Illumina MiSeq-16S of Branta canadensis cloacal swab sample
Roughly 1 ng of template DNA from each sample was amplified using the V4 primer pairV4_515F (5'-GTGYCAGCMGCCGCGGTAA-3') and V4_806R (5'-GGACTACNVGGGTWTCTAAT-3')following the Fluidigm (Standard Biotools, CA) protocol. Barcoded amplicons generated for each sample were quantified with a Qubit fluorometer (ThermoFisher, CA), and fragment sizes were assessed using a Fragment Analyzer (Agilent, CA). Amplicons were then pooled at equimolar concentrations, separated on a 2% agarose Ex-gel (ThermoFisher) to eliminate primer dimers, and purified from the gel with a Qiagen gel extraction kit. The cleaned, size-selected pool was re-quantified and re-evaluated on the Fragment Analyzer to verify fragment size distribution. Afterward, the pooled library was diluted to 5 nM and its concentration further refined via qPCR using a CFX Connect Real-Time system (Bio-Rad, Hercules, CA) to optimize cluster density on the sequencing flow cell. The pooled library was denatured, supplemented with 20% non-indexed PhiX V3 control (Illumina), and then loaded onto a MiSeq V2 flow cell (500-cycle kit) at 8 pM to enable cluster generation and sequencing. Paired-end sequencing was performed, yielding 250-nt reads from both directions. Raw sequencing output was processed with bcl2fastq v2.20 (Illumina, CA) to generate demultiplexed, compressed FASTQ files.