This document summarizes initial analysis of sequencing data from a tumor/normal cell line sample for the Genome in a Bottle Benchmark project. Optical mapping and single cell sequencing show differences in ploidy between the tumor and normal samples. Variant calling identified substantial aneuploidy common in pancreatic tumors in the sample, including large inversions, translocations, and loss of heterozygosity across chromosomes. The tumor contains a known KRAS mutation seen in pancreatic cancer. Ongoing sequencing with multiple technologies aims to further characterize this sample for use as a benchmark.

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