4. Sequencing Library Preparation
Once we established the successful amplification of the V3-V4 regions of the 16S rRNA gene from our DNA extracts using an agarose gel (see above), we further confirmed that our method successfully extracted microbial DNA by sequencing these regions from a small subset of our samples. Specifically, we sent seven PCR samples from two chickadee species (Poecile atricapillus and P. carolinensis) plus the 16S rRNA primers described above to Rush Genomics and Microbiome Core Facility (Chicago, Illinois, USA) for sequencing. Library preparation and sequencing of the seven samples were completed by the sequencing facility using the CS1 (ACACTGACGACATGGTTCTACA CCTACGGGNGGCWGCAG) and CS2 (TACGGT-AGCAGAGACTTGGTCT GACTCHVGGGTATCTAATCC) linkers, indicated by underlining, on the 341F and 806R 16S primers, indicated in bold. The sequencing facility performed Fluidigm amplicon library preparation to ready the samples for next generation sequencing. The samples were normalized, pooled, and sequenced on Illumina MiniSeq using paired end 300 bp reads. With each run, a negative and positive controls were run alongside our samples to control for contaminants at different stages of the sequencing.