Revealing complete complex KIR haplotypes phased by long-read sequencing technology

Abstract

The killer cell immunoglobulin-like receptor (KIR) region of human chromosome 19 contains up to 16 genes for natural killer (NK) cell receptors that recognize human leukocyte antigen (HLA)/peptide complexes and other ligands. The KIR proteins fulfill functional roles in infections, pregnancy, autoimmune diseases and transplantation. However, their characterization remains a constant challenge. Not only are the genes highly homologous due to their recent evolution by tandem duplications, but the region is structurally dynamic due to frequent transposon-mediated recombination. A sequencing approach that precisely captures the complexity of KIR haplotypes for functional annotation is desirable. We present a unique approach to haplotype the KIR loci using single-molecule, real-time (SMRT) sequencing. Using this method, we have—for the first time—comprehensively sequenced and phased sixteen KIR haplotypes from eight individuals without imputation. The information revealed four novel haplotype structures, a novel gene-fusion allele, novel and confirmed insertion/deletion events, a homozygous individual, and overall diversity for the structural haplotypes and their alleles. These KIR haplotypes augment our existing knowledge by providing high-quality references, evolutionary informers, and source material for imputation. The haplotype sequences and gene annotations provide alternative loci for the KIR region in the human genome reference GrCh38.p8.

Haplotype-Based Analysis of KIR-Gene Profiles in a South European Population—Distribution of Standard and Variant Haplotypes, and Identification of Novel Recombinant Structures

“…Existence of two such long and highly similar stretches of sequence favored further asymmetric recombination between the paralogous regions. This resulted in expanded and shortened haplotypes bearing tandem duplications or deletions of the intervening genes, as shown by us and others (9,10,15,(19)(20)(21)(22)(23)(24). Additional consequences of asymmetric recombination are novel fusion genes encoding chimeric KIR that blend structural and functional features of their parent receptors.…”

“…Initial studies of human KIR genotypes faced this vast polymorphism without previous knowledge of the structure and forms of variation of the KIR-gene complex, revealing multiple KIR-gene and allele profiles in different individuals, which could give a false impression of randomness (11,25). Order, in the form of knowledge on common and variant patterns of association between KIR genes and alleles, emerged from subsequent studies of population groups and families; and from phasing and physical mapping of partial and complete KIR-gene haplotypes by DNA sequencing (4,(6)(7)(8)(9)(10)(26)(27)(28)(29). Those studies were complemented by others focused on estimation of CNV and allelic diversity (23,24,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

“…Long-read sequencing technologies have been used to detect chromosomal rearrangements 34 , novel SVs 35,36 , and SVs missed by standard short-read sequencing methods 37,38 , including applications in the complex killer immunoglobulin-like receptors (KIR) 39,40 and human leukocyte antigen (HLA) 34,41 loci. Furthermore, the sensitivity of SV detection is improved by resolving variants in a haplotype-specific manner 38,42 .…”

Accurate and Efficient KIR Gene and Haplotype Inference from Genome Sequencing Reads with Novel K-mer Signatures

“…All full-length, publicly-reported, KIR allele sequences were downloaded from IPD-KIR 2.7.1 [24] and Roe et al 2017. The allele sequences were binned into each gene and intergene region ('Reference Alleles' in Figure 1).…”

“…These receptor-ligand pairs coevolved under selection pressures from reproduction and pathogenic defense [3], and it is believed that KIR genes have undergone a balancing selection via duplications and deletions into two broad categories of haplotypes, in which one category tends to vary more at the allelic level and the other tends to vary more at the structural (gene content and order) level [4][5] [6]. Mostly European-ancestry KIR sequences have been publicly deposited via 34 fullhaplotype sequences and approximately 2500 full-or inter-gene sequences [7][5] [8]. Haplotype structures are divided into two classes.…”