qKAT: a high-throughput qPCR method for KIR gene copy number and haplotype determination

Abstract

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The KIR locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes KIRs technically laborious and expensive to type. To aid the investigation of KIRs in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each KIR locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies KIR haplotype alterations that were not previously visible using other approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0358-0) contains supplementary material, which is available to authorized users.

Novel KIR genotypes and gene copy number variations in northeastern Thais

“…To prepare genomic DNA for KIR copy number identification, 10‐ng samples of genomic DNA (2·5 μl of 4 ng/μl) were aliquoted into 384‐well plates using the Hydra 96 microdispenser (Art Robbins, San Jose, CA). KIR copy number typing was performed using a quantitative multiplex PCR assay described by Jiang et al …”

“…The typing results of 17 KIR genes obtained using PCR‐SSP were previously reported and compared with those obtained with the qPCR method . Copy numbers for all KIR genes ( KIR2DL1–5 , 2DS1–5 , 2DP1 , 3DP1 , 3DL1 , 3DL2 , 3DL3 and 3DS1) were determined in the Thai samples using a Roche LightCycler 480 at Cambridge Institute for Medical Research (CIMR), University of Cambridge, UK.…”

“…The KIR genotypes (IDs) were ascertained according to the Allele Frequency Net Database (http://www.allelefrequencies.net). The KIR haplotypes (centromeric–telomeric motif pairings) in NETs were predicted by the KIR Haplotype Identifier program (http://www.bioinformatics.cimr.cam.ac.uk/haplotypes/) using the determined copy number for each KIR gene . KIR haplotypes were predicted in samples ( n = 73) that had copy number typing for all KIR genes.…”

“…To prepare genomic DNA for KIR copy number identification, 10-ng samples of genomic DNA (2Á5 ll of 4 ng/ll) were aliquoted into 384-well plates using the Hydra 96 microdispenser (Art Robbins, San Jose, CA). KIR copy number typing was performed using a quantitative multiplex PCR assay described by Jiang et al 35 The typing results of 17 KIR genes obtained using PCR-SSP were previously reported 28 and compared with those obtained with the qPCR method. 35 Copy numbers for all KIR genes (KIR2DL1-5, 2DS1-5, 2DP1, 3DP1, 3DL1, 3DL2, 3DL3 and 3DS1) were determined in the Thai samples using a Roche LightCycler 480 at Cambridge Institute for Medical Research (CIMR), University of Cambridge, UK.…”

“…KIR copy number typing was performed using a quantitative multiplex PCR assay described by Jiang et al 35 The typing results of 17 KIR genes obtained using PCR-SSP were previously reported 28 and compared with those obtained with the qPCR method. 35 Copy numbers for all KIR genes (KIR2DL1-5, 2DS1-5, 2DP1, 3DP1, 3DL1, 3DL2, 3DL3 and 3DS1) were determined in the Thai samples using a Roche LightCycler 480 at Cambridge Institute for Medical Research (CIMR), University of Cambridge, UK. Using multiplex assays, the copy number was measured by relative quantification analysis of the target gene (KIR) and reference gene (signal transducer and activator of transcription 6; STAT6) using the comparative Cq method.…”