Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing

Abstract

Clinical applications of precision oncology require accurate tests that can distinguish true cancer specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Reinspection of a Clinical Proteomics Tumor Analysis Consortium (CPTAC) Dataset with Cloud Computing Reveals Abundant Post-Translational Modifications and Protein Sequence Variants

“…This is largely unsurprising today given the mounting evidence, including the results in the original CPTAC study re-evaluated here, that peptide abundance has little to no correlation with transcript abundance or copy number [33]. Current workflows typically rely on quality filters that are ultimately affected by the relative copy numbers of each transcript or genomic read [34]. We report herein the first evidence that proteomic data can be searched independently in a truly global manner to identify sequence variants that are transcribed and translated.…”

“…While we have manually validated a few of these, large-scale verification is not currently feasible for the millions of peptides identified in studies of this size. We do acknowledge that some of these could be false positives, but it is important to note that there are disagreements between WES and RNAseq as well [34,35]. If proteomics is considered with equal latitude, this will only result in the identification of new unique variants.…”

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study

“…The rapid growing number of sample processing protocols, library preparation methods, sequencing platforms, and bioinformatics pipelines to detect mutations in cancer genome, presents great technical challenges for the accuracy and reproducibility of utilizing NGS for cancer genome mutation detections. To investigate how these experimental and analytical elements may affect mutation detection accuracy, recently we carried out a comprehensive benchmarking study 2 using both whole-genome (WGS) and whole-exome sequencing (WES) data sets generated from two well-characterized reference samples: a human breast cancer cell line (HCC1395) and a B lymphocytes cell line (HCC1395BL) derived from the same donor 3 . We generated WGS and WES data using various NGS library preparation protocols, seven NGS platforms (NovaSeq, HiSeq, PacBio, 10X Genomics, Ion Torrent, Miseq, and Affymetrix CytoScan HD) at six centers including Illumina (IL), National Cancer Institute (NC), Novartis (NV), European Infrastructure for Translational Medicine (EA), Fudan University (FD), and Loma Linda University (LL) (Fig.…”

“…Data set described in this paper was mainly used in our two companion studies, to assess the effect of variables during the process of WGS and WES, including biosample types, tumor content, library protocol and DNA inputs, sequencing site and replicates, reads coverage and bioinformatics tools, on the performance of cancer mutation detection 2 and to characterize a pair of tumor-normal cell lines as community reference samples 3 .…”

The Sequencing Quality Control 2 study: establishing community standards for sequencing in precision medicine

“…The SEQC2 consortium performed WGS using the reference tumor samples to understand the variables that impact diagnoses. Although the WGS workflow could be divided into different steps (such as sample and library preparation, sequencing and bioinformatic analysis), the study found that each step is highly integrated and interdependent, and clinical validation is necessary across the entire sample-to-result workflow [ 13 ].…”