Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting

Khan, Tarik A.; Friedensohn, Simon; Vries, Arthur R. Gorter de; Straszewski, Jakub; Ruscheweyh, Hans‐Joachim; Reddy, Sai T.

doi:10.1126/sciadv.1501371

Cited by 110 publications

(148 citation statements)

References 55 publications

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“…1C). Notably, the three "functional" V H s (V H 1-62-1, V H 2-6-8, and V H 7-2) not detected by HTGTS-Rep-seq also were not found by another high-throughput repertoire sequencing method (25), suggesting that they may actually be nonfunctional with respect to the ability to undergo V(D) J recombination. V H -to-DJ H rearrangements occur at the pro-B stage, with only one in three expected to be in-frame (5).…”

Section: Resultsmentioning

confidence: 99%

“…Being DNA-based, HTGTS-Rep-seq also bypasses a major limitation of RNA-based methods for certain applications by quantitatively capturing the frequency of Ig rearrangements in a population regardless of their expression level or whether they are productive or nonproductive. Current means to address biases due to multiplex PCR or varying expression levels between cells include the use of universal identifiers (25,36,37) or single cell methods (38), but HTGTS-Rep-seq can accurately identify a population repertoire profile without the additional cost or steps of synthesizing primers with random barcodes, or sorting for single cells.…”

Section: Resultsmentioning

confidence: 99%

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Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Repseq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJ H intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. 1). In this process, the V, D, and J coding ends are generated as covalent hairpins that must be opened and that are often further processed, before being joined by classical nonhomologous end joining (2). Processing of V, D, J coding ends can involve generation of deletions or insertions of nucleotides at the junction regions (2), including the frequent de novo addition of nucleotides by the terminal deoxynucleotidyl transferase component of the V(D)J recombination process (3). Notably the V(D)J junctional region encodes a major antigen contact region of the antibody variable region, known as complementarity determining region 3 (CDR3), and thus these junctional diversification processes make a huge contribution to antibody diversity.The mouse IgH locus spans 2.7 megabases (Mb). There are 100s of V H s in the several megabase distal portion of the IgH, with the number varying substantially in certain mouse strains (4). The V H s lie ∼100 kb upstream from a 50-kb region containing 13 D H s, which is followed several kilobases downstream by a 2-kb region containing four J H s. The IgH constant region (C H ) exons lie downstream of the J H s. After assembly of a V H DJ H exon, transcription initiates upstream of the V H and terminates downstream of the C H exons, with V(D)J and C H portions being fused...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, mutations were most likely generated during the PCR amplification of the linear donor template despite the use of a high fidelity polymerase (KapaHiFi). Recent studies using high-throughput sequencing on PCR amplicons have shown that reproducible polymerase hotspot errors are more common than previously believed, even with high-fidelity polymerases2829. Therefore, the generation of long donor templates by PCR may result in erroneous variants, which would present a major problem for future therapeutic applications.…”

Section: Resultsmentioning

confidence: 99%

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

Kelton

Waindok

Pesch

et al. 2017

Sci Rep

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The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.

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“…Some of the earlier HTS data was produced in this way 101. This can be overcome using methods that incorporate UMIs at the reverse transcription step so that only one copy of each mRNA molecule is counted 102. This method could also be used to align copies of the same sequence to identify and remove sequencing errors in high read methods.…”

Section: Clonality Analysismentioning

confidence: 99%

Immunoglobulin gene analysis as a tool for investigating human immune responses

Dunn-Walters

Townsend

Sinclair

et al. 2018

Immunological Reviews

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SummaryThe human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self‐reactive B cells removed. Later antigen‐dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen‐specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub‐speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

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Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting

Abstract: A new experimental-bioinformatic method was developed for error and bias correction in high-throughput antibody sequencing.

Cited by 110 publications

References 55 publications

Highly sensitive and unbiased approach for elucidating antibody repertoires

Highly sensitive and unbiased approach for elucidating antibody repertoires

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

Immunoglobulin gene analysis as a tool for investigating human immune responses

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