EukRibo: a manually curated eukaryotic 18S rDNA reference database

EukRibo is a manually curated database of reference small-subunit ribosomal RNA gene (18S rDNA) sequences of eukaryotes, specifically aimed at taxonomic annotation of high-throughput metabarcoding datasets. Unlike other reference databases of ribosomal genes, it is not meant to exhaustively capture all publicly available 18S rDNA sequences from the INSDC repositories, but to represent a subset of highly trustable sequences covering the whole known diversity of eukaryotes, with a focus on protists, manually verified taxonomic identifications, and relatively low genetic redundancy. EukRibo is part of a suite of public resources generated by the UniEuk project (www.unieuk.org), which are all designed to follow a common taxonomic framework for maximal interoperability. The high level of taxonomic accuracy of EukRibo, together with a newly designed, phylogenetically-informed annotation approach, allow high confidence in the taxonomic annotation of environmental metabarcodes, as well as identification of new eukaryotic diversity at various taxonomic levels using a connected components approach. * * * Accompanying preprint available at https://doi.org/10.1101/2022.11.03.515105. * * * <strong>EukRibo ReadMe file, versions 1 and 2</strong> Each EukRibo release consists of <strong>4 files</strong>:<br> - a <strong>tsv table </strong>containing the taxonomic and other information about the 18S rDNA sequences included in the release<br> - a <strong>fasta file </strong>containing the <strong>full sequences </strong>as retrieved from the INSDC repositories (NCBI, EMBL-EBI/ENA, DDBJ)<br> - a <strong>fasta file </strong>containing the <strong>variable region V4 </strong>extracted from all these sequences (based on the fragment amplified with the Tara-Oceans V4 primers)<br> - a <strong>fasta file </strong>containing the <strong>variable region V9 </strong>extracted from the subset of sequences where it is present (based on the fragment amplified with the Tara-Oceans V9 primers) The primary goal of EukRibo was to be used to annotate the EukBank meta-dataset of available V4 metabarcoding datasets, and therefore all sequences included in EukRibo contain the variable region V4.<br> Only a subset of these sequences (about 75%) also contain the variable region V9; this is because many 18S rDNA sequences in the INSDC repositories stop before the V9 fragment. Sequences with slightly incomplete V4 or V9 fragments were kept if phylogenetically useful - i.e. if they are the only available representatives of a certain taxonomic lineage.<br> <strong>V4 </strong>We allowed up to 50 missing positions in the relatively conserved area at the 5' end of the V4 fragment (for an average fragment length of about 380 bp); no sequence incomplete at the 3' end of the V4 fragment is included.<br> <strong>V9 </strong>We allowed up to 30 missing positions in the relatively conserved area at the 3' end of the V9 fragment (for an average length of about 135 bp); no sequence incomplete at the 5' end of the V9 fragment is included.<br> We allowed a higher proportion of missing positions for the V9 region because being more conservative would imply losing too many sequences, including entire taxonomic lineages. <strong>Version 1 of EukRibo</strong><br> This is the starting version of EukRibo that was used for the taxonomic annotation of the EukBank dataset, with taxonomy strings that were fixed as of October 2020.<br> - Contains 46,345 sequences with a sufficiently complete V4 region; 46,299 with the actual complete V4 region and 46 (about 0.1%) with missing positions at the 5' end.<br> - Of these, 34,438 also include a sufficiently complete V9 region; 23,226 with the actual complete V9 region and 11,206 (about 33%) with missing positions at the 3' end. <strong>Version 2 of EukRibo</strong><br> This is a version of EukRibo that was made taxonomically compatible with version 3 of the EukProt database (https://doi.org/10.1101/2020.06.30.180687), with taxonomic revisions as of July 2022 as well as additional information on the included selection of sequences that was not provided in the tsv file of version 1.<br> - Contains the exact same selection of sequences as in version 1, with the addition of genus <em>Meteora</em>, the last remaining known supergroup-level eukaryotic lineage for which an 18S rDNA was not previously available. (The <em>Meteora </em>sequence contains the full V4 fragment but does not include a sufficiently complete V9 fragment.)<br> - Only 34,432 sequences with a sufficiently complete V9 region are now retained because of 6 previously unrecognised chimeric sequences where the V9 fragment does not originate from the same organism as the V4 fragment. <strong>Files in EukRibo version 1</strong>:<br> 46345_EukRibo.tsv.gz<br> 46345_EukRibo_full_seqs.fas.gz<br> 46345_EukRibo_V4.fas.gz<br> 34438_EukRibo_V9.fas.gz The tsv file contains 6 columns:<br> <strong>gb_accession </strong>- INSDC accession number of the sequence<br> <strong>supergroup</strong>, <strong>taxogroup1</strong>, <strong>taxogroup2 </strong>- binning of the taxa into strictly monophyletic clades of evolutionary and/or ecological significance<br> <strong>UniEuk_taxonomy_string </strong>- full UniEuk-compatible taxonomic annotation of the sequence<br> - an unlimited number of levels is allowed (going down to strain for isolated organisms or to clone for environmental sequences)<br> - informal names are used for phylogenetically supported clades without formal name<br> <strong>V9 </strong>- presence ('Y') or absence ('N') of a sufficiently complete V9 fragment in the sequence <strong>Files in EukRibo version 2</strong>:<br> 46346_EukRibo-02.tsv.gz<br> 46346_EukRibo-02_full_seqs.fas.gz<br> 46346_EukRibo-02_V4.fas.gz<br> 34432_EukRibo-02_V9.fas.gz The tsv file now contains 12 columns:<br> <strong>gb_accession</strong>, <strong>supergroup</strong>, <strong>taxogroup1</strong>, <strong>taxogroup2</strong>, <strong>UniEuk_taxonomy_string</strong><br> - same columns as in version 1<br> <strong>alternative_strain_names </strong>(new) - provides alternative strain/isolate names when known to help cross-linking genetic data coming from the same organism<br> <strong>V4 </strong>(new) - indicates whether the V4 fragment is complete ('yes - complete') or missing positions at the 5' end ('yes - partial')<br> <strong>V9 </strong>(emended content) - now contains more precise information than in version 1 about whether it is complete ('yes - complete'), missing positions at the 3' end ('yes - partial'), or was excluded, and the 6 possible reasons why ('no - missing', 'no - too incomplete', 'no - chimera', 'no - bad quality', 'no - deletion in V9', 'no - Ns in V9')<br> <strong>EukProt_ID_same_strain </strong>(new) - accession of EukProt datasets from the same isolate<br> <strong>EukProt_ID_different_strain </strong>(new) - accession of EukProt datasets from a different isolate of the same species<br> <strong>columns_modified_since_previous_version </strong>(new) - lists all of the 6 pre-existing columns that have a modified content compared to version 1<br> <strong>remarks </strong>(new) - additional information such as presence of an intron in the V9 fragment, taxonomic identity of the two parts of chimeric sequences, or the presence of Ns or a deletion in the V4 or the V9 fragment (but insufficient to warrant exclusion)