Figure 1.

(A) Adaptor-ligation PCR for next-generation sequencing-based antibody repertoire analysis. A universal primer specific for the isotype constant region (isotype-specific primer) and an adaptor primer were used for unbiased amplification of antibody genes. IgM-, IgD-, IgG-, IgA-, and IgE-specific primers were used for amplification of respective isotype genes. Amplified gene products were sequenced using an Illumina MiSeq sequencer, and each sequence read was classified into immunoglobulin subclasses by discrimination using the identifying sequence of the constant region. (B) Proportions of IgG and IgA subclasses in healthy individuals. Proportions of sequence reads of IgG subclasses (IgG3, IgG1, IgG2, IgG4, and IgGP) and IgA subclasses (IgA1 and IgA2) are indicated. Total sequence reads excluding unproductive reads (out-of-frame reads) were used for calculation. Each dot represents an individual and bars indicate mean frequencies (n = 12).