Next-generation sequencing (NGS) is a technology used to determine the sequence of many DNA molecules in parallel.
Details
In the context of antibodies, usually NGS is applied to Plasmablasts and Memory B cells since they circulate in peripheral blood. Following exposure to antigens, such as vaccination, the vast majority of plasmablasts (>75%) in circulation make antibodies against the antigen of interest, whereas the proportion of memory B cells making antibodies against any specific antibody of interest doesn’t exceed 1 in 2,500 to 100,00 (1). This means that large populations of cells are required to get antibodies from the latter against specific targets of interest. Additionally, plasmablasts express 100x more mRNA than memory B cells, distorting the proportions of different clones (although this can be fixed by cDNA amplification).
The actual sequencing, as of 2015, involves either linkage PCR (two separate PCRs for heavy and light chain, as well as a linkage PCR reaction) or cell barcoding. Direct sequencing of B cell genomic DNA is challenged by the fact that 50% of B cells have multiple rearranged immunoglobulin gene loci, due to “nonproductive rearrangements and receptor editing”, such as allelic inclusion (1). Direct mRNA sequencing also is frustrated by the fact that pairing must be inferred from the relative proportions of the heavy and light chains (however, mRNA sequencing can be used to track malignancies).
The primers used in PCR have trouble detecting mutations in the N-terminus of V-genes (1).