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.