| Autor: |
Vázquez Bernat N; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden., Corcoran M; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden., Hardt U; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.; Division of Rheumatology, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden., Kaduk M; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden., Phad GE; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden., Martin M; Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden., Karlsson Hedestam GB; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. |
| Abstrakt: |
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. |
