Year of Award

2013

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Individualized Interdisciplinary Doctoral Program

Department or School/College

Interdisciplinary Studies Program

Committee Co-chair

Jesse Johnson, Gordon Luikart

Commitee Members

Jefferey Good, William Holben, Stephen Porcella, Alden Wright

Keywords

DNA Sequencing, Exon Capture, Next-generation Sequencing, Non-model species

Abstract

The efficient method called exon capture provides for sequencing genes genome-wide, targeting candidate genes, and sampling specific exons within genes. Although developed for model species with available whole genome sequences, the method can capture exons in nonmodel species using the genomic resources of a related model species. How close the relatives must be for effective exon capture is not known. The work herein demonstrates cross-taxa capture in ungulates, using the domestic cow genome as a reference. It also describes a computer program designed for collecting exon sequences for exon capture, allowing users to set per-gene and overall base pair (bp) limits, and to prefer internal or external exons. Cross-taxa exon capture was tested with subject-reference divergence times from 0 to ~60 million years. Sequencing success decreased with increasing subject-reference phylogenetic divergence. With the domestic cow genome as reference, American bison exons, at 1-2 million years (MY) of divergence, were captured as successfully as those of a domestic cow. The cow and bison captures each yielded sequence from ~80% of the 3.6 million bp targeted. Two bighorn sheep, 7 mule deer, and 4 pigs at about 20, 30, and 60 MY of divergence from the cow, respectively, yielded averages of ~70%, ~60%, and ~55% of the targeted bp. A gene family with many closely related, duplicated loci was expected to show reduced success compared to the whole collection. This prediction was supported, as 63 exons in the MHC gene family sequences yielded 62% fully sequenced in the cow, and 32%, 20%, and 4% for the bighorn, deer, and pigs, respectively. A comparison of two sequence alignment programs showed that Stampy, designed for high sample-reference divergence, was dramatically better than BWA, designed for low divergence, only in the pig capture, in which Stampy yielded ~30% more bp than did BWA. A universal ungulate exon capture array could be developed using the 8,999 exons that were fully sequenced in all species, including the pig at ~60 MY. As this method helps us understand the genetic basis of evolutionary processes, so it can contribute to an informed study and stewardship of our ecological endowment.

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© Copyright 2013 Ted Cosart