Execution Flags

These are the flags for the execution process of EnTAP. These will be used via the command line (denoted CMD), entap_run.params file (denoted R-ini), or entap_config.ini file (denoted E-ini). Since there are required and recommended flags, these will be repeated throughout the other categories where relevant. All commands may be used through the command line in addition to the recommended usage.

There are a few data types (qualifiers) to keep in mind used throughout these ini files. Anything that is specifies as a ‘multi’ type means that the parameter may be entered multiple times. If it is in an ini file, each parameters must be separated by a comma (‘,’). Example for multi-integer:”1,2,3” (entered without quotes)

Required Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
run	Execute the main EnTAP annotation functionality including, but not limited to, Similarity Searching and Gene Family analysis. Optional Frame Selection, Expression Analysis, and HGT analysis can be added with the appropriate flags.	cmd	flag	run
input / i	Path to the transcriptome file (either nucleotide or protein)	R-ini	string	/path/to/input/transcriptome.fa
database / i	Specify up to 5 DIAMOND indexed (.dmnd) databases to run similarity search against	R-ini	multi-string	/path/to/diamond/database.dmnd
run-ini	Point to the entap_run.params to specify run-specific parameters and paths.	cmd	string	/path/to/entap_run.params
entap-ini	Point to the entap_config.ini to specify database paths and software execution paths.	cmd	string	/path/to/entap_config.ini
entap-db-bin	Path to the entap_database.bin database. Either this or the EnTAP SQL database must be used. The binary database is the default and is recommended	E-ini	string	/path/to/entap_database.bin
diamond-exe	Specify the execution method for DIAMOND. This can be a path to the diamond file generated during installation, or simply the command if installed globally	E-ini	string	diamond
eggnog-map-data	Path to the directory containing the EggNOG SQL database eggnog.db that was downloaded during the Configuration stage. EnTAP will check for the eggnog.db database within this specified directory	E-ini	string	/path/to/eggnog_db_directory
eggnog-map-dmnd	Path to the EggNOG DIAMOND configured database eggnog_proteins.dmnd that was generated during the Configuration stage.	E-ini	string	/databases/eggnog_proteins.dmnd
eggnog-map-exe	Path to the EggNOG-mapper executable, or method of execution. If installed globally, this is simply emapper.py	E-ini	string	emapper.py

Recommended Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
run	Execute the main EnTAP annotation functionality including, but not limited to, Similarity Searching and Gene Family analysis. Optional Frame Selection, Expression Analysis, and HGT analysis can be added with the appropriate flags.	cmd	flag	run
input / i	Path to the transcriptome file (either nucleotide or protein)	R-ini	string	/path/to/input/transcriptome.fa
database / d	Specify up to 5 DIAMOND indexed (.dmnd) databases to run similarity search against	R-ini	multi-string	/path/to/diamond/database.dmnd
run-ini	Point to the entap_run.params to specify run-specific parameters and paths.	cmd	string	/path/to/entap_run.params
entap-ini	Point to the entap_config.ini to specify database paths and software execution paths.	cmd	string	/path/to/entap_config.ini
out-dir	Specify an output directory for all of the files generated by EnTAP	R-ini	string	/path/to/entap_output
frame_selection	Specify if you would like to perform frame selection/gene prediction on your input nucleotide transcriptome. This flag will be ignored if you input protein sequences. If this is set to false with nucleotide input, EnTAP will perform blastx functionality where appropriate.	R-ini	bool	true
entap-db-bin	Path to the entap_database.bin database. Either this or the EnTAP SQL database must be used. The binary database is the default and is recommended	E-ini	string	/path/to/entap_database.bin
entap-graph	Path to the entap_graphing.py EnTAP graphing file. If this is not specified, EnTAP graphics will not be generated	E-ini	string	/path/to/entap_graphing.py
diamond-exe	Specify the execution method for DIAMOND. This can be a path to the diamond file generated during installation, or simply the command if installed globally	E-ini	string	diamond
eggnog-map-data	Path to the directory containing the EggNOG SQL database eggnog.db that was downloaded during the Configuration stage. EnTAP will check for the eggnog.db database within this specified directory	E-ini	string	/path/to/eggnog_db_directory
eggnog-map-dmnd	Path to the EggNOG DIAMOND configured database eggnog_proteins.dmnd that was generated during the Configuration stage.	E-ini	string	/databases/eggnog_proteins.dmnd
eggnog-map-exe	Path to the EggNOG-mapper executable, or method of execution. If installed globally, this is simply emapper.py	E-ini	string	emapper.py

General EnTAP Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
run	Execute the main EnTAP annotation functionality including, but not limited to, Similarity Searching and Gene Family analysis. Optional Frame Selection, Expression Analysis, and HGT analysis can be added with the appropriate flags.	cmd	flag	run
input / i	Path to the transcriptome file (either nucleotide or protein)	R-ini	string	/path/to/input/transcriptome.
database / d	Specify up to 5 DIAMOND indexed (.dmnd) databases to run similarity search against	R-ini	multi-string	/path/to/diamond/database.dmnd
run-ini	Point to the entap_run.params to specify run-specific parameters and paths.	cmd	string	/path/to/entap_run.params
entap-ini	Point to the entap_config.ini to specify database paths and software execution paths.	cmd	string	/path/to/entap_config.ini
out-dir	Specify an output directory for all of the files generated by EnTAP	R-ini	string	/path/to/entap_output
overwrite	All previously ran files will be overwritten. Without this flag, EnTAP will recognize the results from the previous run and skip executing the portions that were already ran	R-ini	bool	true
resume	Set this flag to TRUE to if you would like EnTAP to continue execution if files from a previous run are found. If set to FALSE, EnTAP will stop execution after files from a previous run are detected. Note, ‘overwrite’ flag supersedes this - for example if ‘overwrite’ is set to TRUE and ‘resume’ set to FALSE with files from a previous run detected, they will be deleted and execution will continue.	R-ini	bool	false
graph	Specifying this will check whether or not your system has graphing functionality supported and then exit. System will need Python installed with the Matplotlib module.	cmd	bool	true
threads / t	Specify the number of threads for execution	R-ini	integer	5
no-trim	By default, EnTAP will trim your sequence headers to the first space to maintain compatbility across different software. Using this flag will instead retain the information of the header by removing all spaces. Example: >TRINITY_231.1 protein12312_43 inform >TRINITY_231.1protein12312_43inform	R-ini	bool	true
state	Precise control over Execution of EnTAP. This flag allows for certain parts to be ran while skipping others. More information can be seen in the Execution section.	R-ini	string	‘+’
version	Prints the current EnTAP version you are running	cmd	flag	version
no-check	EnTAP checks execution paths and inputs prior to annotating to prevent finding out your input was wrong until midway through a run. Using this flag will eliminate the check (not advised to use!)	R-ini	bool	true
output-format	Specify multiple output file formats for each stage of the pipeline TSV File (default) CSV File FASTA Protein File (default) FASTA Nucleotide File (default) Gene Enrichment Gene ID + Effective Length Gene Enrichment Gene ID + GO Terms Gene Ontology Terms (Sequence ID,GO Term ID, GO Term, Category, and Sequence Effective Length) TSV format (default)	R-ini	multi-integer	1,3,4,7
entap-db-sql	Path to the entap_database.db database. Either this or the EnTAP binary database must be used	E-ini	string	/path/to/entap_database.db
entap-db-bin	Path to the entap_database.bin database. Either this or the EnTAP SQL database must be used. The binary database is the default and is recommended	E-ini	string	/path/to/entap_database.bin
entap-graph	Path to the entap_graphing.py EnTAP graphing file. If this is not specified, EnTAP graphics will not be generated	E-ini	string	/path/to/entap_graphing.py

Expression Analysis Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
align / a	Path to the alignment file (either SAM or BAM format). Ignoring this flag will skip expression filtering. Be sure to look at the other Expression Analysis flags if using this.	R-ini	string	/path/to/alignment.bam
rsem-calculate-expression	Specify the path to the rsem-calculate-expression file generated during installation of RSEM	E-ini	string	/libs/RSEM-1.3.3/rsem-calculate-expression
rsem-sam-validator	Specify the path to the rsem-sam-validator file generated during installation of RSEM	E-ini	string	/libs/RSEM-1.3.3/rsem-sam-validator
rsem-prepare-reference	Specify the path to the rsem-prepare-reference file generated during installation of RSEM	E-ini	string	/libs/RSEM-1.3.3/rsem-prepare-reference
rsem-convert-sam-for-rsem	Specify the path to the rsem-convert-sam-for-rsem file generated during installation of RSEM	E-ini	string	/libs/RSEM-1.3.3/rsem-convert-sam-for-rsem
fpkm	Specify the FPKM (fragments per kilobase of exon per million mapped fragments) cutoff for Expression Filtering. All transcripts below this number will be filtered out and removed.	R-ini	float	0.5
single-end	Signify your reads are single-end for RSEM execution instead of paired-end (default)	R-ini	bool	true

Frame Selection Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
frame_selection	Specify if you would like to perform frame selection/gene prediction on your input nucleotide transcriptome. This flag will be ignored if you input protein sequences. If this is set to false with nucleotide input, EnTAP will perform blastx functionality where appropriate.	R-ini	bool	true

Frame Selection - TransDecoder Specific Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
transdecoder-m	Specify the minimum protein length for TransDecoder	R-ini	integer	100
transdecoder-no-refine-starts	Specify this flag if you would like to pipe the TransDecoder command ‘–no_refine_starts’ when it is executed	R-ini	bool	false
transdecoder-long-exe	Method to execute TransDecoder.LongOrfs. This may be the path to the executable, or simply, ‘TransDecoder.LongOrfs’ if installed globally	E-ini	string	TransDecoder.LongOrfs
transdecoder-predict-exe	Method to execute TransDecoder.Predict. This may be the path to the executable, or simply, ‘TransDecoder.Predict’ if installed globally	E-ini	string	TransDecoder.Predict

Similarity Search Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
database / d	Specify up to 5 DIAMOND indexed (.dmnd) databases to run similarity search against	R-ini	multi-string	/path/to/diamond/database.dmnd
data-type	Specify which EnTAP database you’d like to use for execution Binary Database (default) - This will be much quicker and is recommended SQL Database - Slower although will be more easily compatible with every system	R-ini	integer	0
contam / c	Specify contaminants to be used during Simlilarity Search best hit selection. Contaminants can be selected by species or through a specific taxon (insecta) from the NCBI Taxonomy Database. If your taxon is more than one word just replace the spaces with underscores (_). Alignments will be flagged as contaminants and will be lower scoring compared to other alignments.	R-ini	multi-string	insecta
taxon	This flag will allow for ‘taxonomic favoring’ of hits that are closer to your target species or lineage. Any lineage can be used as referenced by the NCBI Taxonomic database, such as genus, phylum, or species. Format must replace all spaces with underscores (‘_’)	R-ini	string	homo_sapiens
e	Specify E-value cutoff for Similarity Searching results (in scientific notation format).	R-ini	scientific	10E-5
tcoverage	Specify minimum target coverage for similarity searching	R-ini	float	50
qcoverage	Specify minimum query coverage for similarity searching	R-ini	float	50
uninformative	Comma-deliminated list of terms you would like to be deemed “uninformative”. Any alignments during Similarity Searching tagged as uninformative will be scored lower	R-ini	string	conserved, predicted, unnamed, hypothetical, putative, unidentified, uncharacterized, unknown, uncultured, uninformative
diamond-exe	Specify the execution method for DIAMOND. This can be a path to the diamond file generated during installation, or simply the command if installed globally	E-ini	string	diamond

Similarity Search - DIAMOND Specific Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
diamond-sensitivity	Specify the DIAMOND sensitivity used against input DIAMOND databases (Similarity Searching and HGT Analysis). Sensitivities are based off of DIAMOND documentation with a higher sensitivity generally taking longer but giving a higher alignment rate. Sensitivity options are fast, mid-sensitive, sensitive, more-sensitive, very-sensitive, ultra-sensitive.	R-ini	string	very-sensitive

Ontology Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
ontology_source	Specify which ontology source packages you would like to use. Multiple flags may be used to specify execution of multiple software packages. 0 - EggNOG (default) 1 - InterProScan	R-ini	multi-integer	0

Ontology - EggNOG Specific Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
eggnog-map-data	Path to the directory containing the EggNOG SQL database eggnog.db that was downloaded during the Configuration stage. EnTAP will check for the eggnog.db database within this specified directory	E-ini	string	/path/to/eggnog_db_directory
eggnog-map-dmnd	Path to the EggNOG DIAMOND configured database eggnog_proteins.dmnd that was generated during the Configuration stage.	E-ini	string	/databases/eggnog_proteins.dmnd
eggnog-map-exe	Path to the EggNOG-mapper executable, or method of execution. If installed globally, this is simply emapper.py	E-ini	string	emapper.py
eggnog-contaminant	Specify this to turn on/off EggNOG contaminant analysis (on by default). This leverages the taxon input from the contaminant Similarity Search command to determine if an EggNOG annotation should be flagged as a contaminant. EggNOG contaminant analysis can only be performed alongside Similarity Search contaminant analysis (not on its own) and will only be utilized if no alignments were found for a given transcript during Similarity Searching	R-ini	bool	true
eggnog-dbmem	Specify this to use the ‘–dbmem’ flag with EggNOG-mapper. This will load the entire eggnog.db sqlite3 database into memory which can require up to ~44GB of memory. However, this will significantly speed up EggNOG annotations	R-ini	bool	true
eggnog-sensitivity	Specify the DIAMOND sensitivity used during EggNOG mapper execution against the EggNOG database. Sensitivities are based off of DIAMOND documentation with a higher sensitivity generally taking longer but giving a higher alignment rate. Sensitivity options are fast, mid-sensitive, sensitive, more-sensitive, very-sensitive, ultra-sensitive.	R-ini	string	more-sensitive

Ontology - InterProScan Specific Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
interproscan-db	User this option if you would like to run InterProScan against specific databases. Multiple databases can be selected. tigrfam sfld prodom hamap pfam smart cdd prositeprofiles prositepatterns superfamily prints panther gene3d pirsf coils mobidblite	R-ini	multi-string	pfam
interproscan-exe	Specify the execution method for InterProScan. Commonly this can be the path to the interproscan.sh file	E-ini	string	interproscan.sh

Horizontal Gene Transfer Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
hgt-donor	Specify the DIAMOND configured (.dmnd extension) donor databases for Horizontal Gene Transfer analysis. Separate databases with a comma (‘,’)	R-ini	multi-string	path/to/donor/database1.dmnd,path/to/donor/database2.dmnd
hgt-recipient	Specify the DIAMOND configured (.dmnd extension) recipient databases for Horizontal Gene Transfer analysis. Separate databases with a comma (‘,’)	R-ini	multi-string	path/to/recipient/database1.dmnd,path/to/recipient/database2.dmnd
hgt-gff	Specify path to the GFF file for HGT analysis. The input GFF must satisfy the following: Protein identifiers must match between FASTA and GFF attribute fields Primary transcripts only (longest isoform for each gene) Feature type = ‘transcript’ or ‘mRNA’ Must be in relative order. This can be accomplished if it is ran through software such as agat_sp_keep_longest_isoform (https://agat.readthedocs.io/en/latest/tools/agat_sp_keep_longest_isoform.html)	R-ini	string	path/to/gff/file.gff
diamond-exe	Specify the execution method for DIAMOND. This can be a path to the diamond file generated during installation, or simply the command if installed globally. DIAMOND is leverage for HGT analysis	E-ini	string	diamond

EnTAP API Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
api-taxon	Check whether a species can be found within the specified EnTAP database. Returns a JSON formatted text indicating whether the species was found. Format must replace all spaces with underscores (‘_’) as follows: “- -taxon homo_sapiens” or “- -taxon primates”	cmd	string	homo_sapiens

NCBI API Flags

param	description	location (cmd/R-ini,E-ini)	qualifier	example
ncbi-api-key	Enter your personal NCBI API key, if available. This can be assigned to you through your NCBI account. Although not required, enabling EnTAP to use your API key will allow for much quicker accessions to the NCBI database. Your API key will only be used to access the NCBI database and only stored locally. If you are a contributor to EnTAP, DO NOT accidentally commit your API key to git!	R-ini	string	n/a
ncbi-api-enable	Allow EnTAP to access the NCBI database API to pull additional information for your data during Similarity Searching if searching against a NCBI database.	R-ini	bool	true