Data from: Spider webs capture environmental DNA from terrestrial vertebrates

Published: 20 September 2023| Version 1 | DOI: 10.17632/b4n9vpxzcd.1
Joshua Newton


eDNA sequence data obtained form spider webs, showing there ability to act as passive biofilters capturing eDNA from vertebrates present in the local environment. Raw sequencing results (fastq) and information for demultiplexing reads (.txt) to assess vertebrate DNA extracted from spiderwebs. Metadata from sample sites is also included for analysis.


Steps to reproduce

A total of 49 spider webs were obtained randomly at two sites with the exact location and height of spider webs documented prior to sampling (Karakamia = 23, Zoo = 26; average height = 1.5m). All spider webs were extracted using adapted protocol of the Qiagen PowerLyzer PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany). Lysis was performed by adding 540, 810, 1080 or 1350 μL ATL lysis buffer and 60, 19, 120 or 150 μL proteinase K, respectively to plastic tubes containing individual spider webs, depending on the size and absorbent properties of individual webs (Table S1). Samples were lysed at 56°C for 72 hr with a gentle rotation. After lysis, samples were vortexed at 10,000 g for one minute and 650 μL lysis mixture retrieved. DNA was then isolated using, on the QIAcube automated extraction platform (Qiagen, Hilden, Germany) following manufacturers protocols. Three assays were used to amplify vertebrate DNA from all spider webs collected outdoors, 12S-V5 (F2: 5′-TAGAACAGGCTCCTCTAG-3′; R2: 5′-TTAGATACCCCACTATGC-3′); 16Smam1/2 assay (F: 5’-CGGTTGGGGTGACCTCGGA-3′; R: 5’-GCTGTTATCCCTAGGGTAACT-3′) ; and 16S Reptile assay (F: 5′-AGACNAGAAGACCCTGTG-3′; R: 5′-CCTGATCCAACATCGAGG-3). For DNA metabarcoding, sample dilutions showing optimal levels of amplifiable DNA underwent a single step qPCR, using fusion tagged primers consisting of Illumina compatible sequencing adaptors, a unique index (6-8bp in length) and a respective primer sequence for each assay. All qPCR reactions were prepared in dedicated clean room facilities with 4 μL of template eDNA for reaction volumes totalling 27 μL, which contained the same reagents, controls, and cycling conditions described above. Each eDNA sample was amplified in duplicate PCRs with the same index tag before amplicons were pooled in approximate equimolar concentration based on qPCR Rn values resulting in two Amplicon libraries, a single end library containing pooled 12S-V5 and 16Smam samples and a paired end library containing 16Srep samples. Amplicons in the single end library were then size-selected to 150 – 300 bp and the paired end library size-selected to 200 – 600 bp, using a Pippin-Prep 2% Agarose Gel Cassette (Sage Science, Beverly, USA) to remove any amplicons outside of the target range. Size-selected libraries were then quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, USA) and diluted to 2nM. The single end library was loading onto a 300 cycle MiSeq V2 Standard Flow cell for single end sequencing, and paired end library loaded onto a 500 cycle MiSeq Reagent Nano Kit V2 Standard flow cell for paired end sequencing. Sequencing for both libraries was conducted separately on an Illumina MiSeq platform (Illumina, San Diego, USA), with a final single end library molarity of 10 pM containing 7% PhiX, and paired end library molarity of 7.5 pM containing 5% PhiX.


Curtin University