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 LibbyRun2 which included nemabiome samples from other horse populations, as well as cattle, bison, and domestic sheep. This library was sequenced using an Illumina MiSeq v2 500-cycle kit.