High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis

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.

Polymorphisms in immunoglobulin heavy chain variable genes and their upstream regions

“…We managed to validate a number of novel alleles by targeted amplification of genomic DNA of the same individuals. In addition, we report the presence of G at position 318 instead of A in the gDNA sequence of IGHV3-7*02 , which supports the findings of previous studies 31, 32 .…”

“…Many reference sequences, which were deposited to the IMGT germline database are partial and contain only the V-region sequence. It is surprising that the genetic variation in the upstream regions is overlooked, considering the fact that the leader regions are frequently used as primer binding sites for immunoglobulin repertoire library preparation protocols 32, 36, 37 .…”

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

“…For example, databases of variable region gene segments can be improved using the IgDiscover tool [17]. Targeting the more conserved 5’ UTR of V genes may reduce the amount of primers needed in MPCR designs and yield amplicons covering the entire variable region but still need to be designed for rhesus macaque [33]. New algorithms such as the recently described version of the tool for Ig Genotype Elucidation via Rep-Seq (TIgGER) for identifying human Ig [73] can also improve germline allele assignment if adapted for rhesus macaque.…”

“…The recently developed IgDiscover tool [17] now makes it possible to leverage germline databases of related species to improve those of model organisms, for example using human germline databases to study Indian- and Chinese-origin rhesus macaques. Authors from the same lab also developed a strategy for targeting the 5’ UTR of V genes, often conserved among these gene clusters, thus reducing the number of primers needed to target the variable region in human [33]. However, rhesus MPCR amplification systems are inherently biased to the V and J gene segments they target because the primers are designed based on the consensus of a limited number of reference sequences [34].…”

“…Moreover, even with MPCR or 5’ RACE it is still difficult to capture complete Ig mRNA transcripts with the commonly available sequencing instruments like the 2 x 250 bp HiSeq system and the 2 x 300 bp MiSeq system, in part due to the large length variability of recombined molecules [39] and the potential for non-canonical recombination events [40]. Library preparation techniques have recently been improved to support this capability but only in human [33]. Despite these technical advances, it is still unclear how well these strategies perform in rhesus macaque, where the relatively limited resources preclude similar benchmarking efforts previously done for human Ig repertoires [41] and TCR repertoires [42].…”