DOI: 10.5281/zenodo.6782365
title: Distinguishing between canonical and non-canonical tRNA genes reveals that Thermococcaceae adhere to the standard archaeal tRNA gene set

description: <strong>Abstract</strong> Automated genome annotation is an essential tool for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed – for selected genomes – in the Genomic tRNA database (GtRNAdb). Given the central role of tRNA in molecular biology, the accuracy and proper application of tRNAscan-SE is important for both interpretation of the output, and continued improvement of the software. Here, we report a manual annotation of the predicted tRNA gene sets for 20 complete genomes from the archaeal taxon Thermococcaceae. According to GtRNAdb, these 20 genomes contain a number of putative deviations from the standard set of canonical tRNA genes in Archaea. However, manual annotation reveals that only one represents a true divergence; the other instances are either (i) non-canonical tRNA genes resulting from the integration of horizontally transferred genetic elements, or CRISPR-Cas activity, or (ii) attributable to errors in the input DNA sequence. To distinguish between canonical and non-canonical archaeal tRNA genes, we recommend using a combination of automated pseudogene detection by tRNAscan-SE and the tRNAscan-SE isotype score, greatly reducing manual annotation efforts and leading to improved predictions of tRNA gene sets in Archaea. <strong>Repository contents</strong> <strong>01_workflow_tRNAscanSE_predictions_210archaea.html </strong>contains the workflow and graphical output for tRNA gene set predictions in 20 Thermococcaceae genomes and 210 archaeal genomes. Files 03 to 06 below are the files quoted in this workflow. <strong>02_workflow_tRNAscanSE_predictions_210archaea.Rmd </strong>contains the markdown file associated with 01_workflow_tRNAscanSE_predictions_210archaea.html above. <strong>03_thermo_trnas_GtRNAdb.txt</strong><strong> </strong>contains the predicted tRNA gene sets of 20 Thermococcaceae genomes as listed on GtRNAdb (Data Release 19 (June 2021)). <strong>04_Archaea_genome_list.txt </strong>contains the details of all 217 archaeal genomes listed on GtRNAdb (Data Release 19 (June 2021)). The seven genomes for which the NCBI genome sequences were no longer available are indicated by #### preceding the name. <strong>05_thermo_tRNAs_genome.txt</strong><strong> </strong>contains the predicted tRNA gene sets of 20 Thermococcaceae genomes as predicted by locally run tRNAscan-SE (version 2.0.6), with standard settings for Archaea (option -A). To display the output, options -H and --detail were added. We note that pseudogene detection is active under these conditions. <strong>06_Archaea_210_GtRNAdb_tRNAs.txt </strong>contains the predicted tRNA gene sets of the 210 archaeal genomes as listed on GtRNAdb (Data Release 19 (June 2021)). <strong>07_Archaea_210genomes_tRNAs.txt</strong> contains the predicted tRNA gene sets of the 210 archaeal genomes as predicted by locally run tRNAscan-SE (version 2.0.6), with standard settings for Archaea (option -A). To display the output, options -H and --detail were added. We note that pseudogene detection is active under these conditions. <strong>08_NCBI_genomes.zip</strong> contains the NCBI GenBank genome sequence files used in this study. These include the 20 Thermococcaceae genomes, the wider 210 archaeal genomes, and several others of interest. <strong>09_phylogeny.tar.zip</strong> contains the data used to draw a phylogenetic tree for the 20 Thermococcaceae organisms. The folder includes a file listing the details of all data in the folder (Readme.md), a workflow file (workflow_UndinMarkers_v2.md), and data folders. <strong>Notes</strong> The extended TIGRFAM database referred to in the phylogenetic tree construction process can be found at https://zenodo.org/record/3839790#.YjByaVzMI3g The perl script used during phylogenetic tree construction, catfasta2phyml.pl, is available in the GitHub repository https://github.com/nylander/catfasta2phyml tRNAscan-SE is a freely available resource available online (http://lowelab.ucsc.edu/tRNAscan-SE/) GtRNAdb is a publicly accessible resource available online (http://gtrnadb.ucsc.edu/) NCBI is a publicly accessible resource available online (https://www.ncbi.nlm.nih.gov/) rrnDB is a publicly accessible resource available online (https://rrndb.umms.med.umich.edu/) BLAST is a publicly accessible resource available online (https://blast.ncbi.nlm.nih.gov/Blast.cgi)