Abstract
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.
Highlights
The development of Next generation sequencing (NGS)-based approaches to Ig repertoire analysis offers new opportunities to investigate B cell responses in health and disease [reviewed in [1,2,3,4,5,6,7,8]]
When enumerating the number of IGHV3 and IGHV4 sequences in the library data for six individuals, we found that the relative proportion of IGHV3 sequences was lower in the 5′Rapid Amplification of cDNA Ends (RACE) libraries compared to the 5′ multiplex (5′MTPX) libraries, and we observed the opposite for IGHV4 sequences (Figure 3F)
Since one objective of Repertoire sequencing (Rep-seq) analysis is to identify the specific germline alleles expressed in a given individual using germline gene inference tools, we investigated the output from libraries produced by the 5′ Rapid Amplification of cDNA Ends (5′RACE) or 5′MTPX protocols for the six subjects
Summary
The development of NGS-based approaches to Ig repertoire analysis offers new opportunities to investigate B cell responses in health and disease [reviewed in [1,2,3,4,5,6,7,8]]. Utilized approaches to immune repertoire sequencing analysis involve the production of isotype-specific libraries of the Ig cDNA These libraries are sequenced using NGS protocols that enable the production of amplicons that encompass either partial, in the case of libraries which use framework 1 located primer sequences [10, 11], or full-length sequences of the recombined variable (V), diversity (D), and joining (J) gene segments of Ig heavy chains (HC) or VJ sequences of Ig light chains (LC, kappa or lambda). The use of UMIs allows the identification of sequences arising from the same mRNA molecule and facilitates error correction of Ig sequences This approach comes at the cost of increasing the sequence length of libraries that strain the currently available high-throughput NGS methods. The optimized protocols described here facilitate the production of high-quality libraries for germline genotype inference and Ig Rep-seq expression analysis
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