Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer

Abstract

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Identification of targetable BRAF ΔN486_P490 variant by whole-genome sequencing leading to dabrafenib-induced remission of a BRAF-mutant pancreatic adenocarcinoma

“…The specimens were received as OCT (Optimal Cutting Temperature compound)-embedded tumor tissue from a liver metastasis and peripheral blood as normal. DNA and RNA purification, extraction, and library preparations were performed as previously reported (Wrzeszczynski et al 2018). Whole-genome DNA sequencing was performed on the Illumina HiSeq X 2 × 150 bp paired-end sequencing (Illumina).…”

“…As part of the study, whole-exome DNA sequencing was additionally performed using SureSelectXT Human All Exon V6 + COSMIC capture kit (Agilent) and 2 × 125 bp paired-end sequencing was performed on a Illumina HiSeq 2500 instrument to reach an average read depth of 238× for the tumor and 105× for the normal. The RNA sample was prepared using an mRNA protocol as previously reported (Wrzeszczynski et al 2018), and the library was sequenced on the Illumina HiSeq 2500 2 × 125 bp rapid run platform obtaining approximately 57 million reads. Somatic single-nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), copy-number variants (CNVs), and fusions were called from whole-genome and transcriptome sequencing as previously reported (Wrzeszczynski et al 2017).…”

“…Initial variant detection was performed as part of the NYGC whole genome and transcriptome clinical assay (Wrzeszczynski et al 2018). SNV and indels were annotated via snpEff, snpSift (Cingolani et al 2012), and GATK VariantAnnotator using annotation from ENSEMBL, COSMIC (Tate et al 2019), Gene Ontology, and 1000 Genomes.…”

Clinical validation of Whole Genome Sequencing for cancer diagnostics

“…During the past few years, whole genome sequencing (WGS) and the associated bioinformatic data processing and interpretation has matured from a research-use-only tool to a diagnostic-level technology 24 . Together with the clinical need to screen for an increasing number of (complex) biomarkers in an increased number of tumor types (or even pan-cancer) 1,25 and the availability of limited amounts of biopsy material, the use of a single all-inclusive DNA test is a more than welcome development for efficient molecular diagnostics.…”

“Somatic” and “pathogenic” - is the classification strategy applicable in times of large-scale sequencing?

“…39 The sensitivity of whole genome sequencing is currently in the range of 15-20%, but the technique allows simultaneous identification of structural and copy number variations and the cost of sequencing is decreasing. 40…”