Immunoglobulin library preparation for NGS

Next generation sequencing (NGS) of antibody (Ab) repertoires enables examination of the adaptive immune system at an unprecedented level. A key element in this analysis is the availability of sufficiently comprehensive reference gene databases to allow the identification of as little as single nucleotide changes from germline-encoded Ab sequences. Because genomic sequencing of the immunoglobulin (Ig) loci is challenging and time-consuming, we developed a germline inference tool, IgDiscover, which enables the production of individualized Ab germline gene databases from multiple subjects in just a few days. Here, we provide a detailed protocol for the production of libraries suitable for Next Generation Sequencing (NGS) for germline gene inference and Ab repertoire studies. Various parameters used in the production of the libraries have been tested in order to demonstrate the factors that are critical to the production of high quality libraries that ensure comprehensive coverage of the available repertoire, low error rate and low levels of chimerism.