Dataset Information

Determining immunoglobulin-specific B cell receptor repertoire of murine splenocytes by next-generation sequencing.

ABSTRACT: High-throughput antibody repertoire analysis via next-generation sequencing is a key method in understanding humoral immunity. Errors introduced during library preparation and sequencing can be corrected with molecular amplification fingerprinting using unique molecular identifiers. Here, we provide a step-by-step protocol for laboratory and bioinformatic workflows to perform sequencing in murine cells with isotype-specific primers, obtaining total and isotype-specific B cell receptor repertoires. This enables the examination of isotype-dependent immune responses and improves the understanding of underlying mechanisms in humoral immunity. For complete details on the use and execution of this protocol, please refer to Khan et al. (2016).

SUBMITTER: Werner A

PROVIDER: S-EPMC9010798 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Determining immunoglobulin-specific B cell receptor repertoire of murine splenocytes by next-generation sequencing.

Werner Anja A Schäfer Simon S Gleußner Nina N Nimmerjahn Falk F Winkler Thomas H TH

STAR protocols 20220409 2

High-throughput antibody repertoire analysis via next-generation sequencing is a key method in understanding humoral immunity. Errors introduced during library preparation and sequencing can be corrected with molecular amplification fingerprinting using unique molecular identifiers. Here, we provide a step-by-step protocol for laboratory and bioinformatic workflows to perform sequencing in murine cells with isotype-specific primers, obtaining total and isotype-specific B cell receptor repertoire ...[more]

PMID: 35434659

Similar Datasets

Project description:The impact of natural killer (NK) cell alloreactivity on hematopoietic stem cell transplantation (HSCT) outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR)/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS) technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19) using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci) and a high percentage of mapping (mean 93% for all loci). Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT but also on the role of KIR+ NK cell populations in control of viral infections and diseases.

Dataset Information

Determining immunoglobulin-specific B cell receptor repertoire of murine splenocytes by next-generation sequencing.

Publications

Determining immunoglobulin-specific B cell receptor repertoire of murine splenocytes by next-generation sequencing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets