HAPCAD: An open-source tool to detect PCR crossovers in next-generation sequencing generated HLA data

Area of Science:

• Immunogenetics
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) is crucial for human leukocyte antigen (HLA) genotyping.
• PCR artifacts can be generated during NGS, potentially leading to misidentification of HLA alleles.
• The HLA-DRB1*03:42 allele has been observed as a potential artifact in NGS HLA genotyping.

Purpose of the Study:

• To investigate the origin of the apparent HLA-DRB1*03:42 allele observed in NGS.
• To determine if this allele is a true genetic variant or a PCR-generated artifact.
• To develop a method for distinguishing true alleles from PCR crossover artifacts.

Main Methods:

• Repeat genotyping of 131 samples previously identified as HLA-DRB1*03:01 homozygotes using probe-based methods.
• Analysis of NGS data to identify potential PCR artifacts, specifically "hybrid amplicons".
• Development and application of the Hybrid Amplicon/PCR Crossover Artifact Detector (HAPCAD) program.

Main Results:

• Repeat genotyping revealed heterozygous calls (DRB1*03:01+DRB1*03:42) in samples previously typed as homozygous DRB1*03:01.
• The apparent DRB1*03:42 allele is hypothesized to be a "hybrid amplicon" resulting from PCR crossover.
• HAPCAD was developed to identify potential hybrid amplicons by mimicking PCR crossover in silico.

Conclusions:

• The apparent rare HLA-DRB1*03:42 allele is likely a PCR artifact, not a true genetic variant.
• Distinguishing between true alleles and hybrid amplicons requires careful analysis.
• HAPCAD provides a computational approach to flag potential PCR crossover artifacts in HLA genotyping data.