Novel method for next generation sequencing of KIR by Roche 454

Natural Killer cells are regulated by the polymorphic Killer Ig-like Receptor family. KIR genotype and expression has been associated with HIV-1 disease progression. We developed a high-throughput method to characterize both DNA genotype and RNA expression of KIR using a novel 454 sequencing method (Roche), for study of HIV and innate immunity. This method uses a 299bp and 395bp amplicon to distinguish and assign long - and short-tailed KIR. These amplicons, from motifs common to 3’ UTR of known KIR genes, amplify both KIR DNA and mRNA. We developed a custom bioinformatics pipeline to analyze 454 sequence reads and it compares each sequence (average 3000 reads per specimen) to a reference alignment of KIR genes via the BLAT algorithm. Using this method, we characterized KIR in a cohort of 700 treatment-naïve adults in early HIV-1 disease. Initial results indicate that our KIR DNA 454 sequencing data agree with KIR DNA genotyping results from mass spectrometry-based typing as well as sequence-specific oligonucleotide typing. KIR3DL1 expression on NK cells by flow cytometry shows 80% agreement with KIR mRNA expression from PBMCs by 454. The use of mRNA- and DNA-based next generation sequencing for human immune receptor genes, such as KIR, will accelerate genetic association and functional genetic studies of diseases of the immune system.