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GIAB Tumor Normal ASHG 2023
- 1. Optical mapping and low-pass single cell data shows ploidy differences • Building on germline variant benchmarks for 7 normal DNA reference materials, working towards the first tumor/normal benchmark from the Genome in a Bottle (GIAB) consortium • Making available data from a variety of technologies on Tumor (HG008-T), Normal-Duodenal (N-D), and Normal-Pancreas (N-P) • This work presents initial analysis of copy number, structural, and small variants Overview Sequencing a broadly-consented tumor/normal cell line for a Genome in a Bottle Benchmark Justin Wagner1, Jennifer McDaniel1, Nathan D. Olson1, Vaidehi Patel1, Gail Rosen2, Andrew Liss3, Justin Zook1, and Genome in a Bottle Consortium 1) Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899, 2) Drexel University, EECE, Philadelphia, PA 3) Massachusetts General Hospital, Boston, MA Genome in a Bottle Consortium Acknowledgements Characterizing Variant Calling on Initial Sequencing data Thank you to Baylor College of Medicine, Phase Genomics, NYGC, UCSC, NU, Bionano, and Bioskryb for initial sequencing, and others for on-going sequencing. See consortium updates at www.genomeinabottle.org or email justin.zook@nist.gov • Called Illumina PCR-Free 150-180x on both tumor and normal DNA with Strelka2 and Mutect2 then generated a comparison set of variants with NeuSomatic • Used hap.py to compare VCFs from Strelka2, Mutect2, and NeuSomatic to each other • Plotted an example of Strelka2 calls that matched NeuSomatic to show read support for SNVs • Investigating how somatic variant callers perform in different CNVs and sequence contexts of this tumor/normal pair • Preliminary results identified substantial aneuploidy common in pancreatic tumors, with ~15 large inversions and translocations and ~16 chromosomes with extensive loss of heterozygosity • Most CNVs in all cells, but some CNVs only in a subset of clones • The tumor contains the common G12V mutation in KRAS and the ~2 Mbp region containing this mutation is likely triplicated • Comparing to TCGA analyses in Steele et al., HG008-T deletions appear similar to the chromosomal-scale loss of heterozygosity (cLOH) signatures ~430 Somatic variants in only some cells in haploid regions ~370 Somatic variants in only some cells in diploid regions On-going Sequencing For HG008 Range Tech T N-D N-P Read length Coverage Long ONT (UL) X X X ~100 - 300 kb pending ONT (duplex) X ~10 - 100 kb pending ONT (std) X ~35 kb 45X PacBio HiFi (Revio) X X X ~10 - 20 kb pending Bionano Optical Mapping X 150 kb - multi Mb 400X Arima and PhaseGenomics HiC-Illumina X X 2x150 bp pending Karyologic karyotyping X chomosomal NA Short Illumina WGS X X X 2x150 bp 180X (T), 150X (N) Element X X 75 - 150 bp pending PacBio Onso X X 100 - 200 bp pending Bioskryb-Illumina single-cell WGS X 2x50 bp <1X (120 cells) ~4000 Somatic variants in most cells in diploid regions ~2000 Somatic variants in more cells in haploid (loss of heterozygosity) regions Bioskryb Low-pass Single Cell Bulk Bionano TCGA cLOH Samples Deletions in HG008-T Steele et al. 2022 Nature • Project website includes overall description, links to data, and sequencing updates • Please reach out with any analysis suggestions or results to share https://www.nist.gov/programs-projects/cancer-genome-bottle https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data_somatic