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.

Harnessing the power of RADseq for ecological and evolutionary genomics

“…1). All methods start with relatively high molecular weight genomic DNA 18 and begin by digesting it with one or more restriction enzymes. All methods add specific sequencing adapters , or double-stranded oligonucleotides, that are required by all next-generation sequencing platforms.…”

“…RADseq techniques have been optimized based on starting material comprised of high molecular weight genomic DNA, and thus these techniques may perform poorly with highly degraded genomic DNA 18 . For example, in methods without enzyme-specific adaptors (e.g., ezRAD, CRoPS), smaller fragments of starting genomic DNA not adjacent to cut sites may end up in the sequencing library, thus wasting sequencing effort on non-RAD loci.…”

Molecular variation across populations of a widespread North American firefly, Photinus pyralis, reveals that coding changes do not underlie flash color variation or associated visual sensitivity

“…Library construction followed a 3RAD protocol [87]. 3RAD differs from ddRADseq [48] by using three restriction enzymes, two to digest genomic DNA and one to cut adapter dimers.…”

Future directions and priorities for Arctic bryophyte research

“…The short DNA fragment lengths sequenced on Illumina platforms, however, may be suited for sequencing even degraded DNA. Library generation protocols such as RADseq can accommodate highly degraded DNA (Tin et al 2014;Graham et al 2015), and metabarcoding of Lepidoptera has been accomplished from century-old type specimens yielding 458-610 bp sequences (Prosser et al 2016). Consequently, we suggest that given the low sampling density of Arctic bryophytes, older specimens should still be considered for molecular analysis, especially leveraging next-generation sequencing technologies.…”