Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes.

David Roe,Chad Locklear,Jenny Brouckaert,Nick Downey,Keyton Ivery,Martin Maiers,Jonathan Williams,Rui Kuang

doi:10.3389/fimmu.2020.582927

Abstract

The homology, recombination, variation, and repetitive elements in the natural killer-cell immunoglobulin-like receptor (KIR) region has made full haplotype DNA interpretation impossible in a high-throughput workflow. Here, we present a new approach using long-read sequencing to efficiently capture, sequence, and assemble diploid human KIR haplotypes. Probes were designed to capture KIR fragments efficiently by leveraging the repeating homology of the region. IDT xGen® Lockdown probes were used to capture 2–8 kb of sheared DNA fragments followed by sequencing on a PacBio Sequel. The sequences were error corrected, binned, and then assembled using the Canu assembler. The location of genes and their exon/intron boundaries are included in the workflow. The assembly and annotation was evaluated on 16 individuals (8 African American and 8 Europeans) from whom ground truth was known via long-range sequencing with fosmid library preparation. Using only 18 capture probes, the results show that the assemblies cover 97% of the GenBank reference, are 99.97% concordant, and it takes only 1.8 haplotigs to cover 75% of the reference. We also report the first assembly of diploid KIR haplotypes from long-read WGS. Our targeted hybridization probe capture and sequencing approach is the first of its kind to fully sequence and phase all diploid human KIR haplotypes, and it is efficient enough for population-scale studies and clinical use. The open and free software is available at https://github.com/droeatumn/kass and supported by a environment at https://hub.docker.com/repository/docker/droeatumn/kass.

Highlights

The protein coding killer-cell immunoglobulin-like receptor (KIR) genes span ~10–16 kb each, with pseudogenes that are ~5 and ~13 kb
Average times are reduced when assemblies are run in parallel. These experiments in individuals from diverse populations demonstrate that KIR haplotypes can be efficiently enriched and assembled using an efficient number of capture probes
The workflow successfully reconstructed both haplotypes from targeted sequencing in 16 individuals and from whole genome sequencing (WGS) in 1 individual

Summary

INTRODUCTION

The protein coding killer-cell immunoglobulin-like receptor (KIR) genes span ~10–16 kb each, with pseudogenes that are ~5 and ~13 kb. It is difficult to interpret KIR haplotypes for an individual human genome given the reads from high-throughput sequencing when the structural arrangements are unknown. This is largely due to read lengths from prevailing technologies being too short to map unambiguously to the repetitive and homologous KIR genes. When applied to a cohort of 8 African Americans and a cohort of 8 Europeans, the results demonstrate that every KIR gene and intergene contains constant regions that are targetable by capture probes, and that by targeting the constant regions, the variable regions can be captured and sequenced by standard PacBio workflows Maximizing this paradigm shows that 18 short probe sequences can capture KIR haplotypes and allow. Annotate the assembled sequences with their genes and exon/ intron locations

MATERIALS AND METHODS

Evaluation of the Workflow

RESULTS

DISCUSSION

ETHICS STATEMENT