Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database

Tissue Antigens
Cherie L HolcombH A Erlich

Abstract

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybri...Continue Reading

References

Apr 11, 1990·Nucleic Acids Research·A MeyerhansS Wain-Hobson
Jun 12, 1989·Nucleic Acids Research·A R ShuldinerJ Roth
Dec 8, 2005·Applied and Environmental Microbiology·Kevin E AshelfordAndrew J Weightman
Jun 2, 2012·Tissue Antigens·H Erlich
Sep 21, 2012·Journal of Clinical Microbiology·Astrid GallPaul Kellam
Oct 20, 2012·Nucleic Acids Research·James RobinsonSteven G E Marsh

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Citations

Jun 2, 2015·Human Immunology·Wyatt C NelsonDaniel E Geraghty
Jan 28, 2015·Scientific Reports·Claudia Brandariz-FontesJennifer A Leonard
Jan 23, 2014·Tissue Antigens·C GabrielG F Fischer
Dec 18, 2014·Immunogenetics·Catalina Gonzalez-QuevedoDavid S Richardson
Jan 7, 2017·PloS One·Vladimir Potapov, Jennifer L Ong
Oct 3, 2019·HLA : Immune Response Genetics·Marine CargouJonathan Visentin
Oct 5, 2019·Journal of Clinical Medicine·Iori KisuTakashi Shiina
Jan 24, 2020·HLA : Immune Response Genetics·Eri KikkawaMakoto Onizuka
Mar 8, 2021·Human Immunology·Nicholas K BrownJane Kearns

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