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

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• 作者：Shana L. McDevitt^a ; ^c ; ^{shana.mcdevitt@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Jessen V. Bredeson^b ; ^{jessenbredeson@berkeley.edu" class="auth_mail" title="E-mail the corresponding author} ; Scott W. Roy^c ; ^{scottwroy@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Julie A. Lane^a ; ^{jlane@chori.org" class="auth_mail" title="E-mail the corresponding author} ; Janelle A. Noble^a ; ^{jnoble@chori.org" class="auth_mail" title="E-mail the corresponding author}
• 关键词：HAPCAD ; Hybrid Amplicon/PCR Crossover Artifact Detector ; HLA ; Human Leukocyte Antigen ; NGS ; next-generation sequencing ; SSO ; sequence specific oligonucleotide ; CWD ; Common and Well-Documented ; SBT ; Sequence-Based Typing ; P1 ; parent allele 1 ; P2 ; parent allele 2
• 刊名：Human Immunology
• 出版年：2016
• 出版时间：March 2016
• 年：2016
• 卷：77
• 期：3
• 页码：257-263
• 全文大小：965 K

文摘

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.