A scalable high-throughput targeted next-generation sequencing assay for comprehensive genomic profiling of solid tumors

Abstract

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000TM sequencing run with a turnaround time of <7 days from specimen receipt to report. The results demonstrate that the scalable assay accurately and reproducibly detects small variants, copy number alterations, microsatellite instability (MSI) and tumor mutational burden (TMB) from 40ng DNA, and multiple gene fusions, including known and unknown partners and splice variants from 20ng RNA. 717 tumor samples and reference materials with previously known alterations in 96 cancer-related genes were sequenced to evaluate assay performance. All variant classes were reliably detected at consistent and reportable variant allele percentages with >99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

A Computational Framework for Comprehensive Genomic Profiling in Solid Cancers: The Analytical Performance of a High-Throughput Assay for Small and Copy Number Variants

“…Our results indicate that TSO500 is a reliable test with a robust workflow both for the wet and for the computational steps in the SNV and InDel analyses. All expected alteration were detected, and a good coverage performance was provided: high values of the median coverage, coverage MAD, median insert size, exon 100X and target at 100X and 250X probed the reliability of the bioinformatics pipeline, useful to call SNV and indel variants, for both the exon and target regions [ 1 , 2 ]. Accounting for this, no false negatives were detected in the small variants calling.…”

“…Broad molecular profiling technologies better than organ-based approaches are believed to serve such dynamic purposes. Next-generation sequencing (NGS) approaches have progressively allowed the spread of such determinations, facilitating the execution of comprehensive genomic profiling (CGP) assays on patients’ tumor samples [ 1 , 2 ]. Specifically, CGP identifies molecular alterations that sometimes can be targeted by the available treatments [ 1 , 2 ].…”

An institutional review of genomic sequencing in pediatric solid tumors

“…The company's current test is OmniSeq INSIGHT, which utilizes hybrid capture DNA‐ and RNA‐sequencing technologies to combine next‐generation sequencing of the full coding and junctional regions of 523 genes for mutations and deletions–insertions, copy number alterations in 59 genes and fusions and/or splice variants in 55 genes, microsatellite instability (MSI), tumor mutational burden (TMB), with an immune gene expression profiling panel that evaluates RNA expression of 64 genes and PD‐L1 expression by IHC. Fusions and splice site variants are detected using the hybrid capture of mRNA 10 …”

Tumor Diagnosis of FFPE Sample by Target Genome Sequencing From Multiple Sequencing Platforms

“…The high sensitivity, speci city, precision, and accuracy demonstrated the clinical applicability of this assay for SNVs and InDels/duplications detection. Similarly, on the NovaSeq 6000 platform (Illumina), the TSO500 panel also showed excellent performances and good reproducibility in identifying multiple DNA/RNA variant types and molecular signatures from FFPE tissue specimens [11][12][13] . GenoLab M (GeneMind LTD., China), a novel high-throughput sequencing platform, utilizes the well-recognized sequencing-bysynthesis (SBS) techniques and reversible termination approaches to deliver con dent base-calling accuracy and high yield of error-free reads.…”