Data from: Impacts of degraded DNA on restriction enzyme associated DNA (RAD) sequencing

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 hours incubation at room temperature post-euthanasia. 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 to 48 hours) 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 hour 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.

分子生态学研究中，因野外采样条件不佳导致的DNA降解现象十分普遍。然而，其对基于限制性酶切位点关联下一代测序（restriction site associated next-generation DNA sequencing，RADSeq、GBS）技术的影响尚不明确。本研究通过实验探究了原位DNA降解对改良双酶切RADSeq技术（3RAD）的数据产出效果的影响。我们以8条安乐死后的湖白鲑（Coregonus clupeaformis）肌肉组织为材料，在室温下分别孵育0、12、48和96小时后依次提取基因组DNA，以此构建测序文库。该处理方式使得投入测序的DNA样品质量跨度从近乎完整到高度剪切断裂。所有样品均以多重混样的方式在Illumina MiSeq测序平台上完成测序。由轻度至中度降解DNA（12至48小时组）构建的文库测序效果良好。与之相反，使用高度降解DNA（96小时组）构建的文库，其每个个体的RAD标签数、变异位点数量以及保留的一致RAD标签占比均出现显著下降。该性能下降主要源于测序质量评分不佳导致的原始读段（raw reads）大量意外丢失。在更换限制性酶、调整覆盖度倍数（2至16倍）以及额外进行读段长度修剪后，本研究的结论依然保持一致。本研究得出结论：起始DNA质量是RADSeq技术需要重点考量的因素；不过该技术在基因组DNA出现大规模降解前仍具备良好的稳定性。