High efficiency error suppression for accurate detection of low-frequency variants

• Published in: Nucleic acids research 2019-09, Vol.47 (15), p.e87-e87
• Main Authors: Wang, Ting Ting, Abelson, Sagi, Zou, Jinfeng, Li, Tiantian, Zhao, Zhen, Dick, John E, Shlush, Liran I, Pugh, Trevor J, Bratman, Scott V
• Format: Article
• Language: English
• Subjects: Alleles; Cell Line, Tumor; DNA Barcoding, Taxonomic - methods; Fetal Blood - cytology; Fetal Blood - metabolism; Gene Frequency; HCT116 Cells; High-Throughput Nucleotide Sequencing; Humans; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - pathology; Leukocytes, Mononuclear - metabolism; Leukocytes, Mononuclear - pathology; Methods Online; Mutation; Neoplasm Proteins - genetics; Precision Medicine - methods; Scientific Experimental Error; Sequence Analysis, DNA - methods
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkz474

Abstract Detection of cancer-associated somatic mutations has broad applications for oncology and precision medicine. However, this becomes challenging when cancer-derived DNA is in low abundance, such as in impure tissue specimens or in circulating cell-free DNA. Next-generation sequencing (NGS) is particularly prone to technical artefacts that can limit the accuracy for calling low-allele-frequency mutations. State-of-the-art methods to improve detection of low-frequency mutations often employ unique molecular identifiers (UMIs) for error suppression; however, these methods are highly inefficient as they depend on redundant sequencing to assemble consensus sequences. Here, we present a novel strategy to enhance the efficiency of UMI-based error suppression by retaining single reads (singletons) that can participate in consensus assembly. This ‘Singleton Correction’ methodology outperformed other UMI-based strategies in efficiency, leading to greater sensitivity with high specificity in a cell line dilution series. Significant benefits were seen with Singleton Correction at sequencing depths ≤16 000×. We validated the utility and generalizability of this approach in a cohort of >300 individuals whose peripheral blood DNA was subjected to hybrid capture sequencing at ∼5000× depth. Singleton Correction can be incorporated into existing UMI-based error suppression workflows to boost mutation detection accuracy, thus improving the cost-effectiveness and clinical impact of NGS.