title: Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).
abstract: Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.
authors.affiliation: Department of Biology, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada.
authors.forename: Carly F
authors.initials: CF
authors.lastname: Graham
authors.affiliation: College of Public Health, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Travis C
authors.initials: TC
authors.lastname: Glenn
authors.affiliation: M.G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: McArthur
authors.affiliation: Medical Sciences, Northern Ontario School of Medicine, Greater Sudbury, Ontario, P0M, Canada.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Boreham
authors.affiliation: College of Public Health, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Troy
authors.initials: T
authors.lastname: Kieran
authors.affiliation: Savannah River Ecology Laboratory, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Lance
authors.affiliation: Department of Biology, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Manzon
authors.affiliation: Department of Biology, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Martino
authors.affiliation: College of Public Health, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Pierson
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Rogers
authors.affiliation: Department of Biology, McMaster University, Hamilton, Ontario, L8S 4M1, Canada.
authors.forename: Joanna Y
authors.initials: JY
authors.lastname: Wilson
authors.affiliation: Department of Biology, University of Regina, Regina, Saskatchewan, S4S 0A2, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Somers
mesh_terms: D000818:Animals; D053836:DNA Degradation, Necrotic; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009132:Muscles; D012487:Salmonidae / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D004262:DNA Restriction Enzymes
keywords: Coregonus clupeaformis; SNP; ddRADSeq; reduced representation libraries