title: Exon capture optimization in amphibians with large genomes.
abstract: Gathering genomic-scale data efficiently is challenging for nonmodel species with large, complex genomes. Transcriptome sequencing is accessible for organisms with large genomes, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but relatively few data exist for exon capture experiments in nonmodel organisms with large genomes. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for amphibians and other taxa with large, complex genomes. Increasing the amounts of c0 t-1 repetitive sequence blocker and individual input DNA used in target enrichment reactions reduced the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9% and rendering target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen vertebrates with large genomes, including amphibians.
authors.affiliation: Department of Ecology and Evolutionary Biology, La Kretz Center for California Conservation Science, Institute of the Environment and Sustainability, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Evan
authors.identifier: http://orcid.org/0000-0002-1803-1126
authors.initials: E
authors.lastname: McCartney-Melstad
authors.affiliation: Department of Ecology and Evolutionary Biology, La Kretz Center for California Conservation Science, Institute of the Environment and Sustainability, University of California, Los Angeles, CA, 90095, USA.|Museum of Natural Science, Louisiana State University, Baton Rouge, LA, 70803, USA.|Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.
authors.forename: Genevieve G
authors.initials: GG
authors.lastname: Mount
authors.affiliation: Department of Ecology and Evolutionary Biology, La Kretz Center for California Conservation Science, Institute of the Environment and Sustainability, University of California, Los Angeles, CA, 90095, USA.
authors.forename: H Bradley
authors.initials: HB
authors.lastname: Shaffer
mesh_terms: D000663:Amphibians / Q000145:classification* / Q000235:genetics*; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019143:Evolution, Molecular; D005091:Exons; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*; D059467:Transcriptome
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA
keywords: amphibian; exon capture; large genome; target enrichment