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).