KIR allelic variation in humans with and without atopic dermatitis
Uniquely indexed primers were designed to target KIR exons 3, 4, and 5 (D0, D1 and D2 Domain). Primer mixes were prepared containing 2-4 primers per amplicon to cover all KIR genes in a multiplexed assay with an amplicon size ~400 bp. Five separate PCR reactions were setup for each sample to cover all targeted exons in 12.5 l volume in 96-well plates with GoTaq DNA Polymerase (Promega) and the associated buffer system. As each sample is uniquely indexed during the PCR reactions, all amplicons from the PCR reactions were pooled using volumes appropriate to obtain balanced read coverage for each KIR exon. Each pool was then ligated with unique Illumina indexes and sequencing adapters ready for Illumina sequencing. The products were sequenced on the Miseq using 600V3 chemistry. Post sequencing, quality filtered paired reads were demultiplexed based on UMIs and reads were merged using in-house developed bioinformatic tools. The merged reads were then aligned to a KIR locus reference sequence for all KIR genes using CLCbio genomics workbench. The alignment files (bam files) were then used for assigning the KIR allele calls using VGAS which generates the cluster consensus sequences and All Class which makes the allele calls based on IMGT reference database.