Why do we refer to them as OTUs?

OTU stands for “Operational Taxonomic Unit”. OTU’s are clusters of sequences that are relatively similar and may represent multiple species. Since a single OTU can be multiple species, they are referred to as OTU’s rather than species.

The sample output says that my sample has DNA from a plant genus that isn’t likely to be there, why is this the case?

Even if the genus isn’t found at your site, more than likely some species closely related to species in the listed genus is found at your site. Our database is limited and we cannot identify every plant species (yet).

Why do I not have data from some of my samples?

Some samples just do not amplify well – either do an insufficient amount of plant DNA in the sample or because it was difficult for us to get sufficiently clean DNA. We do our best, but we cannot guarantee data from 100% of samples.

If the match is <100% can it still be a species in that genus?

Yes, but it is less likely to be a species in that genus. The OTU is based on a consensus sequence that represents a most probable sequence and a number of similar sequences that might differ in, say, 1 or 2 base pairs. So, multiple species can be represented by the consensus sequence, even if individuals of the different species do not have exactly the same sequence.

Can you resolve species within a genus?

It depends on how different the species are, but it is unlikely with the current technique…that requires multiple barcodes and/or longer barcodes generally.

What if there is no match between an OTU ID and anything in the library?

If there is no match in the library, this means that no species that have been sampled match the consensus sequences at the 97% level. Generally, this is reported as an “unknown” in a paper, but more work can be done to narrow down what species they might represent. First, we’ll supply a “fasta” file, which has the actual consensus sequences. You can go to the NCBI website and look to see what species have the closest match and try to interpret that in the context of your samples. Or, you can collect leaves of species that are likely to match that unknown. We’ll sequence these and rerun the bioinformatics to see if the unknowns now match.

I sent plant samples to have run for the reference library. How do I interpret these data?

We sequence these samples similar to how we sequence diet samples. As such, you’ll see a percentage of reads assigned to each OTU. Often more than one OTU will have sequences. In short, the OTU that is assigned to this sample can now be interpreted to include this species if the OTU shows up in the diet. For example, if voucher sample X shows that 95% of the reads are OTU y in the data we send you, then when you see OTU y in the diet, you can assume that the species that was provided for sample X could be part of the diet. It can be helpful to compare the species already assigned to an OTU in the closed reference library with the identity of the voucher specimen you provided. They should be closely related.