BAGS: Barcode, Audit & Grade System

Firstly, to use the application you should make sure the taxonomic group or groups you want to annotate are present at the BOLD Systems database, considering that intermediate taxa are usually the most likely to be absent. Additionally, the spelling of the taxa should be identical to the spelling according to the information at BOLD.

The app has three main options: download a tsv library for every species belonging to the taxa, download a tsv library for only marine species belonging to the taxa or download a tsv library for only non-marine species belonging to the taxa.

Then, after you enter the name of the taxonomic group/groups or enter a file with a list of species names, a data set will be created and curated following these steps:

1. Downloading the data set in tsv file format, consisting of specimen data and its respective COI-5P sequence belonging to the chosen taxa, from the BOLD Public Data Portal.


2. Filtering out the following from the data set:
   
   
   • Specimens with sequences of length below the threshold chosen by the user
   
   
   • Specimens without data on species name, BIN, lattitude or country of origin
   
   
   • Ambiguous characters occasionally present in the species name and COI-5P sequences
   
   
   • Specimens with sequences consisting of > 1% Ns, which are usually the most commonambiguous character


3. In the case of the marine or non-marine taxa options, the data set is filtered once again, retaining or excluding only the species known to be from marine or brackish habitats, using their species name as reference. This is achieved using the WoRMS database, therefore, the download and annotation will take longer.


4. Lastly, according to the quality and availability of the data of each specimen, qualitative grades from A-E are assigned to each species present in the data set. Then, several reference libraries in fasta format are created, which can be downloaded individually.

The assignment of each grade is based on the quality, availability and replicability of the data and metadata for each species, as well as the quality and congruence of the COI-5P sequences, evaluated in accordance to their Barcode Index Number (BIN).

The BIN System is an online framework at BOLD that generates Operational Taxonomic Units (OTUS) by clustering barcode sequences algorithmically, grouping them in a manner that ideally, mirrors their respective specimen morphological identification.

The grades are attributed to each species according to the following criteria:

• Grade A Consolidated concordance The morphospecies is assigned a unique BIN, which is also assigned uniquely to that species, plus the species has more than 10 specimens present in the library


• Grade B Basal concordance The morphospecies is assigned a unique BIN, which is also assigned uniquely to that species, plus the species has 10 or less specimens present in the reference library


• Grade C Multiple BINs The morphospecies is assigned more than one different BINs, but each of those BINs are assigned exclusively to that species


• Grade D Insufficient data Species is not assigned discordantly, but it has less than 3 specimens available in the reference library


• Grade E Discordant species assignment Species assigned to a BIN that is assigned to more than one different species. The specimen may match with a different species or display paraphyly or polyphyly

• Costa, Filipe O., Landi, M., Martins, R., Costa, M. H., Costa, M. E., Carneiro, M., . Carvalho, G. R. (2012). A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal. PloS One, 7(4), 1-9. doi: 10.1371/journal.pone.0035858


• Oliveira, L. M., Knebelsberger, T., Landi, M., Soares, P., Raupach, M. J., & Costa, F. O. (2016). Assembling and auditing a comprehensive DNA barcode reference library for European marine fishes. Journal of Fish Biology, 89(6), 2741-2754. doi: 10.1111/jfb.13169