Feaces-08-Extraction_Control
DNA was extracted from 164 samples (74 saliva, 74 faecal, 8 environmental, and 4 water controls) at the Globe Institute of the University of Copenhagen (Denmark). DNA was extracted from the saliva using a PowerSoil DNA isolation kit (Qiagen) following the manufacturer's protocol with some modifications. DNA from the faecal samples was extracted from FTA cards following. Each extraction round included 12-24 samples, including an extraction blank. The final DNA extracts (50 L) were stored at -20C before further processing. All DNA extractions were performed in a flow hood in a dedicated pre-PCR laboratory to minimise the risk of contamination. The following PCR conditions were used: a pre-cycle of 95C for 10 minutes, followed by 30 cycles of 95C for 15 seconds, 53C for 20 seconds and 72C for 40 seconds. PCRs were run in a reaction volume of 25 L on an Applied Biosystem 2720 Thermal Cycler. Metabarcoding PCR amplicons were purified using the DNA Purification Ampure Bead protocol with in-house SPRI beads. They were then pooled based on their amplicon band strength score, with low-scoring samples assigned 10 L, medium-scoring 8 L, and high-scoring 5 L. Amplicon pools were purified using SPRI beads in a 1:1 bead:DNA volume ratio to remove non-target DNA and primer dimers and two aliquots were made from the purified pools. The libraries were constructed using a modified version of the Blunt-End-Single-Tube (BEST) method with the adaptor concentration doubled. Whereafter, the libraries were quantified on Agilent 2100 Bioanalyzer and sequenced on the Illumina MiSeq platform using PE300 bp chemistry.