SREL Reprint #3338

Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

Carly F. Graham1, Travis C. Glenn2, Andrew G. McArthur3, Douglas R. Boreham4, Troy Kieran2, Stacey Lance5, Richard G. Manzon1, Jessica A. Martino1, Todd Pierson2, Sean M. Rogers6, Joanna Y. Wilson7, and Christopher M. Somers1

1Department of Biology, University of Regina, Regina, Saskatchewan S4S 0A2, Canada
2College of Public Health, University of Georgia, Athens, GA 30602, USA
3M.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
4Medical Sciences, Northern Ontario School of Medicine, Greater Sudbury, Ontario, P0M, Canada
5Savannah River Ecology Laboratory, University of Georgia, Athens, GA 30602, USA
6Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
7Department of Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada

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 situ DNA 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.

Keywords: Coregonus clupeaformis, ddRADSeq, reduced representation libraries, SNP

SREL Reprint #3338

Graham, C. F., T. C. Glenn, A. G. McArthur, D. R. Boreham, T. Kieran, S. L. Lance, R. G. Manzon, J. A. Martino, T. Pierson, S. M. Rogers, J. Y. Wilson, and C. M. Somers. 2015. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq). Molecular Ecology Resources 15(6): 1304-1315.

This information was provided by the University of Georgia's Savannah River Ecology Laboratory (srel.uga.edu).