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Cost‐effective and fast KIR gene‐content genotyping by multiplex melting curve analysis
Author(s) -
Amorim Leonardo M.,
Santos Tiago H. S.,
Hollenbach Jill A.,
Norman Paul J.,
Marin Wesley M.,
Dandekar Ravi,
Ribeiro Enilze M. S. F.,
PetzlErler Maria L.,
Augusto Danillo G.
Publication year - 2018
Publication title -
hla
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.347
H-Index - 99
eISSN - 2059-2310
pISSN - 2059-2302
DOI - 10.1111/tan.13430
Subject(s) - genotyping , biology , melting curve analysis , gene , computational biology , genotype , polymerase chain reaction , genetics
Killer cell immunoglobulin‐like receptor ( KIR ) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene‐content haplotypes in worldwide populations. KIR gene‐content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene‐content level, even with the advent of next‐generation sequencing (NGS) methods. Gene‐content determination is generally expensive and/or time‐consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR‐SSP) followed by melting curve analysis that allows cost‐effective, precise and fast generation of results. Our method was 100% concordant with a gel‐based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 μM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user‐friendly Java‐based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time.