Nemabiome sequencing of a horse from Alberta, Canada
DNA from L3 larvae isolated from coproculture 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. This sample was included in a pooled sequencing library which included nemabiome samples from different horse populations. This library was sequenced using an ILLUMINA miseq v2 500 cycle kit.