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

Abstract

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.

Keywords: Human Leukocyte Antigen; IMGT/HLA Database; Next-generation sequencing; Open-source tools; PCR crossovers.

Figure 1

“Jumping” PCR/PCR Crossover Schematic. Figure 1 displays the “Jumping” PCR mechanism used to explain the in vitro production of DRB1*03:42 from partial DRB3*01:01:02:01 and DRB1*03:01:01:01 alleles often found on a DR52 haplotype [4].

Figure 2

Motif-Specific Primer Schematic. Figure 2 displays a schematic of the amino acid sequence and annealing location the motif-specific primer targets in comparison to the Roche DRB general primers. Relevant alleles are also listed with their appropriate forward and reverse motif-specific primers, which are designed to exclusively amplify the target allele.

Figure 3

Motif-Specific Primer Experiment Results. Figure 3 displays the amplification results from the Motif-Specific Primer Experiment. 2% Agarose electrophoresis was used to visualize the amplification products for the twelve noted primer pair combinations. If DRB1*03:42 were amplified, as detailed in Table 1, amplification (~350 bp) would be expected in gel lane A4.

Figure 4

HAPCAD in silico PCR Crossover Mechanism. Figure 4 shows the basic mechanism by which HAPCAD generates putative hybrid amplicons with the pair-wise manipulation of IMGT/HLA Database allele sequences. Two parent alleles are selected for which the polymorphic positions are determined. Hybrid sequences are then constructed using the 5′ sequence of parent allele 1 (P1) up to the base before each polymorphism and the remaining sequence of parent allele 2 (P2), including the polymorphic position through the end of the sequence. A second hybrid is then generated corresponding to the same polymorphic position consisting of the 5′ P2 sequence up to the base before each polymorphism and P2 sequence starting at the polymorphism until the end of the sequence. Parent allele sequences contributing to each hybrid are differentiated by a “|” before the polymorphic position in the figure. HAPCAD does not consider the first polymorphic position, as the resultant hybrids will always regenerate a parent allele. The putative hybrid sequences are then compared against the remaining alleles in the HLA alignment file to determine if any of the hybrid sequences correspond to an HLA allele. If a match is identified, the PCR crossover range will be reported between the polymorphic position at which the matched hybrid was generated and the previous polymorphic position, indicating the region of sequence homology between the two parent alleles.