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

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          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.

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          Most cited references103

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          Performance comparison of benchtop high-throughput sequencing platforms.

          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).
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            Detection and quantification of rare mutations with massively parallel sequencing.

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

              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.
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                Author and article information

                Contributors
                gg@che.utexas.edu
                Journal
                Nat Biotechnol
                Nat. Biotechnol
                Nature Biotechnology
                Nature Publishing Group US (New York )
                1087-0156
                1546-1696
                19 January 2014
                2014
                : 32
                : 2
                : 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
                BFnbt2782
                10.1038/nbt.2782
                4113560
                24441474
                98f3f468-e475-4a17-bcf8-a4ed5a0d1b3c
                © Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2014

                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
                : 10 June 2013
                : 4 December 2013
                Categories
                Article
                Custom metadata
                © The Author(s), under exclusive licence to Springer Nature America, Inc. 2014

                Biotechnology
                immunogenetics,b cells,applied immunology,antibody isolation and purification
                Biotechnology
                immunogenetics, b cells, applied immunology, antibody isolation and purification

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