A pipeline for identification of olfactory receptor(OR) gene family

1. Quick start
2. Installation
3. Usage
4. Example workflows

1. Install

   git clone git@github.com:jianzuoyi/orfam.git
   cd orfam
   make
   

2. Run the example script

   cd example
   ./run_orfam
   

• Python 2.7 (https://www.python.org)
  • Biopython

System paths to orfam's component software are specified in the [orfam.config] (bin/orfam.config) file, which should reside in the same directory as the orfam executable (for alternate locations use the -K flag). Upon installation, orfam attempts to automatically generate this file, but manual editing may be necessary.

• Bioawk (https://github.com/lh3/bioawk)
• bedtools (https://github.com/arq5x/bedtools2)
• tblastn (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/)
• Exonerate (http://www.ebi.ac.uk/about/vertebrate-genomics/software/exonerate)
• MAFFT (http://mafft.cbrc.jp/alignment/software/)
• MEGACC (http://www.megasoftware.net/)

If any components already exist on the system, their paths should be manually specified by editing orfam.config.

orfam is a modular framework with three components:

• orfam align - Search against the target genome with known OR protein sequences as query and produce a alignment result file that can be processed with other orfam modules.
• orfam func - Identification of intact OR genes.
• orfam pseudo - Identification of truncated OR genes and pseudogenes.

orfam align Search against the target genome with known OR protein sequences as query and produce a alignment result file that can be processed with other orfam modules.

Internally, orfam align runs the following steps to produce a output file (BLAST format 6):

1. Discard the query sequences which length is less than 250
2. Alignment with TBLASTN

usage:	orfam align [options]

-q FILE olfactory receptor proteins (FASTA)
-s FILE subject genome (FASTA)
-o STR 	output file [.align]
-T DIR 	temp directory [./tmpXXXXXXXX]
 

-e FLOAT evalue for hits
-t INT 	threads [1]

-K FILE path to orfam.config file (default: same directory as orfam)
-v 	verbose
-h 	show this message

orfam align produces a single output file (BLAST format 6):

• outprefix.tblastn
  • The alignment result file. This file serve as input for orfam func

orfam func identifies intact OR genes from the target genome.

usage:	orfam func [options]

-R FILE reference file (fasta) (required)
-r FILE reference olfactory receptor (fasta) (required)
-B FILE BED file represents the regions of reference olfactory receptor (required)
-A FILE tblastn output (tabular) (required)
-O FILE olfactory receptor for outgroup (fasta) (required)
-S FILE MAO file, setting used to the construction of phylogenetic tree (generated by megaproto) (required)
-o STR 	output prefix [required]
-t INT  threads [1]
-T DIR 	temp directory [./tmpXXXXXXXX]
-k 		keep temporary files
-K FILE path to orfam.config file (default: same directory as orfam)
-v 		verbose
-h 		show this message

orfam func produces two output file:

• outprefix_best_hit.gff
  • This GFF file contains all OR candicate sequences which can be classified into three types: Intact OR genes, Truncated OR genes and OR pseudogenes.
• outprefix_intact.fa
  • This FASTA file contains all Intact OR gene sequences.

orfam pseudo identifies truncated OR genes or OR pseudogenes.

usage:	orfam pseudo [options]

-s FILE subject genome (fasta) (required)
-q FILE query olfactory receptor proteins (fasta) (required)
-b FILE best hits (gff) (required)
-i FILE intact olfactory receptor (fasta) (required)
-o STR 	output prefix
-T DIR 	temp directory [./tmpXXXXXXXX]
-k 	keep temporary files
-K FILE path to orfam.config file (default: same directory as orfam)
-v 	verbose
-h 	show this message"

orfam pseudo produces five output files:

• outprefix_truncated.gff
  • This GFF file contains truncated OR genes.
• outprefix_pseudo.gff
  • This GFF file contains OR pseudogenes.
• outprefix_pseudo_nonsense.fa
  • This FASTA file contains olfactory receptors with nonsense mutations.
• outprefix_pseudo_frameshift.fa
  • This FASTA file contains olfactory receptors with frame shift mutations.
• outprefix_pseudo_others.fa
  • This FASTA file contains olfactory receptors with other mutations.

1. Use orfam align to produce a alignment result file.

   orfam align \
   	-q data/ORs/ORs.fa \
   	-s data/mm10/mm10.fa \
   	-o mm10 \
   	-e 1e-10 \
   	-t 20 \
   	-T temp \
   	-v \
   	-k
   

2. Use orfam func to identify intact OR genes.

   orfam func \
   	-R data/mm10/mm10.fa \
   	-r data/ORs/O43749.fasta \
   	-B data/ORs/O43749.bed \
   	-O data/ORs/outgroup.fa \
   	-S bin/infer_NJ_protein.mao \
   	-A mm10.tblastn \
   	-o mm10 \
   	-t 20 \
   	-T temp \
   	-k \
   	-v
   

3. Use orfam pseudo to identify truncated OR genes and OR pseudogenes.

   orfam pseudo \
   	-s data/mm10/mm10.fa \
   	-q intact/mm10_intact.fa \
   	-b mm10_best_hit.gff \
   	-i mm10_intact.fa \
   	-o mm10 \
   	-T temp \
   	-k \
   	-v