9-OR: GENERATION OF HLA DIVERSITY IN VITRO

Aim As HLA studies expand across the globe, novel HLA alleles are identified. HLA genotyping with next generation sequencing (NGS) technology suggests the possibility that at least some of the reported 7,527 alleles may result from erroneous amplification of genomic DNA. Methods DRB loci, including DRB1, DRB3, DRB4, and DRB5, were genotyped for 1,534 European American, 708 African American, and 239 Bangladeshi samples with Roche 454 technology, which produces “clonal” sequencing, originating from single molecules. Genotype interpretation was performed with Conexio ATF and SCORE software. A subset of samples was previously genotyped with SSO methods. Results 59 of 131 samples genotyped as DRB1 ∗ 03:01, DRB1 ∗ 03:01 by SSO were genotyped as DRB1 ∗ 03:01:01:01, DRB1 ∗ 03:42 by 454. Sequences for DRB1 ∗ 03:01:01:01 and DRB3 ∗ 01:01:02:01 (commonly found in cis with DRB1 ∗ 03:01) show that DRB1 ∗ 03:42 is identical to DRB1 ∗ 03:01:01:01 at its 5′end and to DRB3 ∗ 01:01:02:01 at its 3′end. Thus, a PCR product (amplicon) could be generated in the initial amplification reaction by extension of partial amplicon from DRB1 ∗ 03:01:01:01 on DRB3 ∗ 01:01:02:01 in what is termed “jumping PCR.” Notably, DRB1 ∗ 03:42 was only seen in combination with DRB1 ∗ 03:01 and only when DRB3 ∗ 01:01 was present. In fact, of 131 samples SSO typed as DR3, DR3 homozygous, 110 showed evidence of jumping PCR when re-genotyped by 454. Inspection of other genotypes showed additional, non-DR3 alleles that could result from jumping PCR. The appearance of these alleles in specific genotypes contributes to individual genotype- and population-level Hardy-Weinberg deviations, further suggesting that they are spurious. Conclusions The massively parallel, clonal nature of 454 NGS genotyping can reveal sequence that would be missed with other technology. Appearance of rare DRB alleles in specific genotypic contexts suggests that these sequences are generated in vitro by jumping PCR, and that some recognized HLA alleles have origins in PCR rather than the human genome.