Amoebozoan barcoding marker cytochrome c oxidase (Cox1), RNA editing and issues in creating a public reference sequence database

Abstract

Summary DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene is a promising tool not only in animals but also for many groups of protists, including Amoebozoa. To use this tool, we need a reference database for the comparison and assignment of newly obtained sequences. As NCBI/GeneBank® is the most complete molecular sequence database to date, it is logical to use it as a reference database. In fact, it is used as such, when the newly obtained sequences are checked against this database using BLAST. Yet, a quarter of all available barcoding Cox1 sequences of Amoebozoa would not be seen in the BLAST results, as they are deposited with the status ‘UNVERIFIED’. Some of these sequences show reading frame shifts due to multiple single nucleotide deletions. These deletions, seen at the genomic level, may indicate presence of insertional RNA editing in this gene. This phenomenon was experimentally proven only in myxomycetes and Arcellinida among Amoebozoa. Interestingly, many sequences marked as UNVERIFIED do not show frame shifts or other signs of RNA editing, while some of the sequences that are not assigned this status, do. For the sequence database to be fully searchable, new sequences have to be properly accessioned. A recent communication with NCBI confirms that when a sequence has putative editing sites, the submitter should provide a note on this feature and references to appropriate papers. In this case, the sequence can be accessioned normally.