Package: woltka
WoltkaWoltkais a versatile program for determining the structure and functional capacity of microbiomes. It mainly works with shotgun metagenomic data. It bridges first-pass sequence aligners with advanced analytical platforms (such as QIIME 2). It takes full advantage of, and is not limited by, theWoLreference database. Its scope and highlights are:Woltka ships with aQIIME 2 plugin.See here for instructions.ContentsOverviewInstallationExample usageTutorialsWorking with WoL,The OGU analysis,Sequence alignmentMain workflowInput files,Output files,Classification systems,Classification methods,"Coord-match" functional profiling,StratificationTable utilitiesCollapse,Coverage,Normalize,Filter,MergeFor users ofQIIME 2,Qiita,Bowtie2,SHOGUN,RefSeq,GTDB,MetaCyc,KEGGReferencesCommand-line interface,Test datasets,Computational efficiencyFAQsCitationContactOverviewWhere does Woltka fit in a pipelineWoltka is aclassifier. It fits in between sequence alignment and microbiome analyses.What does Woltka doWoltka processesalignments-- the mappings of microbiome sequencing data against reference sequences (such as genomes or genes), andinfers the best placementof the queries in a hierarchicalclassification system. One query could have simultaneous matches in multiple references. Woltka finds the most suitable classification unit(s) to describe the query accordingly the criteria specified by the user. Woltka generatesprofiles(feature tables) -- the abundances of classification units which describe the structure or function of microbial communities.What else does Woltka doWoltka provides several utilities for handling feature tables, includingnormalizingdata,collapsinga table to higher-level features, calculating feature groupcoverage,filteringfeatures based on per-sample abundance, andmergingtables.What does Woltka not doWoltka does NOTalignsequences. You need to align your sequencing data (FastQ, etc.) against a reference database (we recommendWoL) using an aligner of your choice (e.g.,Bowtie2). The resulting alignment files can be fed into Woltka.Woltka does NOTanalyzeprofiles. We recommend usingQIIME 2for robust downstream analyses of the profiles to decode the relationships among microbial communities and with their environments.Flowchart of Woltka's main classification workflow:InstallationRequirement:Python3.6 or above.pipinstallwoltkaSee more details aboutinstallation.Example usageWoltka provides several small test datasets underwoltka/tests/data. To access them,downloadthis GitHub repo, unzip, and navigate to this directory.One can execute the following commands to make sure that Woltka functions correctly, and to get an impression of the basic usage of Woltka.(Note: a more complete list of commands is providedhere. Alternatively, you can skip this test dataset and check out theinstructionfor working with WoL.)1. OGU (operational genomic unit) table generation (details):woltkaclassify-ialign/bowtie2-oogu.biomThe input path,align/bowtie2, is a directory containing five Bowtie2 alignment files (S01.sam.xz,S02.sam.xz,...S05.sam.xz) (SAM format, xzipped), each representing the mapping of metagenomic sequencing reads per sample against a reference genome database (here areguidlinesfor performing alignment).The output file,table.biom, is a feature table in BIOM format, which can then be analyzed using various bioformatics programs such asQIIME 2.2. Taxonomic profiling at the ranks of phylum, genus and species (details):woltkaclassify\--inputalign/bowtie2\--maptaxonomy/taxid.map\--nodestaxonomy/nodes.dmp\--namestaxonomy/names.dmp\--rankphylum,genus,species\--outputoutput_dirThe mapping file (taxid.map) translates genome IDs to taxonomy IDs, which then allow Woltka to classify query sequences based on the NCBI taxonomy (nodes.dmpandnames.dmp).The output directory (output_dir) will contain three feature tables:phylum.biom,genus.biomandspecies.biom, each representing a taxonomic profile at one of the three ranks.3. Functional profiling by UniRef entries, then by GO molecular processes (details):woltkaclassify\--inputalign/bowtie2\--coordsfunction/coords.txt.xz\--mapfunction/uniref/uniref.map.xz\--mapfunction/go/process.tsv.xz\--rankuniref,process\--outputoutput_dirHere, the input files are stillread-to-genomealignments, rather than read-to-gene ones. Woltka matches reads with genes based on their coordinates on genomes using an efficient algorithm ("coord-match"). The gene coordinates are given by the database filecoords.txt(seedetails). The read coordinates are extracted from the alignment files. This ensures consistency between structural and functional analyses.Subsequently, Woltka is able to assign query sequences to functional units, as defined in mapping files (uniref.mapandprocess.tsv). As you can see, compressed files are supported and auto-detected.Similarly, the output files are two functional profiles:uniref.biomandprocess.biom.4. Combined taxonomic/functional profiling by GO molecular processes of individual genera of organisms (details).Two steps.First, perform taxonomic classification. The--outmapparameter writes a read-to-genus mapping file per sample to the directorygenus_map/. The--name-as-idflag replaces NCBI TaxIDs with real taxon names in the output.woltkaclassify\--inputalign/bowtie2\--maptaxonomy/taxid.map\--nodestaxonomy/nodes.dmp\--namestaxonomy/names.dmp\--name-as-id\--rankgenus\--outputgenus.biom\--outmapgenus_mapSecond, perform functional classification. The--stratifyparameter imports the genus mappings from the last analysis, and groups functional units (GO processes) by the genus of the source genome.woltkaclassify\--inputalign/bowtie2\--stratifygenus_map\--coordsfunction/coords.txt.xz\--mapfunction/uniref/uniref.map.xz\--mapfunction/go/process.tsv.xz\--rankprocess\--outputgenus_by_process.biomIn the output profile (see below), each feature is a combination of taxonomy and function. This "stratified" profile lets the researcher explore the functional capacities of individual microbial components.Feature IDS01S02S03S04S05Aeromonas|GO:0000917420307Aeromonas|GO:0005975012520Bacteroides|GO:00062601050000Bacteroides|GO:0006281106203Lactobacillus|GO:0045454200343Lactobacillus|GO:005508500700...CitationThe first paper describing Woltka was published at:Zhu Q, Huang S, Gonzalez A, McGrath I, McDonald M, Haiminen N, Armstrong G, et al.Phylogeny-aware analysis of metagenome community ecology based on matched reference genomes while bypassing taxonomy.mSystems. 2022. e00167-22.Note: This paper focuses on theOGU analysis. Although it does not discuss other functions of Woltka, it is so far the only citable paper if you use Woltka in your studies.ContactPlease forward any questions to the project leader:Dr. Qiyun Zhu(qiyunzhu@gmail.com).