Revisiting the functional annotation of TriTryp using sequence similarity tools

Trypanosomatids are the causative agents of deadly diseases in humans and livestock. Given the high phylogenetic distance of trypanosomatids from model organisms, these organisms have ample unannotated genes. Manual functional annotation is time-consuming, highlighting the importance of automated functional annotation tools. The development of automated functional tools is a hot research topic, and multiple tools have been developed for the task. PANNZER2 is an automated functional annotation tool that merely relies on the sequence similarity of the query to the annotated proteins. We tried PANNZER2 on <i>Trypanosoma brucei</i>, the most studied organism among trypanosomatids, to see if it could improve our knowledge of the functions of the genes. Even with the availability of automated annotation tools like InterPro2GO in databases such as TriTrypDB, PANNZER2 has made surprisingly confident predictions for some hypothetical proteins in <i>T. brucei</i>. In this study, we identify gaps in such annotations because of not employing pairwise sequence alignment tools in TriTrypDB's automated annotation process. Our findings demonstrate that even the use of stringent cutoffs can successfully annotate a significant number of proteins. Additionally, we discovered that adjusting the open reading frames in certain genes leads to sequences with increased sequence signature coverage-characterized by the length covered by at least one sequence signature-compared to the original sequences. This enhanced sequence signature coverage suggests these genomic fragments could be pseudogenes. To facilitate further exploration, we developed a script to help identify potential pseudogenes within an organism's genome, offering researchers a new tool for genomic analysis and understanding. We extended all our analysis to <i>Trypanosoma cruzi</i> and <i>Leishmania major</i> to assess the impact of this approach across different species. Our study demonstrates that by utilizing pairwise sequence similarity alignment, even with stringent cutoffs, we can attribute 2986, 3953, and 3798 new GO terms to the genomes of <i>T. brucei</i>, <i>T. cruzi</i>, and <i>L. major</i>. Additionally, we found that 210, 239, and 29 genes exhibit increased sequence signature coverage following frame correction, suggesting the presence of pseudogenes.