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High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis
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Number of Authors: 72019 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 10, article id 660Article in journal (Refereed) Published
Abstract [en]

Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5'RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5' multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.

Place, publisher, year, edition, pages
2019. Vol. 10, article id 660
Keywords [en]
next generation sequencing, immunoglobulin, antibody, repertoire, library, germline gene, inference, database
National Category
Biological Sciences Microbiology in the medical area
Identifiers
URN: urn:nbn:se:su:diva-168620DOI: 10.3389/fimmu.2019.00660ISI: 000463564400001OAI: oai:DiVA.org:su-168620DiVA, id: diva2:1314925
Available from: 2019-05-10 Created: 2019-05-10 Last updated: 2019-05-10Bibliographically approved

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Martin, Marcel
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Department of Biochemistry and BiophysicsScience for Life Laboratory (SciLifeLab)
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