CONSTAX (CONSensus TAXonomy) is a tool, written in Python 3, for improved taxonomic resolution of environmental fungal ITS sequences. Briefly, CONSTAX compares the taxonomic classifications obtained from RDP Classifier, UTAX or BLAST, and SINTAX and merges them into an improved consensus taxonomy using a 2 out of 3 rule (e.g. If an OTU is classified as taxon A by RDP and UTAX/BLAST and taxon B by SINTAX, taxon A will be used in the consensus taxonomy) and the classification p-value to break the ties (e.g. when 3 different classification are obtained for the same OTU). This tool also produces summary classification outputs that are useful for downstream analyses. In summary, our results demonstrate that independent taxonomy assignment tools classify unique members of the fungal community, and greater classification power (proportion of assigned operational taxonomic units at a given taxonomic rank) is realized by generating consensus taxonomy of available classifiers with CONSTAX.
CONSTAX v.2 improves upon v.1 with the following features:
conda install -c bioconda constaxThis uses conda installations, including vsearch instead of usearch. You can modify SINTAXPATH in pathfile.txt if you have a usearch installation.
gunzip usearch<version>.gz to unzip.chmod +x usearch<version> to make the file executable.apt install openjdk-8-jdk-headless
apt install antgit clone https://github.com/rdpstaff/RDPTools.git
cd RDPTools
git submodule init AlignmentTools ReadSeq classifier TaxonomyTree
git submodule update
sed -i 's/1.5/1.6/' AlignmentTools/nbproject/project.properties ReadSeq/nbproject/project.properties classifier/nbproject/project.properties
sed -i 's/basedir="."/basedir="." xmlns:unless="ant:unless"/' classifier/build.xml
sed -i 's/name="download-traindata" unless="offline"/name="download-traindata" unless="skip_td_download"/' classifier/build.xml
sed -i 's+move file="${dist.dir}/data.tgz"+move unless:set="skip_td_download" file="${dist.dir}/data.tgz"+' classifier/build.xml
cd classifier
ant jar -Dskip_td_download=true
cp dist/classifier.jar ../conda install -c bioconda blast
tar -xzvf ncbi-blast-<version>+-x64-<system>64.tar.gz (replace version and system).blastn and makeblastdb executables to your path. You can do this by moving them to your bin directory.git clone https://github.com/liberjul/CONSTAXv2.gitconstax.sh executable.
cd CONSTAXv2
chmod +x constax.sh
ln -s constax.sh constaxDependent on where your sequences originate, you will need to have an appropriate database with which to classify them.
For Fungi or all Eukaryotes, the UNITE database is preferred.
For Bacteria and Archaea, we recommend the SILVA database. The SILVA_XXX_SSURef_tax_silva.fasta.gz file can be gunzip-ped and used.
Note: SILVA taxonomy is not assigned by Linnean ranks (Kingdom, Phylum, etc.), so instead placeholder ranks 1-n are used. Also, the size of the SILVA database means that a server/cluster is required to train the classifier (128GB RAM for RDP). If you have a computer with 32GB of RAM, you may be able to train using the UNITE database. If you cannot train locally for UNITE, the RDP files can be downloaded from here. The genus_wordConditionalProbList.txt.gz file should be gunzip-ped after downloading.
We have included a script for filtering the databases, which can create a Bacteria-only database, for example. The -k or --keyword argument is a substring of the record header.
python fasta_select_by_keyword.py -i SILVA_138_SSURef_tax_silva.fasta \
-o SILVA_138_SSURef_tax_silva_bact.fasta -k " Bacteria;"constax --helpusage: constax [-h] [-c CONF] [-n NUM_THREADS] [-m MHITS] [-e EVALUE] [-p P_IDEN] [-d DB] [-f TRAINFILE] [-i INPUT] [-o OUTPUT] [-x TAX] [-t] [-b]
[--select_by_keyword SELECT_BY_KEYWORD] [-s] [--make_plot] [--check] [--mem MEM] [--sintax_path SINTAX_PATH] [--utax_path UTAX_PATH]
[--rdp_path RDP_PATH] [--constax_path CONSTAX_PATH] [--pathfile PATHFILE] [--isolates ISOLATES] [--isolates_query_coverage ISOLATES_QUERY_COVERAGE]
[--isolates_percent_identity ISOLATES_PERCENT_IDENTITY] [--high_level_db HIGH_LEVEL_DB] [--high_level_query_coverage HIGH_LEVEL_QUERY_COVERAGE]
[--high_level_percent_identity HIGH_LEVEL_PERCENT_IDENTITY] [-v]
optional arguments:
-h, --help show this help message and exit
-c CONF, --conf CONF Classification confidence threshold (default: 0.8)
-n NUM_THREADS, --num_threads NUM_THREADS
Number of threads to use for parallel computing steps (default: 1)
-m MHITS, --mhits MHITS
Maximum number of BLAST hits to use, for use with -b option (default: 10)
-e EVALUE, --evalue EVALUE
Maximum expect value of BLAST hits to use, for use with -b option (default: 1.0)
-p P_IDEN, --p_iden P_IDEN
Minimum proportion identity of BLAST hits to use, for use with -b option (default: 0.0)
-d DB, --db DB Database to train classifiers, in FASTA format (default: )
-f TRAINFILE, --trainfile TRAINFILE
Path to which training files will be written (default: ./training_files)
-i INPUT, --input INPUT
Input file in FASTA format containing sequence records to classify (default: otus.fasta)
-o OUTPUT, --output OUTPUT
Output directory for classifications (default: ./outputs)
-x TAX, --tax TAX Directory for taxonomy assignments (default: ./taxonomy_assignments)
-t, --train Complete training if specified (default: False)
-b, --blast Use BLAST instead of UTAX if specified (default: False)
--select_by_keyword SELECT_BY_KEYWORD
Takes a keyword argument and --input FASTA file to produce a filtered database with headers containing the keyword with name --output (default:
False)
-s, --conservative If specified, use conservative consensus rule (2 False = False winner) (default: False)
--consistent If specified, show if the consensus taxonomy is consistent with the real hierarchical taxonomy (default: False)
--make_plot If specified, run R script to make plot of classified taxa (default: False)
--check If specified, runs checks but stops before training or classifying (default: False)
--mem MEM Memory available to use for RDP, in MB. 32000MB recommended for UNITE, 128000MB for SILVA (default: 32000)
--sintax_path SINTAX_PATH
Path to USEARCH/VSEARCH executable for SINTAX classification (default: False)
--utax_path UTAX_PATH
Path to USEARCH executable for UTAX classification (default: False)
--rdp_path RDP_PATH Path to RDP classifier.jar file (default: False)
--constax_path CONSTAX_PATH
Path to CONSTAX scripts (default: False)
--pathfile PATHFILE File with paths to SINTAX, UTAX, RDP, and CONSTAX executables (default: pathfile.txt)
--isolates ISOLATES FASTA formatted file of isolates to use BLAST against (default: False)
--isolates_query_coverage ISOLATES_QUERY_COVERAGE
Threshold of sequence query coverage to report isolate matches (default: 75)
--isolates_percent_identity ISOLATES_PERCENT_IDENTITY
Threshold of aligned sequence percent identity to report isolate matches (default: 1)
--high_level_db HIGH_LEVEL_DB
FASTA database file of representative sequences for assignment of high level taxonomy (default: False)
--high_level_query_coverage HIGH_LEVEL_QUERY_COVERAGE
Threshold of sequence query coverage to report high-level taxonomy matches (default: 75)
--high_level_percent_identity HIGH_LEVEL_PERCENT_IDENTITY
Threshold of aligned sequence percent identity to report high-level taxonomy matches (default: 1)
-v, --version Display version and exit (default: False)If using a database for the first time, you will need to use the -t or --train flag to train the classifiers on the dataset.
In the directory with your OTU/zOTU/ASV/ESV FASTA file:
/path/to/CONSTAXv2/constax -i <input file> \
-o <output_directory> \
-f <training_files_directory> \
-x <taxonomy_assignments_directory> \
-d <database_file> \
--sintax_path <path/to/usearch> \
--rdp_path <path/to/RDPTools/classifier.jar> \
--constax_path <path/to/CONSTAXv2> \
-c 1.0 -b -tThe classification results are in the output directory. The file consensus_taxonomy.txt can be read in to R for microbiome analysis.
-c, --conf=0.8Classification confidence threshold, used by each classifier (0,1]. Increase for improved specificity, reduced sensitivity.
-n, --num_threads=1Number of threads to use for parallelization. Maximum classification speed at about 32 threads. Training only uses 1 thread.
-m, --mhits=10 Maximum number of BLAST hits to use, for use with -b option. When classifying with BLAST, this many hits are kept. Confidence for a given taxa is based on the proportion of these hits agree with that taxa. 5 works well for UNITE, 20 with SILVA (standard, not NR).
-e, --evalue=1Maximum expect value of BLAST hits to use, for use with -b option. When classifying with BLAST, only hits under this expect value threshold are used. Decreasing will increase specificity, but decrease sensitivity at high taxonomic ranks.
-p, --p_iden=0.8Minimum proportion identity of BLAST hits to use, for use with -b option. Minimum proportion of conserve bases to keep hit.
-d, --dbDatabase to train classifiers. UNITE and SILVA formats are supported. See Datasets.
-f, --trainfile=./training_filesPath to which training files will be written.
-i, --input=otus.fastaInput file in FASTA format containing sequence records to classify.
-o, --output=./outputsOutput directory for classifications.
-x, --tax=./taxonomy_assignmentsDirectory for taxonomy assignments.
-t, --trainComplete training if specified. Cannot run classification without training files present.
-b, --blastUse BLAST instead of UTAX if specified. If installed with conda, this in the option that will work by default. UTAX is available from USEARCH. BLAST classification generally performs better with faster training, similar classification speed, and greater accuracy.
--select_by_keywordTakes a keyword argument and --input FASTA file to produce a filtered database with headers containing the keyword with name --output. Helpful for limiting search database to Bacteria, Archaea, Fungi, or other group.
--conservativeIf specified, use conservative consensus rule (2 null = null winner). Works better for SILVA to use this option.
--make_plotIf specified, run R script to make plot of classified taxa.
--checkIf specified, runs checks but stops before training or classifying.
--memMemory available to use for RDP, in MB. 32000MB recommended for UNITE, 128000MB for SILVA.
--sintax_pathPath to USEARCH/VSEARCH executable for SINTAX classification. Can also be vsearch if already on path.
--utax_pathPath to USEARCH executable for UTAX classification.
--rdp_pathPath to RDP classifier.jar file, or classifier if on path from RDPTools conda install.
--constax_pathPath to CONSTAX scripts.
--pathfileFile with paths to SINTAX, UTAX, RDP, and CONSTAX executables.
--isolatesFASTA formatted file of isolates to use BLAST against.
--high_level_dbFASTA database file of representative sequences for assignment of high level taxonomy. The SILVA NR99 database for SSU/16S/18S sequences and the UNITE Eukayotes database.