Method Study

High-throughput sequencing (HTS) of antibody amplicon libraries has become a powerful method in the emerging field of systems immunology. However, numerous sources of bias in HTS workflows may affect antibody repertoire data. A crucial step in antibody amplicon library preparation is the addition of adapter sequences, which are platform-specific short nucleotide sequences. In addition to conventional ligation-based adapter addition, it is also possible to use PCR-based methods such as Direct Addition (one-step adapter addition, DA) and Primer Extension (two-step adapter addition, PE), which have led to the lack of a consensus method and uncertainty regarding the impact of adapter addition on repertoire HTS data. Therefore, we compared all three methods by performing HTS with the Illumina MiSeq platform using total RNA originating from mouse antibody-secreting cells. We used technical replicate-based validation and clonal overlap and rank statistics to demonstrate that the two PCR-based methods produced HTS repertoires equivalent to ligation. Specifically: Nine BALB/c mice (8-10 weeks old, Charles River, specific-pathogen free) were immunized with 50 ug alum-precipitated chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl (NP, NP-CGG, BioCat). Mice were sacrificed 14 days post-immunization (dpi) and their spleens and bone marrow (from femurs and tibia) were harvested. Antibody-secreting CD138-positive cells were enriched from spleen and bone marrow 14 dpi as previously described (Reddy, 2010, Nat Biotech).