Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes
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,...
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| Published in: | Frontiers in immunology Vol. 11; p. 582927 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Switzerland
Frontiers Media S.A
09-10-2020
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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
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. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 This article was submitted to NK and Innate Lymphoid Cell Biology, a section of the journal Frontiers in Immunology Reviewed by: Stephen K. Anderson, National Cancer Institute at Frederick, United States; Lisbeth Guethlein, Stanford University, United States Edited by: Lutz Walter, Deutsches Primatenzentrum, Germany |
| ISSN: | 1664-3224 1664-3224 |
| DOI: | 10.3389/fimmu.2020.582927 |