Family reunion via error correction: an efficient analysis of duplex sequencing data

Nicholas Stoler,Kateryna D Makova,Barbara Arbeithuber,Irene Tiemann-Boege,Renato Salazar,Gundula Povysil,Anton Nekrutenko,Monika Heinzl

doi:10.1186/s12859-020-3419-8

Nicholas Stoler, Kateryna D Makova + Show 6 more

Open Access

https://doi.org/10.1186/s12859-020-3419-8

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Abstract

BackgroundDuplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost—sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are thrown away.ResultsIn this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows “reuniting” these reads with their respective families increasing the output of the method and making it more cost effective.ConclusionsWe combine an error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0. It is written in Python, C, AWK, and Bash. It is open source and readily available through Galaxy, Bioconda, and Github: https://github.com/galaxyproject/dunovo.

Highlights

Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies
The first dataset was produced by Schmitt et al [9], who employed Duplex Sequencing (DS) to identify a rare mutation at the ABL1 locus responsible for resistance to a chronic myeloid leukemia therapeutic compound imatinib
Since each DNA fragment is labeled by two tags, one at each end, the theoretical upper bound for the number of unique combinations is 4(12 + 12)

Summary

Introduction

Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost—sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. The descendants of each original DNA fragment are identified and grouped together using tags—one sorts tags in sequencing reads lexicographically and all reads containing the same tag are bundled into a family.

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