ITS2 DNA metabarcoding of strongyle parasites from Sable Island horse feces
DNA from L3 larvae isolated from coprocultures was extracted using an in-house buffer (described in Avramenko et al. 2015) and proteinase K. PCR amplification of the ITS2 gene region was conducted on a 1:10 dilution of the DNA extract (in water) using the NC1 and NC2 primers (with 0-3 random bases) for 25 cycles, followed by a second PCR adding unique combinations of Illumina indexes for 7 cycles. PCR product for both PCRs were purified using magnetic AMPure XP magenetic beads at a ratio of 1:1. DNA concentration of the purified second PCR product was estiamted using a microvolume spectrophotometer (Nanodrop) and 100ng of the sample was pooled into a library. DNA concentration of pooled libraries were estimated using qPCR, and diluted to 12 pM. Pooled libraries were sequenced on an Illumina MiSeq platform with 20% phiX spike-in. This sample was included in the pooled sequencing library SableIsland2014 which only included nemabiome samples from Sable Island. This library was sequenced using an Illumina MiSeq v3 600-cycle kit.