KSGP 3.1

KSGP 3.1 integrates GTDB, PR2, and SILVA rRNA sequences to improve taxonomic assignment of Archaea metabarcoding data through a database and annotation pipeline.

Key Features:

• GTDB+ Database: Combines GTDB with PR2 and MIDORI mitochondrial databases after removing incongruent, chimeric, and duplicate SSU sequences.
• KSGP Database: Expands GTDB+ with near full-length rRNA sequences and prokaryote SSU sequences from SILVA, re-annotated using three approaches including lowest common ancestor, SINTAX, and similarity-based clustering.
• LotuS2 Integration: Embeds the databases into the LotuS2 metabarcoding pipeline for automated analysis.
• Multiple Annotation Approaches: Supports conservative lowest common ancestor annotation, SINTAX-based classification, and similarity-based clustering to group database sequences into putative taxa at all taxonomic levels.

Scientific Applications:

• Archaea Metabarcoding: Assigns taxonomy to Archaea Operational Taxonomic Units (OTUs) with increased Class and Order assignments by 2.7 and 4.2 times over SILVA using SINTAX.
• Putative Taxa Assignment: Assigns 60% and 41% of Archaea OTUs to putative family and genus level taxa respectively through similarity-based clustering.
• Metatranscriptomic Data Annotation: Annotates Archaea in RNAseq and metatranscriptomic datasets.
• Estuarine Microbiome Studies: Improves annotation of estuarine Archaea communities compared to standard reference databases.

Methodology:

KSGP 3.1 constructs reference databases by combining GTDB with eukaryote PR2 and MIDORI mitochondrial databases, followed by removal of incongruent, chimeric, and duplicate SSU sequences. Near full-length rRNA sequences and prokaryote SSU sequences from SILVA are added and re-annotated using lowest common ancestor, SINTAX, and similarity-based clustering approaches. The databases are integrated into the LotuS2 metabarcoding pipeline.