Studying Antibody Repertoires with Next-Generation Sequencing

Lees, William D.; Shepherd, Adrian J.

doi:10.1007/978-1-4939-6613-4_15

Cited by 5 publications

(3 citation statements)

References 65 publications

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“…After the initial alignment and assembly of reads, the antibody sequences can be clustered based on their similarity to the known gene segments encoding heavy or light antibody chains. Clustering these antibody sequences allows researchers to understand the lineage structure of all antibodies produced in individual B cells [10–12].…”

Section: Resultsmentioning

confidence: 99%

Advancing computational biology and bioinformatics research through open innovation competitions

et al. 2019

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Open data science and algorithm development competitions offer a unique avenue for rapid discovery of better computational strategies. We highlight three examples in computational biology and bioinformatics research in which the use of competitions has yielded significant performance gains over established algorithms. These include algorithms for antibody clustering, imputing gene expression data, and querying the Connectivity Map (CMap). Performance gains are evaluated quantitatively using realistic, albeit sanitized, data sets. The solutions produced through these competitions are then examined with respect to their utility and the prospects for implementation in the field. We present the decision process and competition design considerations that lead to these successful outcomes as a model for researchers who want to use competitions and non-domain crowds as collaborators to further their research.

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Section: Resultsmentioning

confidence: 99%

Advancing computational biology and bioinformatics research through open innovation competitions

et al. 2019

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“…Even then, there were still issues with matching PCR conditions and efficiency. Methods to overcome the biases detected in amplification have been developed, such as the use of 5’ RACE and using molecular barcodes or identifiers . However, these methods are expensive and have their own draw backs.…”

Section: Discussionmentioning

confidence: 99%

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing

2018

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Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our laboratory compared unamplified sequencing results from the splenic heavy‐chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique variable (V)‐gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high‐frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V‐gene segments, and detects high‐frequency clones in the repertoire.

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“…Separation of the naïve repertoire from the memory repertoire is clearly an essential requirement for establishing the real diversity of the naïve repertoire, but is a non-trivial exercise, as indicated by Lees & Shepherd. 56…”

Section: The Observed Diversity Of Antibody-antigen Responsesmentioning

confidence: 99%

Understanding the human antibody repertoire

Rees¹

2020

mAbs

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The origins of the various elements in the human antibody repertoire have been and still are subject to considerable uncertainty. Uncertainty in respect of whether the various elements have always served a specific defense function or whether they were co-opted from other organismal roles to form a crude naïve repertoire that then became more complex as combinatorial mechanisms were added. Estimates of the current size of the human antibody naïve repertoire are also widely debated with numbers anywhere from 10 million members, based on experimentally derived numbers, to in excess of one thousand trillion members or more, based on the different sequences derived from theoretical combinatorial calculations. There are questions that are relevant at both ends of this number spectrum. At the lower bound it could be questioned whether this is an insufficient repertoire size to counter all the potential antigen-bearing pathogens. At the upper bound the question is rather simpler: How can any individual interrogate such an astronomical number of antibody-bearing B cells in a timeframe that is meaningful? This review evaluates the evolutionary aspects of the adaptive immune system, the calculations that lead to the large repertoire estimates, some of the experimental evidence pointing to a more restricted repertoire whose variation appears to derive from convergent 'structure and specificity features', and includes a theoretical model that seems to support it. Finally, a solution that may reconcile the size difference anomaly, which is still a hot subject of debate, is suggested.

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Studying Antibody Repertoires with Next-Generation Sequencing

Cited by 5 publications

References 65 publications

Advancing computational biology and bioinformatics research through open innovation competitions

Advancing computational biology and bioinformatics research through open innovation competitions

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing

Understanding the human antibody repertoire

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