Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform

Schirmer, M., Ijaz, U. Z. , D'Amore, R., Hall, N., Sloan, W. T. and Quince, C. (2015) Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Research, 43(6), e37. (doi: 10.1093/nar/gku1341) (PMID:25586220) (PMCID:PMC4381044)

Abstract

With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Sloan, Professor William and Quince, Dr Christopher and Ijaz, Dr Umer
• Authors: Schirmer, M., Ijaz, U. Z., D'Amore, R., Hall, N., Sloan, W. T., and Quince, C.
• College/School: College of Science and Engineering > School of Engineering; College of Science and Engineering > School of Engineering > Infrastructure and Environment
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Copyright Holders: Copyright © 2015 The Authors
• First Published: First published in Nucleic Acids Research 43(6):e37
• Publisher Policy: Reproduced under a Creative Commons License

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Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
50335	1	Pioneering the genomics era of environmental microbiology	Christopher Quince	Engineering & Physical Sciences Research Council (EPSRC)	EP/H003851/1	ENG - ENGINEERING INFRASTRUCTURE & ENVIR