Figure 2.

Library preparation of immunoglobulin (Ig) heavy-chain genes for high-throughput sequencing (Ig-seq) using molecular amplification fingerprinting (MAF). (A) In step 1, reverse transcription (RT) is performed to generate first-strand cDNA with a gene-specific (IgM or IgG) primer, which includes a unique reverse molecular identifier (RID) and partial Illumina adapter (IA) region. This results in single-molecule labeling of each cDNA with an RID. In step 2, several cycles of multiplex-PCR are performed using a forward primer set with gene-specific regions targeting heavy-chain variable (V_H) framework region 1 (FR1), with overhang regions comprised of a forward unique molecular identifier (FID) and partial IA. In step 3, singleplex-PCR is used to extend the partial IAs. The result (Step 4) is the generation of antibody amplicons with FID, RIDs, and full IA ready for Ig-seq and subsequent MAF-based error and bias correction. (B) List of oligonucleotides sequences annealing to the V_H FR1 used in multiplex-PCR (Step 2) of the MAF library preparation protocol. The nearest germline IGHV segment(s) likely to be amplified by the respective primer are listed in the rightmost column. (C) The estimated melting temperature distribution of the V_H FR1 forward primer set.