orthofisher enables researchers to conduct high-throughput identification of orthologous genes using Hidden Markov Models (HMM). This tutorial covers the easy-to-implement workflow needed for using orthofisher.

1) Download the test data

For ease of use, this tutorial will rely on a small dataset, which can be downloaded using the following link:

Download test data: tutorial dataset

Next, unzip the downloaded directory and change directory to the newly downloaded directory.

$ cd path_to_unzipped_directory/orthofisher_tutorial

2) Run orthofisher

Two arguments are required when using orthofisher.

The first arument, -f/–fasta, points to a two column tab delimited file that specifies the location of fasta files that will be searched using HMMs. Typically, these are protein fasta files from the entire genome/transcriptome of an organism. Additionally, the second column of the file specifies the identifier for the organism. This will be used when representing sequences from a given proteome in a multi-fasta file. In this tutorial, this is file fasta_arg.txt.

The second argument, -m is a file that points to the location of HMMs that you wish to identify or fish out of a given proteome. In this tutorial, this is file hmms.txt.

$ orthofisher -m hmms.txt -f fasta_arg.txt

3) Examine output

In the current working directory, a subdirectory will be made titled orthofisher_output. Each subdirectory therein contains desirable output, which is briefly desired as:

  1. all_sequences: multi-fasta file sequences of every hit identified during sequence similarity search.

  2. hmmsearch_output: output files generated during hmmsearches

  3. scog: a directory of single copy orthologous HMMs identified in the various fasta files

Also, two text files are made with helpful information that summarizes all the searches:

1. long_summary.txt: Hits identified during sequence similarity search per fasta file per HMM. HMMs are considered single-copy, multi-copy, or absent in a given fasta file.

$ cat orthofisher_output/long_summary.txt
GCF_010094145.1_Didex1_protein.faa      718307-1.fa.mafft.hmm single-copy      1       XP_033451061.1
GCF_010094145.1_Didex1_protein.faa      1001705at2759.hmm     single-copy      1       XP_033445010.1
GCA_011032825.1_Masph1_protein.faa      718307-1.fa.mafft.hmm single-copy      1       KAF2869753.1
GCA_011032825.1_Masph1_protein.faa      1001705at2759.hmm     single-copy      1       KAF2868776.1
no_copy.faa     718307-1.fa.mafft.hmm   absent  0       NA
no_copy.faa     1001705at2759.hmm       absent  0       NA
multi_copy.faa  718307-1.fa.mafft.hmm   multi-copy      2     Massariosphaeria_phaeospora
multi_copy.faa  718307-1.fa.mafft.hmm   multi-copy      2     Didymella_exigua
multi_copy.faa  1001705at2759.hmm       absent  0       NA

col. 1: Query proteome fasta file.
col. 2: HMM file used during sequence similarity search.
col. 3: The sequence represented by the HMM is considered single_copy, multi-copy, or absent in a query proteome.
col. 4: Absolute copy number of hits from the sequence similarity search.
col. 5: The fasta entry identifier of the gene identified.

2. short_summary.txt: Summary of the absolute number and percentage of single-copy, multi-copy, or absent HMMs per fasta file.

$ cat orthofisher_output/short_summary.txt
file_name       single-copy     multi-copy      absent  per_single-copy        per_multi-copy  per_absent
GCF_010094145.1_Didex1_protein.faa      2       0       0     1.0      0.0     0.0
GCA_011032825.1_Masph1_protein.faa      2       0       0     1.0      0.0     0.0
no_copy.faa     0       0       2       0.0     0.0     1.0
multi_copy.faa  0       1       1       0.0     0.5     0.5

col. 1: Query proteome fasta file.
col. 2-4: Absolute number of sequences represented by HMMs that are present in single-copy, multi-copy, or absent.
col. 5-7: Percetange of sequences represented by HMMs that are present in single-copy, multi-copy, or absent.