KSGP 3.1: improved taxonomic annotation of Archaea communities using LotuS2, the genome taxonomy database and RNAseq data.

Taxonomic annotation is a substantial challenge for Archaea metabarcoding. A limited number of reference sequences are available; a substantial fraction of phylogenetic diversity is not fully characterized; widely used databases do not reflect current archaeal taxonomy and contain mislabelled sequences. We address these gaps with a systematic and tractable approach based around the Genome Taxonomy Database (GTDB) combined with the eukaryote PR2 and MIDORI mitochondrial databases. After removing incongruent, chimeric and duplicate SSU sequences, this combination (GTDB+) provides a small improvement in annotation of a set of estuarine Archaea Operational Taxonomic Units (OTUs) compared to SILVA. We add to this a collection of near full length rRNA sequences and the prokaryote SSU sequences in SILVA, creating a new reference database, KSGP ( K arst, S ilva, G TDB, and PR2). The additional sequences are (re-)annotated using three different approaches. The most conservative, using lowest common ancestor, gives a further small improvement. Annotation using SINTAX increases Class and Order assignments by 2.7 and 4.2 times over SILVA, although this may include some "lumping" of un-named and named clades. Still further improvement can be made using similarity based clustering to group database sequences into putative taxa at all taxonomic levels, assigning 60% and 41% of Archaea OTUs to putative family and genus level taxa respectively. GTDB without cleaning and GreenGenes2 both perform poorly and cannot be recommended for use with Archaea. We make the GTDB+ and KSGP databases available at ksgp.earlham.ac.uk; integrate them into a metabarcoding pipeline, LotuS2 and outline their use to annotate Archaea OTUs and metatranscriptomic data.