="16S sequencing of ovis aries gut microbiome: sample "&A15
16S rRNA sequencing was performed through a PCR dual-indexing strategy (Kozich et al., 2013) which was conducted over two separate reactions to prepare the weaner lamb sample library. The first round amplified the Variable region 4 (V4) using universal primers 515F and 806R (Apprill et al., 2015) in the Escherichia coli 16S rRNA gene and a mixture of partial adaptors for Illumina sequencing, which allowed for diversity between sequence clusters. Negative controls of no-template extraction and no-template PCR controls were included, along with a mock community of 19 bacteria species (Zymo) used as a positive control. The second round amplified products from the first PCR and attached Illumina flow cell adaptors, and dual sample indices with unique barcodes. Following PCR all amplification products were examined with agarose gel electrophoresis and purified using JetSeq Clean (Bioline) paramagnetic beads to remove small sized fragments (primer dimers) and unused PCR reagents.