Sanger sequencing-
We used isolate descriptions to select morphologically diverse isolates to send for Sanger sequencing because we wanted to describe the diversity within our collection. We identified 52 isolates for Sanger sequencing (~48% of our collection) and we performed DNA extractions using the Qiagen PowerLyzer Microbial DNA Kit (Germantown, MD, USA). We then amplified the 16S rRNA region with primers 27F and 1492R, which span nearly the entire length of the gene (Weisburg et al. 1991). Amplicons were prepared with 2µl of template, 39.75µl of molecular grade water, 5µl of DreamTaq® buffer, 1µl of 10xDNTP mix, 1µl forward primer (27F), 1µl reverse primer (1492R), and 0.25µl DreamTaq®. Thermocycler conditions for bacterial PCR amplification were 95°C for 5 minutes, followed by 33 cycles of 95°C for 30 seconds, 53°C for 30 seconds, 72°C for 2 minutes, with a final extension of 72°C for 3 minutes. PCR products were purified using Sera-Mag Speedbeads carboxylated magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) and eluted in molecular grade water. Samples were submitted to the Washington State University Laboratory for Biotechnology and Bioanalysis (LBB) for Sanger sequencing using the ABI 3730 DNA Analyzer (ThermoFisher Scientific, Waltham, MA, USA). Isolates that sequenced poorly were run twice. Forward and reverse sequences from three isolates did not pass quality thresholds and were not included for subsequent analyses.
Sanger sequences were trimmed to ‘max ambiguities’= 2 and consensus sequences were made by combining forward and reverse reads with Geneious Primer (v. 2023.2.1, Biomatters, Inc., CA, USA) with the “high sensitivity/slow” method. For taxonomic identification, sequences were used as BLAST queries to the NCBI 16S rRNA database with the following settings: “low complexity filter, max e-value =0.05, word size= 28, gap cost= linear, program= megaBLAST.” The species with the lowest E value and highest percentage identity were selected as the identity of the isolate. Here we present taxa to the genus level, but putative species IDs can be found in Table S3.