title: eDNA of amphibians
design: We amplified the extracted DNA from the 50 water samples. The PCR mix included 1x AmpliTaqTM Gold 360 mix (Applied BiosystemTM), 0.5 M of each dual combinatorial indexing tagged forward and reverse BATR01 primers (i.e., primers with eight variable nucleotides added to their 5 end, allowing further sample identification) and 2 M of human-blocking primer. The final volume was 20 L including 2 L of DNA template. We replicated each sample amplification 12 times in 12 separate PCR plates. In each PCR plate, we set 12 blanks corresponding to empty wells and allowed to estimate the proportion of sequence leaking and tag switches (i.e., false combination of tags, generating chimeric sequences occurring during the sequencing process), seven negative extraction controls (extraction performed on Milli-Q(R) water), seven negative PCR controls (water) and seven positive controls containing an equimolar assembly of DNA from three exotic species (P. nigromaculatus, Pseudacris maculata and R. arvalis). We then pooled the PCR products from the eleven remaining plates, and purified amplicons using a MinElute PCR purification kit (Qiagen). We size-selected BATR01 amplicons on a 2% agarose gel and purified them using MinElute Gel Extraction kit (Qiagen). Library preparation was performed using TruSeq DNA PCR-Free Library Prep (Illumina) with the following modifications to ensure a maximal yield of small-size DNA (amplicon size is on average 110 bp including primers): (i) we skipped the Remove large fragments step; (ii) we added 100 L of undiluted SPB to the 100 L of end-repaired sample; (iii) and we followed the protocol starting from step three of the Remove small fragments step. The final library was quantified by qPCR using the KAPA Library Quantification Kit (Roche). Sequencing of the library was carried out at the Genomic Technologies Facility (University of Lausanne, Switzerland). A 100 cycles paired-end sequencing was performed on a single Illumina HiSeq 2500 lane.