Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods

Title: Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods
Authors: Bashford-Rogers, RJM
Palser, AL
Idris, SF
Carter, L
Epstein, M
Callard, RE
Douek, DC
Vassiliou, GS
Follows, GA
Hubank, M
Kellam, P
Item Type: Journal Article
Abstract: Background Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers. Results Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5’ Rapid amplification of cDNA ends (5’RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA. Conclusions Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.
Issue Date: 5-Aug-2014
Date of Acceptance: 15-Jul-2014
ISSN: 1471-2172
Publisher: BioMed Central
Journal / Book Title: BMC Immunology
Volume: 15
Issue: 1
Copyright Statement: © 2014 Bashford-Rogers et al.; licensee BioMed Central Ltd. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.
Keywords: Science & Technology
Life Sciences & Biomedicine
Genetic Variation
Receptors, Antigen, B-Cell
Sequence Analysis, DNA
Stochastic Processes
1107 Immunology
Publication Status: Published
Article Number: 29
Online Publication Date: 2014-08-05
Appears in Collections:Department of Medicine

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