InsectOR package

Authors: Snehal Dilip Karpe and Vikas Tiwari

This package helps to annotate genes coding for any specific family of proteins from a medium sized genome (currently tested on insect olfactory receptors / ORs).

The web version of the package can be accessed at http://caps.ncbs.res.in/insectOR/

More details about the package can be found at - http://caps.ncbs.res.in/cgi-bin/gws_ors/about.py?help=about&help_t=About%20insectOR

This package is under development.

Operating system requirements:

This package is made for use on unix systems and requires python2.7 and Perl.

Installation:

Please follow these instructions for installing and using this package -

1. Extract the contents of this folder and ensure that all the files in the folder have executable permissions.

2. Make sure that Perl (tested on v5.26.1) and Python (tested on 2.7) are installed. Make sure that the following perl modules are installed -

	Getopt::Long qw(GetOptions)	
	Tie::IxHash
	HTML::Template
	File::Basename
	File::Spec
	IO::File
	GD::Graph::bars
	GD::Graph::Data
	IO::Handle
	Cwd

3. Install following packages and keep their respective folders/binaries inside insectOR/tools or insectOR-main/toolsfolder-

   A) GeneWise ( e.g. wise2.4.1 - https://www.ebi.ac.uk/~birney/wise2/)

   • Copy the wiseX.X.X folder inside insectOR/tools
   • Change the $wiseconfigdir_path in the bin/scoreGenesOnScaffold.pl accordingly (If the GeneWise package wise2.4.1 is installed at the correct location as given above there is no need to edit $wiseconfigdir_path).

   B) GFFtools-GX (e.g. GFFtools-GX-master - https://github.com/vipints/GFFtools-GX)

   • copy the GFFtools-GX-master folder inside insectOR/tools

   C) TMHMM2 (e.g. tmhmm-2.0c - https://services.healthtech.dtu.dk/software.php)

   • mandatory if using option -tmhmm or -tmh1
   • change the $tmhmmpath in the bin/scoreGenesOnScaffold.pl accordingly (If the TMHMM2 package tmhmm-2.0c is installed at the correct location as given above there is no need to edit $tmhmmpath).
   • make sure to give executable permission

   D) HMMTOP2 (e.g. hmmtop_2.1 - http://www.enzim.hu/hmmtop/html/download.html)

   • mandatory if using option -hmmtop or -tmh2
   • change the $hmmtoppath in the bin/scoreGenesOnScaffold.pl accordingly (If the HMMTOP2 package hmmtop_2.1 is installed at the correct location as given above there is no need to edit $hmmtoppath).
   • make sure to give executable permission

   E) Phobius (http://phobius.sbc.su.se/data.html)

   • mandatory if using option -phobius or -tmh3
   • check of the $phobiupath is set correctly in bin/scoreGenesOnScaffold.pl
   • make sure to give executable permission

   F) HMMER (e.g. hmmer-3.1b2 - http://hmmer.org/download.html)

   • mandatory if using option -hmmsearch or -p
   • change the $hmmsearchpath in the bin/scoreGenesOnScaffold.pl accordingly (If the hmmerpackage hmmer-3.1b2 is installed at the correct location as given above there is no need to edit $hmmsearchpath).

   G) MEME (e.g. meme_4.10.2 - http://meme-suite.org/doc/download.html)

   • mandatory if using option -mast or -m
   • change the $mastpath and $mast_xslt_path in the bin/scoreGenesOnScaffold.pl accordingly (If the MEME suit meme_4.10.2 is installed at the correct location as given above there is no need to edit $hmmsearchpath).

4. Change the $basepath in bin/scoreGenesOnScaffold.pl as per the location of insectOR folder on your system.

5. Download and keep '7tm_6.hmm' inside insectOR/hmm folder (https://pfam.xfam.org/family/PF02949/hmm)

   • mandatory if using option -hmmsearch or -p

You are ready to use insectOR!

Notes

1. The exonerate is run using following parameters -

exonerate --model protein2genome --maxintron <max intron size dependent on the organism> <protein query sequence fasta file> <genome fasta file> --showtargetgff TRUE

2. The exonerate alignment contains 'Command line' and 'exonerate' in first line.

Example command

perl (path_to_insectOR/)insectOR/bin/scoreGenesOnScaffold.pl -i exonerate.txt -s seq.fasta -q query.fasta 
perl (path_to_insectOR/)insectOR/bin/scoreGenesOnScaffold.pl -i exonerate.txt -s seq.fasta -q query.fasta -g ncbi.gff -tmh1 -tmh2 -tmh3 -p -m -mf motif.txt 

Input

• Mandatory

-exonerate_file|-i - Exonerate alignment file 

(Sample exonerate file is provided in the test folder - exonerate.txt)

-seq|-s            - Genome sequence (FASTA) file which was used to generate above exonerate file 

(Sample genome sequence file is provided in the test folder - seq.fasta. Please note that this is a Habropoda laboriosa genome scaffold sequence NCBI Genbank accession LHQN01028732.1.)

-queryseq|-q       - OR/query sequence (FASTA) file which was used for generating above exonerate alignment 

(Sample OR sequence file is provided in the test folder - query.fasta)

• Optional

-gff_file|-g       - User provided gene annotations (GFF format) with which InsectOR output will be compared 

(Sample genome annotation file is provided in the test folder - ncbi.gff. Please note that this is a Habropoda laboriosa scaffold gene annotation file in GFF format for NCBI Genbank sequence LHQN01028732.1.)

-cutoff|-c         - Alignment clusters are identified based on this cutoff. 
                     This is the minimum number of alignments needed at a nucleotide position for its inclusion into an alignment cluster. (Default: 1)
-lengthCutoff|-l   - Predicted proteins can be classified as complete or partial based on this cutoff. (Default: 300 amino acids)
-hmmsearch|-p      - Perform HMMSEARCH for insect olfactory receptor signature (PFAM 7tm_6 family) as additional validation.
-tmhmm|-tmh1       - Search for Transmembrane Helices (TMH) using TMHMM2 as additional validation.
-hmmtop|-tmh2      - Search for Transmembrane Helices (TMH) using HMMTOP2 as additional validation.
-phobius|-tmh3     - Search for Transmembrane Helices (TMH) using Phobius as additional validation.

(If all three TMH predictiors are selected, consensus TMH prediction will be performed.)

-mast|-m           - Search for known OR motifs in the predicted proteins using MAST motif tool

-motif_file|-mf    - Users can provide their own motifs (PSPM format of MEME) for MAST motif search into the predicted OR proteins. 

(If MAST option is checked without any file mentioned with this parameter, then default AfOR motif file will be used)
(Sample motif file is provided in the test folder - motif.txt)

-helpmessage|-h    - Print this help message

Output

...predictedORs.summary.txt    - Summary file of insectOR results
...OR_final_insector_table.txt - Table providing detailed information on each gene/fragment predicted by insectOR
...final_proteins.pep          - Protein sequence file of insectOR predicted ORs/fragments in FASTA format
...ORs.starRemoved.pep         - Protein sequence file of insectOR predicted ORs/fragments without any pseudogenizing elements in FASTA format
...ORs.cds                     - Nucleotide sequence file of insectOR predicted OR CDSs in FASTA format
...final_gff_file.gff          - Detailed gene annotations by insectOR in GFF format
...ORs_sorted.bed12            - Detailed gene annotations by insectOR in BED format

If gene annotation file from another resource is provided, following files are also generated -

...gffcomparison                                 - Comparison of gene annotations from insectOR along with overlapping user provided gene annotations. 
...ORrelated_genesFromUserProvidedAnnotation.gff - A shorter version of user provided GFF file containing only overalapping annotations with those from this tool.
...gff.ORs_sorted.bed12                          - sorted BED formatted version of transcripts in the user-provided GFF file.

If HMMSEARCH option against 7tm_6 is selected, following files are also generated -

...ORs.starRemoved.pep.hmmsearchout              - HMMSEARCH output against 7tm_6 HMM file

If either of TMH prediction tools are used, following files are also generated -

...ORs.starRemoved.pep.tmhmmout if '-tmh1' is selected   - Output of TMHMM2 tool
...ORs.starRemoved.pep.hmmtopout if '-tmh2' is selected  - Output of HMMTOP2 tool
...ORs.starRemoved.pep.phobiusout if '-tmh3' is selected - Output of Phobius tool
...ORs.starRemoved.pep.consensusTMHpredout and ...ORs.starRemoved.pep.consensusTMHpred.html if all the three '-tmh1 -tmh2 -tmh3' are selected 
                                                         - Output of consensus TMH prediction tool.

If MAST motif search is selected, following files are also generated -

MAST.html - Motif search output file in HTML format
MAST.xml  - Motif search output file in HTML format

Suggestions and known bugs

• Please use simple names for your genome sequences. Issues might occur if longer sequence lengths and special characters including _ or - are part of these names.
• The sequence names should match across the Exonerate and the sequence files for genome and protein queries.
• InsectOR performs several parallel executions (upto 6 threads) of the validation packages near the end of the execution.
• Please be warned that this might need computational resources.
• Run the InsectOR in a folder with just input files and avoid having multiple runs of InsectOR within the same folder.

Please cite

• Karpe SD, Tiwari V, Ramanathan S (2021) InsectOR—Webserver for sensitive identification of insect olfactory receptor genes from non-model genomes. PLoS ONE 16(1): e0245324. https://doi.org/10.1371/journal.pone.0245324 (Updated from : Karpe SD, Tiwari V & Sowdhamini R. InsectOR - webserver for sensitive identification of insect olfactory receptor genes from non-model genomes. bioRxiv doi: https://doi.org/10.1101/2020.04.29.067470)
• Please also cite respective papers for tools used herewith - e.g. GeneWise, GFFtools, TMH prediction methods, 7tm_6 hmmsearch, MAST motif search tool, etc.

Contact

Please contact karpesnehal@gmail.com or vikast@ncbs.res.in to report any bugs.