The promise and challenge of high-throughput sequencing of the antibody repertoire

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Georgiou and colleagues discuss rapidly evolving methods for high-throughput sequencing of the antibody repertoire, and how the resulting data may be applied to answer basic and translational research questions.

Abstract

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

Related collections

Most cited references 103

Record: found
Abstract: found
Article: not found

Detection and quantification of rare mutations with massively parallel sequencing.

Isaac Kinde, Jian Wu, Nick Papadopoulos … (2011)

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

0 comments Cited 481 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Performance comparison of benchtop high-throughput sequencing platforms.

Nicholas Loman, Raju V. Misra, Timothy Dallman … (2012)

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

0 comments Cited 473 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Detection of ultra-rare mutations by next-generation sequencing.

Michael W Schmitt, Scott Kennedy, Jesse Salk … (2012)

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

0 comments Cited 413 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

George Georgiou: gg@che.utexas.edu

Journal

Journal ID (nlm-ta): Nat Biotechnol

Journal ID (iso-abbrev): Nat. Biotechnol

Title: Nature Biotechnology

Publisher: Nature Publishing Group US (New York )

ISSN (Print): 1087-0156

ISSN (Electronic): 1546-1696

Publication date (Electronic): 19 January 2014

Publication date (Print): 2014

Volume: 32

Issue: 2

Pages: 158-168

Affiliations

[1 ]GRID grid.89336.37, ISNI 0000 0004 1936 9924, Department of Chemical Engineering, , University of Texas at Austin, ; Austin, Texas USA

[2 ]GRID grid.89336.37, ISNI 0000 0004 1936 9924, Department of Biomedical Engineering, , University of Texas at Austin, ; Austin, Texas USA

[3 ]GRID grid.89336.37, ISNI 0000 0004 1936 9924, Department of Molecular Biosciences, , University of Texas at Austin, ; Austin, Texas USA

[4 ]GRID grid.89336.37, ISNI 0000 0004 1936 9924, Institute for Cell and Molecular Biology, University of Texas at Austin, ; Austin, Texas USA

[5 ]GRID grid.168010.e, ISNI 0000000419368956, Department of Bioengineering, , Stanford University, ; Stanford, California USA

[6 ]GRID grid.168010.e, ISNI 0000000419368956, Howard Hughes Medical Institute, Stanford University, ; Stanford, California USA

[7 ]GRID grid.418159.0, ISNI 0000 0004 0491 2699, Max Planck Institute for Infection Biology, ; Berlin, Germany

[8 ]GRID grid.168010.e, ISNI 0000000419368956, Biophysics Graduate Program, Stanford University, ; Stanford, California USA

[9 ]GRID grid.168010.e, ISNI 0000000419368956, Department of Applied Physics, , Stanford University, ; Stanford, California USA

Article

Publisher ID: BFnbt2782

DOI: 10.1038/nbt.2782

PMC ID: 4113560

PubMed ID: 24441474

SO-VID: 98f3f468-e475-4a17-bcf8-a4ed5a0d1b3c

License:

This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

History

Date received : 10 June 2013

Date accepted : 4 December 2013

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: immunogenetics,b cells,applied immunology,antibody isolation and purification

Data availability:

ScienceOpen disciplines: Biotechnology

Keywords: immunogenetics, b cells, applied immunology, antibody isolation and purification

The promise and challenge of high-throughput sequencing of the antibody repertoire

Read this article at

Abstract

Abstract

Related collections

Journal of Applied Biology and Biotechnology

Most cited references 103

Detection and quantification of rare mutations with massively parallel sequencing.

Performance comparison of benchtop high-throughput sequencing platforms.

Detection of ultra-rare mutations by next-generation sequencing.

Author and article information

Contributors

Journal

Affiliations

Article

History

Categories

Custom metadata

Comments

Comment on this article

Cited by 287

Most referenced authors 2,691