Abstract
The mOTU profiler, or mOTUs for short, is a software tool that enables the profiling of microbial communities in terms of their taxonomic composition, relative abundance of metabolically active members, and diversity of strain populations. To this end, it maintains a database of single-copy phylogenetic marker gene sequences, which are used as a reference to which short read metagenomic and metatranscriptomic reads are mapped for the identification and quantification of microbial taxa. Here, we describe the most common use cases of the mOTU profiler in two basic protocols. Additional supporting protocols provide information on its installation and in-depth guidance on adjusting its settings for increasing or decreasing the stringency with which taxa are detected and quantified, as well as for customizing the output file format. Guidelines for understanding the profiling results are provided, along with additional information on unique features, methodological details, and the development history of the tool. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metagenomic and metatranscriptomic mOTU profiling Basic Protocol 2: Metagenomic SNV profiling Support Protocol 1: Installing mOTUs Support Protocol 2: Profiling pipeline-step by step Support Protocol 3: The mOTUs profiling routine using advanced parameters Support Protocol 4: Metagenomic SNV calling: advanced parameters.
Highlights
Microorganisms and the complex communities they form have an impact on the health status of humans, animals, and plants
The mOTUs approach is based on a sequence database of singlecopy phylogenetic marker genes (MGs) that are present in all organisms (Ciccarelli et al, 2006; Sorek et al, 2007)
Species-level clusters of MGs that were identified in both sequenced reference genomes and in metagenomes can be used as operational taxonomic units
Summary
Microorganisms and the complex communities they form have an impact on the health status of humans, animals, and plants. These profiles are subsequently used to produce between-sample distances for individual mOTUs, providing access to strain population diversity patterns for as many as 33,750 species To this end, mOTUs will first align metagenomic sequencing reads against its marker gene database and process the alignments using the metaSNV package (Costea et al, 2017). Metagenomic SNV profiling is a pipeline split into two parts: motus map_snv requires paired-end or single-end short read sequencing data (see Strategic Planning for more details) in FASTQ format The BAM alignment files produced by motus map_snv serve as input for the motus snv_call routine. The intermediate output files produced by mOTUs contain: alignments of reads against the mOTUs database in BAM format marker gene abundances in a two-column, tab-separated table mkdir support_protocol_2 cd support_protocol_2 wget https://zenodo.org/record/5012695/files/protocol_profile.zip unzip protocol_profile.zip cd protocol_profile. Precomputed results of all steps shown in this protocol can be found in the protocol_profile/sp3_precomputed folder
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