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HAPCAD: An open-source tool to detect PCR crossovers in next-generation sequencing generated HLA data #MMPMID26802209
McDevitt SL; Bredeson JV; Roy SW; Lane JA; Noble JA
Hum Immunol 2016[Mar]; 77 (3): 257-63 PMID26802209show ga
Next-generation sequencing (NGS) based HLA genotyping can generate PCR artifacts corresponding to IMGT/HLA Database alleles, for which multiple examples have been observed, including sequence corresponding to the HLA-DRB1*03:42 allele. Repeat genotyping of 131 samples, previously genotyped as DRB1*03:01 homozygotes using probe-based methods, resulted in the heterozygous call DRB1*03:01+DRB1*03:42. The apparent rare DRB1*03:42 allele is hypothesized to be a ?hybrid amplicon? generated by PCR crossover, a process in which a partial PCR product denatures from its template, anneals to a different allele template, and extends to completion. Unlike most PCR crossover products, ?hybrid amplicons? always corresponds to an IMGT/HLA Database allele, necessitating a case-by-case analysis of whether its occurrence reflects the actual allele or is simply the result of PCR crossover. The Hybrid Amplicon/PCR Crossover Artifact Detector (HAPCAD) program mimics jumping PCR in silico and flags allele sequences that may also be generated as hybrid amplicon.