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).