Data from: Evaluation of extraction methods for the recovery of vertebrate DNA from spider webs

Description

eDNA sequence data obtained form DNA extraction tests run on bulk spider web sample for the recovery of vertebrate DNA. Raw sequencing results (fastq) and information for demultiplexing reads (.txt) to assess DNA extraction methods. Metadata also included for analysis.

Steps to reproduce

Sample collection and preparation A bulk spider web sample with total weight of 2.129 grams and was homogenized and separated into 60 sub-samples all weighing between 0.032 - 0.035 grams. These sub-samples were then randomly assigned to one of four sample preparation methods 1) short chemical and enzymatic digest, 2) long chemical and enzymatic digest, 3) PBS wash + short chemical and enzymatic digest, and 4) mechanical lysis . Their resulting digests were then assigned to one of three DNA extraction kits the Blood & Tissue Kit (Qiagen), PowerLyzer PowerSoil Kit (Qiagen), or MagMAX Microbiome Kit (Applied Biosystems), with five replicates for each of the 12 treatments (i.e., preparation method with DNA extraction combination). The general vertebrate primer 12S-V5 (F2: 5′-TAGAACAGGCTCCTCTAG-3′; R2: 5′-TTAGATACCCCACTATGC-3′) (Riaz et al., 2011) was used to amplify vertebrate DNA from all spider web extracts. The qPCRs were performed using a StepOnePlusTM Real-Time PCR system (Applied BioSystems, Waltham, USA) on neat extracts, 1:10 and 1:100 dilutions with PCR reaction volumes totalling 25 μL. Each reaction contained 14.45 µL ddH20, 2 µL MgCl2 (2.5 mM), 2.5 µL 1× PCR Gold buffer, 0.25 µL dNTPs (0.25 mM of each), 1 µL BSA (0.4 mg/ml), 0.6 μL 5X SYBR® Green, 0.2 µL AmpliTaq Gold® DNA Polymerase, 1 µL of each primer (10 µM) and 2 μL of template eDNA. The cycling conditions included initial denaturation for 5 min at 95°C, followed by 50 cycles of 30 s at 95°C, then 30 s at 52°C annealing temperature, and 45 s at 72°C, with a final elongation step of 10 min at 72°C. Each qPCR run incorporated both a non-template control (reagents only), and a positive control (Isoodon obesulus). Library preparation and sequencing For DNA metabarcoding, sample dilutions demonstrating optimal DNA amplification underwent a single step qPCR, using fusion tagged primers consisting of Illumina compatible sequencing adaptors, a unique index (7-8 bp in length) and assay specific primer sequences. Reaction contained the same reagents, controls, and cycling conditions described above. Each eDNA sample underwent duplicate PCR amplifications with the same index tag. The indexed duplicates were then combined and qPCR Rn values use to blend samples in approximate equimolar concentrations into ‘minipools’, with no more than 10 samples included in each. All minipools were quantified (ng/µL) using a QiAxcel Advanced System (Qiagen) and blended in equimolar concentrations to create a single-end amplicon library. Amplicons were then size-selected to 150 – 300 bp, using a Pippin-Prep 2% Agarose Gel Cassette (Sage Science, Beverly, USA) before being quantified using a Qubit 4 Fluorometer (Invitrogen, Carlsbad, USA). Sequencing was then conducted on an Illumina MiSeq platform (Illumina, San Diego, USA) using a MiSeq Reagent Nano Kit v2 (300-cycles), with a final library molarity of 10 pM containing 7% PhiX.

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