After installation is complete, EnTAP must be configured for use. This stage will simply download and configure the necessary databases for full functionality. Configuration can be ran at anytime by a user or the system admin if they would like to update databases. It is in the installation section because it might be a bit easier to setup common databases beforehand that can be shared by multiple users.

I’ll break this up into two sections, Ini File and Usage. The Ini File section will just describe how to ensure EnTAP is reading from the correct paths, which can be easily changed in the entap_config.ini (more on that later!). It will also go over the directories included in the installation. The Usage sections will go over the basic usage during the Configuration stage of EnTAP and how to setup reference databases.

Ini File

From here on out, the “execution”, or “EnTAP”, directory will refer to the directory containing the EnTAP install (or binary file). Typically, this will just be at the root directory that was downloaded from the repository. All paths mentioned in this documentation will be relative to this directory.

Why is this important? EnTAP relies on several accompanying software packages and databases in order to run properly. Correct recognition of these paths is crucial and, as such, needed an entire section! The entap_config.ini is the answer to this pathing issue. It contains all of the necessary paths required for EnTAP (among many other commands) to run and can be configured as seen fit.

When a user is trying to execute EnTAP, they must specify the path to this ini file with the ini flag. By default, the ini file comes with some preset paths based on the installation directory. However, these should be checked for validity. If the ini file is not specified and there is not one in the working directory, an empty entap_config.ini will be generated with the following presets for execution paths. The ini file contains many other commands, but only the execution paths are required for configuration (specifically DIAMOND), so I will get to the others later on.

  • diamond-exe=/EnTAP/libs/diamond-0.9.9/bin/diamond
  • rsem-sam-validator=/EnTAP/libs/RSEM-1.3.0/rsem-sam-validator
  • rsem-calculate-expression=/EnTAP/libs/RSEM-1.3.3/rsem-calculate-expression
  • rsem-prepare-reference=/EnTAP/libs/RSEM-1.3.3/rsem-prepare-reference
  • rsem-convert-sam-for-rsem=/EnTAP/libs/RSEM-1.3.3/convert-sam-for-rsem
  • genemarkst-exe=/EnTAP/libs/gmst_linux_64/
  • transdecoder-long-exe=EnTAP/libs/TransDecoder-v5.3.0/TransDecoder.LongOrfs
  • transdecoder-predict-exe=EnTAP/libs/TransDecoder-v5.3.0/TransDecoder.Predict

If something is globally installed, such as “interproscan-exe” above, put how you’d normally run the software after the ‘=’. As an example, running DIAMOND through a global installation may simply be “diamond”. The Ini File line for DIAMOND will simply read:

  • diamond-exe=diamond


Be sure to at least set the DIAMOND path before moving on

Below is a sample ini file with all of the defaults that should be included in the EnTAP repository.

# [ini_instructions]
#When using this ini file keep the following in mind:
#	1. Do not edit the input keys to the left side of the '=' sign
#	2. Be sure to use the proper value type (either a string, list, or number)
#	3. Do not add unecessary spaces to your input
#	4. When inputting a list, only add a ',' between each entry
# [configuration]
#Specify which EnTAP database you would like to download/generate or use throughout execution. Only one is required.
#    0. Serialized Database (default)
#    1. SQLITE Database
#It is advised to use the default Serialized Database as this is fastest.
#type:list (integer)
# [entap]
#Path to the EnTAP binary database
#Path to the EnTAP SQL database (not needed if you are using the binary database)
#Path to the EnTAP graphing script (
# [expression_analysis]
#Specify the FPKM threshold with expression analysis. EnTAP will filter out transcripts below this value. (default: 0.5)
#Specify this flag if your BAM/SAM file was generated through single-end reads
#Note: this is only required in expression analysis
#Default: paired-end
#type:boolean (true/false)
# [expression_analysis-rsem]
#Execution method of RSEM Calculate Expression.
#Example: rsem-calculate-expression
#Execution method of RSEM SAM Validate.
#Example: rsem-sam-validator
#Execution method of RSEM Prep Reference.
#Example: rsem-prepare-reference
#Execution method of RSEM Convert SAM
#Example: convert-sam-for-rsem
# [frame_selection]
#Select this option if all of your sequences are complete proteins.
#At this point, this option will merely flag the sequences in your output file
#type:boolean (true/false)
#Specify the Frame Selection software you would like to use. Only one flag can be specified.
#Specify flags as follows:
#    1. GeneMarkS-T
#    2. Transdecoder (default)
# [frame_selection-genemarks-t]
#Method to execute GeneMarkST. This may be the path to the executable.
# [frame_selection-transdecoder]
#Method to execute TransDecoder.LongOrfs. This may be the path to the executable or simply TransDecoder.LongOrfs
#Method to execute TransDecoder.Predict. This may be the path to the executable or simply TransDecoder.Predict
#Transdecoder only. Specify the minimum protein length
#Specify this flag if you would like to pipe the TransDecoder command '--no_refine_starts' when it is executed. Default: False
#This will 'start refinement identifies potential start codons for 5' partial ORFs using a PWM, process on by default.' 
#type:boolean (true/false)
# [general]
#Specify the output format for the processed alignments.Multiple flags can be specified:
#    1. TSV Format (default)
#    2. CSV Format
#    3. FASTA Amino Acid (default)
#    4. FASTA Nucleotide (default)
#    5. Gene Enrichment Sequence ID vs. Effective Length TSV (default)
#    6. Gene Enrichment Sequence ID vs. GO Term TSV (default)
#type:list (integer)
# [ontology]
# Specify the ontology software you would like to use
#Note: it is possible to specify more than one! Just usemultiple --ontology flags
#Specify flags as follows:
#    0. EggNOG (default)
#    1. InterProScan 
#type:list (integer)
#Specify the Gene Ontology levels you would like printed
#A level of 0 means that every term will be printed, while a level of 1 or higher
#means that that level and anything higher than it will be printed
#It is possible to specify multiple flags as well
#Example/Defaults: --level 0 --level 1
#type:list (integer)
# [ontology-eggnog]
#Path to the EggNOG SQL database that was downloaded during the Configuration stage.
#Path to EggNOG DIAMOND configured database that was generated during the Configuration stage.
# [ontology-interproscan]
#Execution method of InterProScan. This is how InterProScan is generally ran on your system.  It could be as simple as '' depending on if it is globally installed.
#Select which databases you would like for InterProScan. Databases must be one of the following:
#    -tigrfam
#    -sfld
#    -prodom
#    -hamap
#    -pfam
#    -smart
#    -cdd
#    -prositeprofiles
#    -prositepatterns
#    -superfamily
#    -prints
#    -panther
#    -gene3d
#    -pirsf
#    -coils
#    -morbidblite
#Make sure the database is downloaded, EnTAP will not check!
#--protein tigrfam --protein pfam
#type:list (string)
# [similarity_search]
#Method to execute DIAMOND. This can be a path to the executable or simply 'diamond' if installed globally.
#Specify the type of species/taxon you are analyzing and would like alignments closer in taxonomic relevance to be favored (based on NCBI Taxonomic Database)
#Note: replace all spaces with underscores '_'
#Select the minimum query coverage to be allowed during similarity searching
#Select the minimum target coverage to be allowed during similarity searching
#Specify the contaminants you would like to flag for similarity searching. 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 (_).
#Note: since hits are based upon a multitide of factors, a contaminant might end up being the best hit for an alignment. In this scenario, EnTAP will flag the contaminant and it can be removed if you would like.
#type:list (string)
#Specify the E-Value that will be used as a cutoff during similarity searching.
#List of keywords that should be used to specify uninformativeness of hits during similarity searching. Generally something along the lines of 'hypothetical' or 'unknown' are used. Each term should be separated by a comma (,) This can be used if you would like to tag certain descriptions or would like to weigh certain alignments differently (see full documentation)
#Example (defaults):
#conserved, predicted, unknown, hypothetical, putative, unidentified, uncultured, uninformative, unnamed
#type:list (string)

Preparing Your Reference Databases

All source databases must be provided in FASTA format (protein) so that they can be indexed for use by DIAMOND. This can be completed independent of EnTAP with DIAMOND (- - makedb flag) or as part of the Configuration phase of EnTAP. This section will focus on downloading and preparing some of the more common FASTA source databases. If you already have DIAMOND databases configured, you can skip to Running Configuration. Even if you have a DIAMOND database already configured, Configuration must still be ran!

While any protein FASTA database can be used, it is recommended to use NCBI (Genbank) sourced databases such as RefSeq databases or NR. In addition, EnTAP can easily accept EBI databases such as UniProt/SwissProt.

EnTAP can recognize the species information from these header formats ONLY (NCBI and UniProt):

  • [homo sapiens]
  • OS=homo sapiens

If the individual FASTAs in a custom database you create do not adhere to one of these two formats, it will not be possible to weight taxonomic or contaminant status from them. You will need to change the headers to ensure they align.

The following FTP sites contain common reference databases that EnTAP can recognize:

Both Uniprot databases (SwissProt and TrEMBL) can be downloaded on a Unix system through the following command:


Or, for the TrEMBL database:


Alternatively, the NCBI databases must be downloaded in separate, smaller files, and concatenated together. As an example, the following commands will download and combine the NR database files:




tar -xvzf nr.*

cat nr.* > nr_database.fasta

It is generally recommended that a user select at least three databases with varying levels of curation. Unless the species is very non-model (i.e. does not have close relatives in databases such as RefSeq, it is not necessary to use the full NR database which is less curated). Once your FASTA databases are ready, move on to Running Configuration.

Running Configuration

Once you have your protein FASTA database ready, you can begin to run the Configuration stage. As mentioned before, Configuration will only need to be run once prior to Execution unless you would like to configure/update more databases.

To run configuration with a FASTA database to output directory path/to/output (default is current working directory), the command is as follows (additional databases can be specified if necessary with the -d flag and threads with the -t flag):

EnTAP --config -d path/to/database.fasta -d path/to/database2.fasta --out-dir path/to/output -t 8 --ini path/to/ini

If your databases are already indexed for DIAMOND, you can simply provide the paths in the .ini file and run the following command with 8 threads:

EnTAP --config -t 8 --ini path/to/ini


This is the only stage that requires connection to the Internet.

In both cases, the following databases will be downloaded and configured:


Either the EnTAP binary database (default) or the EnTAP SQL database is required for execution. Both are not needed.

The EnTAP Binary Database is downloaded from the FTP addresses below. By default, the binary version will be downloaded and used. Only one version is required. If you experience any trouble in downloading, you can simply specify the - - data-generate flag during Configuration to configure it locally (more on that later). The database for the newest version of EnTAP will always reside in the “latest” FTP directory. Keep in mind, if you are using an older version of EnTAP, you do not want to download from the “latest” directory. Instead, you will need to consider the version you are using. The FTP will always be updated only when a new database version is created. For example, if you see v0.8.2 and v0.8.5 on the FTP while you are using v0.8.3, you will download the database located in the v0.8.2 directory.


DIAMOND databases must be configured and eventually executed with the same version of DIAMOND.