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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Publications at this Location » Publication #339299
Title: Haplotype resolution of leukocyte receptor complex in cattle through targeted enrichment and SMRT sequencing

Author
item HEIMEIER, DOROTHEA - The Pirbright Institute
item SCHWARTZ, JOHN - The Pirbright Institute
item Bickhart, Derek
item Smith, Timothy - Tim
item HAMMOND, JOHN - The Pirbright Institute

Submitted to: International Society for Animal Genetics (ISAG)
Publication Type: Abstract Only
Publication Acceptance Date: 4/14/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The highly repetitive nature of cattle leukocyte receptor complex (LRC) has made it difficult to assemble and fully characterize this region with short reads used by second-generation sequencing. Previously, we reported the first two cattle killer immunoglobulin-like receptors (KIR) haplotypes; one complete and framed by leukocyte immunoglobulin-like receptor (LILR) and Immunoglobulin alpha Fc receptor (FCAR) genes (263kb), the other shorter and incomplete, that were resolved from combined Sanger and 454-pyrosequencing sequencing of BAC clones. Through subsequent targeted genome enrichment with Roche Nimblegen probes and Illumina sequencing of different cattle breeds and related species, the haplotype variability and gene polymorphism of further haplotypes has been predicted to be gene-variable with additional KIR genes. These data have now been combined and validated with another complete and larger KIR haplotype (350kb) that has been assembled using long-read single molecule real time (SMRT) sequencing with Pacific Bioscience technology. More recently, we have developed this targeted sequencing approach for use with SMRT sequencing to resolve the KIR region in more detail from two more individuals. Initial results showed between 87 and 91% average base coverage when mapped to the shorter complete KIR haplotype at a maximum divergence of 10%. Average coverage was more than 20x, with certain regions reaching more than 100x, which could be indicating additional or missing genes in the investigated individuals. These preliminary data are clearly indicative of significant structural variation, as well as polymorphisms. We are currently developing a bioinformatics pipeline to de novo assemble and phase each haplotype, so we can process a large number of individuals and identify gene-variable haplotypes and polymorphic variants with confidence.