,574-36,269,924 NT_187661.1 100 Kb deletion on chromosome Steinberg et al., 2014 http://dx.doi.org/10.1101/006841
- 7. 7 Alternate Loci and Genes 3.6 Mb of novel sequence 153 genes not on primary assembly Unique sequence in alternate loci Total: 3.6 Mb; 153 genes only on alts
- 8. Personalis, Inc.8 Alt Loci and Genes 25% Medically Interpretable Genes (MIG) Primary Assembly Alt Locus 6.4% 6.2%0.18%
- 9. Personalis, Inc.9 Alt Loci and Genes NT_167246.2: MHC alternate locus No SNP annotationSparse SNP annotation
- 10. Personalis, Inc.10 Analysis challenges Primary Assembly Paralogous duplication Allelic duplication Alt Locus MapQ https://github.com/GenomeRef/SoftwareDevTracking
- 11. Personalis, Inc.11 Analysis challenges: variant representation Primary Assembly Alt Locus G>C 1/1 Only valid if homozygous for Alt 1/. Correct if heterozygous for Alt
- 12. Personalis, Inc.12 Waiting for graph representations? Credit: UC Santa Cruz Genomics Institute
- 13. Personalis, Inc.13 Analysis challenges chr19 vs 19 GenBank: CM00681.2 RefSeq: NC_000019.10
- 14. Personalis, Inc.14 Analysis challenges chr19_KI270938v1_alt CHR_HSCHR19KIR_G248_BA2_HAP_CTG3_1 GenBank: KI270886.1 RefSeq: NT_187640.1
- 15. Personalis, Inc.15 Analysis challenges MICB Reporting formats (GFF, VCF, etc) don’t manage multiple locations easily
- 16. Personalis, Inc.16 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
- 17. Personalis, Inc.17 Analysis challenges • Need aligners that can distinguish allelic and paralogous duplication • Need variant callers/modules than can correctly assign genotypes in complex regions • Need to extend file formats to accommodate new assembly model
Editor's Notes
- Missing and misassembled sequence in the reference assembly can have dire consequences to genome interpretation. In this example, Gene2 is missing from the reference, but present in the sample we are analyzying. Regardless of whether gene2 is missing because of an assembly error, or because it is polymorphic in the population the outcome can be the same. In the best case scenario, reads from gene2 don’t align to the reference and we just can’t analyze gene2. However, if gene2 is related to gene1, we can get off targets alignments that can confound analysis of Gene1 as well, either leading to under calling in the region, or possibly leading to inappropriately calling paralogous sequence variants as allelic sequence variants. If we take sequences we know to be missing in GRCh37, simulate reads and then align these reads to GRCh37, we see that 75% of these find an off target alignment, regardless of the alignment method used. This is why Heng Li created decoy sequences for the 1000 genomes project- in an effort to reduce off-target alignments. However, we still lack the ability to analyze gene2 in this scenario. This underscores the importance of representing all common human sequences in the reference assembly.
- 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
- The CCL3 region on chromosome 17 allows us to explore two major updates seen in GRCh38, and hopefully will underscore the importance of representing missing paralogs in the reference. This region is known to be copy number variant, with individuals having 0-4 copies of a 90Kb repeat unit. In GRCh37, the region was assembled from several sources that contain different structural variants. This led to the creation of a false gap, and a genomic representation that does not likely exist in anyone on the planet. Being able to correctly represent the genomic architecture of this regions is important as there is some, albeit conflicting evidence, of the correlation of the number of copies of CCL3L1 with HIV infection and progression to AIDs.
- To better represent this region, the GRC made a new clone tiling path in this region from a single haplotype resource derived from a hyaditiform mole. An additional allele, representing a 100 Kb insertion was also generated and placed in the assembly as an alternate locus. The reference assembly now has two correct representations of this region – though we may need more.
- For this reason, many people have just ignored these sequences, but doing so in GRCh38 means losing 3.6 Mb of sequence unique to the alternate loci- sequence containing 153 genes. This graph shows the distribution of the amount of unique sequence per alternate locus- so while it is clear they do not all contribute equal amounts of novel sequence, in aggregate the amount is significant. The GRC recently held a workshop to encourage development of new tools that can handle the full assembly, and Heng Li has already distributed a version of BWA-MEM that is alt-locus aware, we need to do considerable testing and additional development to make sure we are using these sequences correctly. We also need to assess the ramifications of this new structure on other parts of the tool chain.