Mutation analysis of IGH associated with different isotypes

Immunoglobulin heavy chain rearrangements were sequences from PBMCs of 8 individuals using IGHC primers for all isotypes bar IgE and IgD. Sequencing was carried out on the 454 pyrosequencing platform and rearranged immunoglobulin heavy chain sequences were partitioned against germline IGHV, IGHD and IGHJ using iHMMune-align.

The datasets for each individual was filtered to remove non-productive rearrangements (frame shift of the IGHJ or stop codons), those with greater than 45 IGHV mutations, sequences which contained indels in the V or J, and those with ambiguities. Within each data only unique sequences were reatained (100% identical).

Clonally-related sequences within each subject's dataset were also identified. Following germline reversion of all non-CDR3 nucleotide positions, sequence were clustered using the vmatch package (http://www.vmatch.de/) allowing up to 3 differences. A single representative for each cluster (representing a likely clonal set) was retained within the dataset. Representative sequences were selected based on the sequence with the highest copy number, or in cases where sequences shared the highest copy number, the least mutated sequence. If a cluster spanned multiple isotypes then a representative for each isotype was kept.