Human DNA
When analyzing metabarcoding data for profiling vertebrate diversity, we remove all human DNA reads under the assumption that they are contamination. However, we found a disproportionate amount of the DNA sequences from mosquito pools was identified to be human and the total human read abundance in samples was well above the level of human DNA found in the extraction and PCR negative controls. Thus, we reexamined levels of human DNA found in all three iDNA metabarcoding datasets. We first eliminated non-human contaminants and non-amplifying samples (replicates with less than 500 total reads). With the remaining dataset, we culled all non-human read sequences. We closely examined the amount of human DNA in the extraction and PCR negative controls to determine a read threshold for a human positive sample. To be extremely conservative in what we deemed as a sample positive for human, we set the threshold as the highest read count in any negative control replicate for each iDNA dataset. For example, if the highest read count for human DNA in a negative control for the carrion fly metabarcoding data was 25,000 then the threshold to be counted as a sample positive for human DNA was 25,000 reads in a replicate. Additionally, we also eliminated samples where human DNA was not present in both sample replicates. A relative abundance index (RAI) was calculated as the number of samples positive for human DNA at a site divided by the total number of samples at a site.