A benchmark study on error-correction by read-pairing and tag-clustering in amplicon-based deep sequencingReportar como inadecuado

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BMC Genomics

, 17:108

Comparative and evolutionary genomics


BackgroundThe high error rate of next generation sequencing NGS restricts some of its applications, such as monitoring virus mutations and detecting rare mutations in tumors. There are two commonly employed sequencing library preparation strategies to improve sequencing accuracy by correcting sequencing errors: read-pairing method and tag-clustering method i.e. primer ID or UID. Here, we constructed a homogeneous library from a single clone, and compared the variant calling accuracy of these error-correction methods.

ResultWe comprehensively described the strengths and pitfalls of these methods. We found that both read-pairing and tag-clustering methods significantly decreased sequencing error rate. While the read-pairing method was more effective than the tag-clustering method at correcting insertion and deletion errors, it was not as effective as the tag-clustering method at correcting substitution errors. In addition, we observed that when the read quality was poor, the tag-clustering method led to huge coverage loss. We also tested the effect of applying quality score filtering to the error-correction methods and demonstrated that quality score filtering was able to impose a minor, yet statistically significant improvement to the error-correction methods tested in this study.

ConclusionOur study provides a benchmark for researchers to select suitable error-correction methods based on the goal of the experiment by balancing the trade-off between sequencing cost i.e. sequencing coverage requirement and detection sensitivity.

KeywordsDeep sequencing Amplicon sequencing Error-correction Tag-clustering Read-pairing Error rate AbbreviationsNGSNext-generation sequencing

PCRPolymerase chain reaction


Scheme 1Raw reads

Scheme 2Read-pairing consensus

Scheme 3Tag-clustering Primer ID consensus

Scheme 4Combined consensus read-pairing consensus, followed with tag-clustering consensus

Electronic supplementary materialThe online version of this article doi:10.1186-s12864-016-2388-9 contains supplementary material, which is available to authorized users.

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Autor: Tian-Hao Zhang - Nicholas C. Wu - Ren Sun

Fuente: https://link.springer.com/

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