Pooled High throughput metabarcoding of Cuyahoga River water sample: Sample DS42
"We amplified DNA extracted from each sample using the following conditions: initial denaturation of 95C for 15 minutes followed by 50 cycles of 95C for 30 sec., 55C for 30 sec. 72C for 30 sec. Overall, we performed, on each sample, 13 independent DNA amplifications targeting Bacteria, Archaea, Mammals, Algae, Amphibians, Birds, Fish, Bryophytes, Arthropods, Copepods, Diatoms, Fungi and vascular plants (Baker et al. 2003; Bissett et al. 2005; Epp et al. 2012; Sherwood & Presting 2007; Sundquist et al. 2007; Taberlet et al. 2007; Taylor 1996; Thomsen et al. 2012; Zeale et al. 2011; Zimmermann et al. 2011). Each amplification primer included a 5'tail for barcoding and Illumina sequencing (see below). We then pooled 12 amplification products (all except the bacterial 16S rRNA amplicons which were sequenced separately) obtained from each water sample and labeled each sample with an individual oligonucleotide barcode by performing 10 cycles of PCR using primers targeting the 5'oligonucleotide tail and containing the Illumina adapter sequence and the unique barcode sequence. We then pooled all barcoded samples together. The bacterial 16S rRNA genes were individually barcoded using the same approach and the products from all samples pooled into a single library. Finally, we sequenced the two libraries on an Illumina MiSeq (150bp PE)."