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.