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.