4. Sequencing Library Preparation
To confirm the amplification of microbial rRNA gene regions from our DNA
extracts, we sequenced PCR amplification products from seven individuals
of two chickadee species (Poecile atricapillus and P.
carolinensis ) and their hybrids. Once we established successful
amplification of the V3-V4 regions of the 16S rRNA gene using an agarose
gel (see above), we sent seven PCR products and the 16S rRNA primers
mentioned 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
(ACACTGACGACATGGTTC-TACA CCTACGGGNGGCWGCAG) and CS2
(TACGGTAGCAGAGACTTGGTCT GACT-CHVGGGTATCTAATCC) 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 at
Rush Genomics and Microbiome Core Facility. Quality checks were run on
the MiniSeq samples using FastQC. Our seven microbial DNA extracts were
sequenced with an Illumina MiSeq using paired end 300 bp reads. With
each run, a negative and positive sample were run alongside the samples
to control for contaminants at different stages of the sequencing.