Results
Our microbial DNA extraction method was successful from both fecal and
preen oil samples collected across multiple avian taxa (e.g., songbirds,
waterfowl, scavengers, and birds of prey), covering a broad range of
feeding guilds (Tables 1 and 2; Figures 1, 2 and 3). 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 (Figures 2 and 3; Supplemental Figures 1-4). Out of the
fecal collections from 15 species and preen collections from 11 species,
only the preen sample from the song sparrow did not amplify or show on a
gel (Tables 3 and 4).
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. 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).
As mentioned above, FastQC was used to determine the quality of the
reads and maintained a minimum phred score of 25 on both the forward and
reverse reads. This resulted in trimming the forward read at 275 bp and
the reverse read at 225 bp, allowing some overlap in the merge and
higher accuracy on the reads for each sequenced sample. Subsequent
bioinformatic analysis of the sequencing results revealed that our
extraction method was successful in extracting both gram-positive and
gram-negative bacteria (Table 5). 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 3).