scholarly journals Comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing

2019 ◽  
Author(s):  
Collin Joyce ◽  
Dennis R. Burton ◽  
Bryan Briney
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Great Promise ◽  
Antibody Diversity ◽  
Model Animal ◽  
Kappa Chain ◽  
Antibody Repertoires

ABSTRACTThe humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoire of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.

scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain transcripts using single side unique molecular identifiers on an Ion Torrent PGM

2017 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Axel R. S. X. Dubois ◽  
Regina Sinner ◽  
Oliver Hunewald ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Gene Segment ◽  
Personal Genome ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Indel Detection ◽  
Processing Strategies ◽  
Nucleotide Insertions

AbstractWith the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT efficiently detects 98% of the introduced indels through gene-segment frameshifts. Undetected indels are either located at the ends of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. IMGT’s indel correction algorithm resolves up to 87% of the tested insertions, but no deletions. The complementary determining regions 3 (CDR3s) are returned 100% correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers results in highly accurate HTS datasets if combined with the presented data processing. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. The protocol and sample processing strategies described in this study will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

scholarly journals The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

2015 ◽  
Vol 370 (1676) ◽  
pp. 20140236 ◽  
Author(s):  
Andrew M. Collins ◽  
Yan Wang ◽  
Krishna M. Roskin ◽  
Christopher P. Marquis ◽  
Katherine J. L. Jackson
Keyword(s):  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Inbred Mouse ◽  
Inbred Strains ◽  
Mouse Strains ◽  
Germline Genes ◽  
Nucleotide Addition ◽  
Antibody Repertoires ◽  
Antibody Heavy Chain ◽  
Mouse Antibody

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.

scholarly journals A multi-objective based clustering for identifying clonally-related sequences from high-throughput B cell repertoire data

2021 ◽  
Author(s):  
Nika Abdollahi ◽  
Anne Langlois De Septenville ◽  
Hugues Ripoche ◽  
Frederic Davi ◽  
Juliana Silva Bernardes
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Clustering Algorithms ◽  
Objective Functions ◽  
Cell Repertoire ◽  
Multi Objective ◽  
B Cell Repertoire ◽  
Attractive Option

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clones. A high number of clones in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity within clones can be related to antigen's selective pressure. Identifying clones is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial clones and iteratively applies two objective functions that optimize cohesion and separation within clones simultaneously. We show that under simulations with varied mutation rates, our method greatly improves clonal grouping as compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools. The method based on multi-objective clustering can accurately identify clone members, has fewer parameter settings and presents the lowest running time among existing tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context to unravel the mechanisms involved in the development and evolution of B cell malignancies.

scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain repertoires using single side unique molecular identifiers on an Ion Torrent PGM

Oncotarget ◽  
2018 ◽  
Vol 9 (54) ◽  
pp. 30225-30239 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Danielle E. Mustin ◽  
Axel R.S.X. Dubois ◽  
Regina Sinner ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Immunoglobulin Heavy Chain ◽  
Ion Torrent ◽  
Ion Torrent Pgm ◽  
Single Side

scholarly journals High-throughput sequencing analysis of genes encoding the B-lymphocyte receptor heavy-chain CDR3 in renal and peripheral blood of IgA nephropathy

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Dapeng Chen ◽  
Zheng Zhang ◽  
Yue Yang ◽  
Quan Hong ◽  
Wenge Li ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
High Throughput ◽  
Heavy Chain ◽  
Peripheral Blood ◽  
High Throughput Sequencing ◽  
Kidney Tissue ◽  
Renal Tissue ◽  
Chronic Glomerulonephritis ◽  
Sequencing Analysis ◽  
Variant Frequency

Abstract Aim: IgA nephropathy (IgAN) is one of the most common chronic glomerulonephritis. Its etiology and pathogenesis remain unclear. We thus explored the immune repertoire of the B-cell receptor (BCR) and the heavy-chain complementarity-determining region 3 (CDR3) in renal tissue and peripheral blood of IgAN patients. Method: Total RNAs extracted from renal tissues and peripheral blood of patients and peripheral blood of healthy controls (HCs) were analyzed via high-throughput multiplex PCR sequencing. We amplified and sequenced BCR heavy-chain CDR3 regions to explore repertoire diversity, V/J gene family distribution, CDR3 lengths, BCR heavy-chain variants, consistency between tissue and peripheral blood data, and clones ‘shared’ by these bodily compartments. Results: We identified the renal tissue and peripheral blood BCR heavy-chain CDR3 immune repertoires of 15 IgAN patients. Top1 could be more readily cloned from peripheral blood of patients than from controls (P<0.05), the average CDR3 length was significantly shorter in patients than in HCs (P<0.05), the variant frequency of the gene encoding the BCR heavy chain was higher in patients than in HCs (P<0.05), and the BCR variant frequency was highest in IgAN kidney tissue. Preliminary screening for ‘shared’ clones showed that, in at least 13 patients, the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ CDR3 sequences were evident in peripheral blood samples from patients, but not HCs. Conclusions: We found that the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ clonal sequences may be useful for noninvasive diagnosis and treatment planning in IgAN.

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