Evaluation of Extraction Methods for the Recovery of Vertebrate DNA From Spider Webs

Spider webs have recently been recognized as an excellent source of vertebrate eDNA. Here we compare four sample preparation methods and three commercially available DNA extraction kits to optimize vertebrate eDNA recovery. We assessed DNA yield, purity, and fragment length before amplification, as well as vertebrate ZOTU richness and community composition after sequencing. Our results demonstrate that both sample preparation and extraction kit significantly influence DNA recovery from spider webs. Digestion of webs in ATL buffer with proteinase K for 72 h, followed by extraction with a column-based Blood & Tissue kit, yielded the highest mean DNA concentration (10.6 ng/μL) with a mean ZOTU richness of 11.8 ZOTUs per sample. The same digestion method paired with magnetic bead-based extraction produced lower mean DNA concentrations (6.19 ng/μL) but achieved the highest mean ZOTU richness (14.8 ZOTUs per sample). Increased proteinase K concentrations and longer digest times did not significantly improve DNA yield or species richness. Washing webs with phosphate buffer solution combined with PowerLyzer PowerSoil column-based extraction kit produced the lowest total DNA yield (0.16 ng/μL) and richness per sample (1 ZOTU). Although the bead-beating preparation method produced high total DNA yield, it often recovered the lowest mean ZOTU richness, indicating the presence of DNA from non-target taxa such as bacteria. Overall, digestion with ATL buffer and proteinase K (irrespective of digest time), combined with either the Blood & Tissue or MagMAX Microbiome kits, increased vertebrate species detections from spider webs.