Using de novo assembly to identify structural variation of eight complex immune system gene regions

Abstract

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

Local adaptation and archaic introgression shape global diversity at human structural variant loci

“…However, it exhibits a highly skewed allele balance (centered on 0.2, in contrast to the expectation of 0.5) – a symptom of reference bias in read mapping ( Chen et al, 2019 ) – as well as low genotyping rates (31% for CDX in 1KGP, compared with 85% in our study). These biases result in lower estimates of the insertion’s allele frequency in East Asian populations (AF = 0.73 in CDX) and underscore the technical challenges of genotyping at this locus with traditional short-read approaches ( Chin et al, 2020 ; Zhang et al, 2021 ).…”

“…The variants identified by Browning et al, 2018 are located in a subregion of the broader IGH locus, downstream of the introgressed SVs, and segregate at high allele frequency in East Asian, European, and American populations ( Figure 4—figure supplement 3 ). The southeast Asian-specific haplotype we identify, which includes the IGHG4 insertion and nearby deletion, may have been challenging to discover due to the difficulties of short-read alignment and genotyping in this region of the genome ( Zhang et al, 2021 ). Indeed, 80.7% of the sequence in the broader IGH locus was filtered out by Browning et al, 2018 through strict masking of aDNA genotypes to remove low-coverage, poorly mapping, or repeat-associated reads ( Figure 4—figure supplement 4 ).…”

“…The challenge of dense genotyping and phasing within the broader IGH region ( Zhang et al, 2021 ) hinders haplotype-based approaches for inferring the timing of selection. Nevertheless, the global patterns of allele frequencies we observe ( Figure 5A,B ) can be interpreted in the context of known population demographic histories, providing a rough estimate of such timing.…”

“…In developing lymphocytes, this locus undergoes somatic V(D)J recombination and hypermutation to produce antibodies that drive the immune response—processes that may be influenced by nearby germline variation ( Watson et al, 2017 ). The combination of these forms of variation makes the region difficult to probe with traditional sequencing methods ( Chin et al, 2020 ; Zhang et al, 2021 ), in turn highlighting the power of long-read sequencing and graph genotyping.…”

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“…The extent of this under-representation has recently been surveyed by Mikocziova et al 24 Recent efforts to generate accurate and complete assemblies of immunoglobulin, T-cell receptor, and natural killer cell receptors in a single individual have exploited state of the art long-read and other mapping and assembly approaches. 25 They have concluded that a full accurate and complete assembly of variation in some of these regions in a single individual is a technical challenge that has not yet been overcome and will require further methodological improvements. These problematic regions of the genome, and others of perhaps similar complexity and biological importance, underscore that the notion that SNP-based GWAS is a comprehensive tool for uncovering genetic variation contributing to disease traits is one in need of re-assessment.…”