Results
Our extraction method was successful in extracting microbial DNA from
both fecal and preen oil samples collected across a diverse range of
avian taxa (e.g., songbirds, waterfowl, scavengers, and birds of prey),
covering a broad range of feeding guilds (Tables 1 and 2; Figure 1). The
success of our extraction method was evident by our consistent ability
to PCR amplify the V3-V4 region of the microbial 16S rRNA gene,
indicated by the presence of bands on agarose gels representing PCR
products of the expected length (Figure 1). Microbial DNA was only
extracted once from each preen and fecal sample; however, multiple PCRs
were occasionally necessary (Figure 1) to visibly see bands on a 2%
agarose gel indicating the presence of the microbial DNA. Out of 25
fecal collections from 15 species and 12 preen oil collections from 12
species, only the extraction from the single song sparrow preen oil
sample did not produce a band on an agarose gel (Figure 1).
Our successful amplification of the targeted 16S rRNA microbial gene
regions was further confirmed by sequencing a subset of our PCR
products. Specifically, we sequenced 16S rRNA V3-V4 regions in microbial
DNA extractions done with fecal material from black-capped and Carolina
chickadees (n=7). We obtained consistently high-quality reads with a
mean of 108,000 reads per sample. We obtained an initial count of 417
ASVs. After trimming and merging the reads, five of seven samples had at
least 90% reads retained, while the remaining two had approximately
85% reads retained (see supplemental). Additionally, after trimming and
merging and getting rid of contaminants, there remained a count of 244
ASVs. Subsequent bioinformatic analysis of the sequencing results
revealed that our extraction method was successful in extracting both
gram-positive and gram-negative bacteria (Table 3). Further, our results
identifying the bacterial classes present in each extraction, as well as
the relative abundance of different bacterial classes, suggests that the
gut microbiome can vary across different chickadee individuals (Figure
2).