Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair derived artifacts as residual errors

Abstract

To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strands-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 6 bp of the library fragments in the 5'-NpCpA-3' or 5'-NpCpT-3' trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 6 bp, PECC-Seq could reduce the sequencing error frequency to mid-10 -7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10 -8 . In mutagen-treated TK6, slight increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights in further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

Detection and analysis of chemical-induced chromosomal damage for public health: integrating new approach methodologies and non-animal methods

“…Mutation signatures associated with cancers have been intensively investigated. Errorcorrected sequencing technologies have evolved to a level that can now be used in genotoxicity evaluations (Matsumura et al, 2019;Valentine et al, 2020;You et al, 2020;Abascal et al, 2021). However, these modalities cannot yet detect structural aberrations, including large deletions.…”

Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

“…To circumvent this problem, error-corrected sequencing (ecNGS) techniques have been developed to improve accuracy and specificity for detecting true, low frequency mutations [6][7][8] . The highest resolution of ecNGS methods, such as Duplex Sequencing (DuplexSeq; TwinStrand Biosciences, Seattle, WA), involves labeling both strands of a DNA duplex with one or more forms of a unique molecular index (UMI) that both relates and distinguishes the sequences of the two strands from each other and from those of other molecules.…”

Estimating somatic mutation rates by Duplex Sequencing in non-model organisms: Daphnia magna as a case study

“…Next generation sequencing (NGS) has been used extensively to identify germline variants, but the variant allele fraction (VAF) of many somatic mutations (in some cases <0.01%) is well below the error rates associated with standard NGS, making the detection of somatic mutations challenging [1,[3][4][5]. Single-molecule sequencing (SMS) technologies, however, dramatically reduce the error rates associated with high-throughput sequencing, potentially enabling the interrogation of rare, subclonal variation by NGS (e.g., Safe-SeqS [1], Duplex Sequencing [6,7], smMIP [2], BotSeqS [8], Hawk-Seq [9], PECC-Seq [10], and NanoSeq [11]).…”

“…Second, in addition to filtering out artifactual mutations resulting from library preparation and sequencing, the inference of mutations is only as robust as the reference genome to which read families are compared. To date, only a few studies have utilized bottlenecked Duplex Sequencing to estimate genome-wide mutation rates, and these studies have been limited to humans and mice [6,[8][9][10]. By virtue of their focus on model organisms, all genome-wide Duplex Sequencing studies have had at their disposal high-quality, chromosome-level reference genomes.…”