PANAS v.1 is a pipeline built for Linux.
Various external programs are required to be in the global directory and accesible from any working directory.
- PAML, available at http://abacus.gene.ucl.ac.uk/software/paml.html (version 4.9j is used). Click to download.
- ParaAT, available at https://github.com/wonaya/ParaAT (version 2.0 is used). Click to download.
ParaAT requires having at least one of the following sequence aligners clustalw2, t_coffee, mafft or muscle. Some of them can be installed from Linux command line. Example:
-bash-4.4$ sudo apt-get install mafft
To make the program accessible from any working directory, add it to your PATH variable. In bash you can add ~/.bash_profile using the following commands:
-bash-4.4$ nano ~/.bash_profile
Type and save the following lines:
export PATH=$PATH:~/your/path/directory/paml4.9j/src/
export PATH=$PATH:~/your/path/directory/ParaAT-master/
Then run on the command line:
-bash-4.4$ source ~/.bash_profile
Import note: PAML may have compile time problems if you have gcc 11.2.0. To fix the problem, you can install the gcc 9.4.0 version which can be installed with the following command:
-sudo apt-get install -y gcc-9
Then, modify the src/Makefile text file by replacing "cc" with "gcc-9" on line 2. With this change PAML should be correctly installed.
-pDirectory containing the protein file(s) in Fasta format. If you ran OMA to get ortholog information, the "DB" file can be used, only with the Fasta. Required parameter.-nDirectory containing the nucleotide file(s) in Fasta format. Required parameter.-oDirectory containing information on orthologous groups. If you ran OMA, you can use the "PairwiseOrthologs" file. However, you can use tab-delimited text files in which each row represents an orthologous group with two columns. See example in the Input data section. Required parameter.-zProcess number used during alignment. It is used by the ParaAT program. Required parameter.-aAligner used. Depending on the alignment program you have installed, you can choose one of the following: "clustalw2", "t_coffee", "mafft" or "muscle"). Default: mafft. Optional parameter.-gGenetic Code used. Default value: 1 (The Standard Code). For more information see the documentation of ParaAT. Optional parameter.-tFormat type of the file(s) containing orthologous group information. You can choose one of the following: "OMA" or "other". If the data of the orthologous groups correspond to the PairwiseOrthologs format of OMA, choose "OMA". Required parameter.-sYou can choose between the parameters: "single-copy" or "multiple-copy". Allows you to choose wheter to perform the analysis using only the information from orthologous genes with a 1:1 ratio ("single-copy"), or orthologous groups of multiple copies, such as 1:many, many:1, and many:many ("multiple-copy"). Useful parameter if your information comes from OMA. If your information is not in the OMA "PairwiseOrthologs" format, you can choose either. Required parameter.-hHelp
Import note: Each gene must have the same code in its nucleotide and amino acid sequence. However, the code from one gene should not be repeated in another gene. Don't use "@" in sequence codes.
The pipeline requires four files. See Examples.
- Directory with file(s) containing multiple amino acid sequences.
- Directory with file(s) containing multiple nucleotide sequences.
- Control files that specify the models and options for the analysis. No need to modify anything. Modifying some parameters in the control file can an error.
- Directory with file(s) containing orthologous groups.
- Example of OMA "PairwiseOrthologs" format, for the option "-t OMA":
-bash-4.4$ head -n 10 genes_speciesA-genes_speciesB.txt
# Format: Protein 1<tab>Protein 2<tab>Protein ID1<tab>ProteinID2<tab>Orthology type<tab>OMA group (if any)
# Every pair is listed only once, and in no particular order.
# The map between sequence number and ID are given
# in the file "Map-SeqNum-ID.map
1 1 atp6_Echinococcus_multilocularis_AB018440 atp6_Eudiplozoon_nipponicum_MW704020 1:1 11
2 2 cox1_Echinococcus_multilocularis_AB018440 cox1_Eudiplozoon_nipponicum_MW704020 1:1 1
3 3 cox2_Echinococcus_multilocularis_AB018440 cox2_Eudiplozoon_nipponicum_MW704020 1:1 1
4 4 cox3_Echinococcus_multilocularis_AB018440 cox3_Eudiplozoon_nipponicum_MW704020 1:1 8
5 5 cytb_Echinococcus_multilocularis_AB018440 cytb_Eudiplozoon_nipponicum_MW704020 1:1 2
6 6 nad1_Echinococcus_multilocularis_AB018440 nad1_Eudiplozoon_nipponicum_MW704020 1:1 5
- Example of another format, for the option "-t other":
-bash-4.4$ head -n 6 genes_speciesA-genes_speciesB.txt
atp6_Echinococcus_multilocularis_AB018440 atp6_Eudiplozoon_nipponicum_MW704020
cox1_Echinococcus_multilocularis_AB018440 cox1_Eudiplozoon_nipponicum_MW704020
cox2_Echinococcus_multilocularis_AB018440 cox2_Eudiplozoon_nipponicum_MW704020
cox3_Echinococcus_multilocularis_AB018440 cox3_Eudiplozoon_nipponicum_MW704020
cytb_Echinococcus_multilocularis_AB018440 cytb_Eudiplozoon_nipponicum_MW704020
nad1_Echinococcus_multilocularis_AB018440 nad1_Eudiplozoon_nipponicum_MW704020
- Directory
Your directory must contain the following files before running.
-bash-4.4$ tree
.
├── protein_directory
│ ├── speciesA.fa
│ ├── speciesB.fa
│ └── speciesC.fa
├── nucleotide_directory
│ ├── speciesA.fa
│ ├── speciesB.fa
│ └── speciesC.fa
├── codeml.ctl
└── Orthologs
├── genes_speciesA-genes_speciesB.txt
├── genes_speciesA-genes_speciesC.txt
└── genes_speciesB-genes_speciesC.txt
or
-bash-4.4$ tree
.
├── protein_directory
│ └── speciesABC.fa
├── nucleotide_directory
│ └── speciesABC.fa
├── codeml.ctl
└── Orthologs
├── genes_speciesA-genes_speciesB.txt
├── genes_speciesA-genes_speciesC.txt
└── genes_speciesB-genes_speciesC.txt
- Running PANAS
-bash-4.4$ ~/path/PANAS.sh -p protein_directory/ -n nucleotide_directory/ -o Orthologs/ -z 4 -a mafft -g 1 -t OMA -s single-copy
- Output
If you choose the "-s single-copy" option, the key output files include:- single-copy directory.
- All_results_single-copy file.
If you choose the "-s multiple-copy" option, the key output files include: - multiple-copy directory.
- All_results_multiple-copy file.
The single-copy or multiple-copy directory has information for each pairwise species comparison. For example:
If the analysis performed pairwise comparison of genes from three species (genes_speciesA vs genes_speciesB; genes_speciesB vs genes_speciesC; genes_speciesA vs genes_speciesC), the directory would contain the following subdirectories "genes_speciesA-genes_speciesB", "genes_speciesB-genes_speciesC" and "genes_speciesA-genes_speciesC".
After running PANAS.sh, your directory should look like this:
-bash-4.4$ tree
.
├── all.cds
├── all.pep
├── codeml.ctl
├── nucleotide_directory
│ └── speciesABC.fa
├── Orthologs
│ ├── genes_speciesA-genes_speciesB.txt
│ ├── genes_speciesA-genes_speciesC.txt
│ └── genes_speciesB-genes_speciesC.txt
├── proc
├── protein_directory
│ └── speciesABC.fa
├── single-copy
│ ├── genes_speciesA-genes_speciesB.txt.carpeta
│ │ ├── codeml.ctl
│ │ ├── genes_speciesA-genes_speciesB.txt.homologos
│ │ ├── guia.ctl
│ │ ├── output
│ │ │ ├── nad4_Echinococcus_multilocularis_AB018440-nad4_Eudiplozoon_nipponicum_MW704020.cds_aln.paml
│ │ │ ├── nad4_Echinococcus_multilocularis_AB018440-nad4_Eudiplozoon_nipponicum_MW704020.cds_aln.paml.mlc
│ │ │ ├── nad4L_Echinococcus_multilocularis_AB018440-nad4L_Eudiplozoon_nipponicum_MW704020.cds_aln.paml
│ │ │ ├── nad4L_Echinococcus_multilocularis_AB018440-nad4L_Eudiplozoon_nipponicum_MW704020.cds_aln.paml.mlc
│ │ │ ├── nad5_Echinococcus_multilocularis_AB018440-nad5_Eudiplozoon_nipponicum_MW704020.cds_aln.paml
│ │ │ ├── nad5_Echinococcus_multilocularis_AB018440-nad5_Eudiplozoon_nipponicum_MW704020.cds_aln.paml.mlc
│ │ │ ├── nad6_Echinococcus_multilocularis_AB018440-nad6_Eudiplozoon_nipponicum_MW704020.cds_aln.paml
│ │ │ └── nad6_Echinococcus_multilocularis_AB018440-nad6_Eudiplozoon_nipponicum_MW704020.cds_aln.paml.mlc
│ │ ├── Results.genes_speciesA-genes_speciesB.txt.vic
│ │ └── Results.genes_speciesA-genes_speciesB.txt.vic2
│ ├── genes_speciesA-genes_speciesC.txt.carpeta
│ │ ├── codeml.ctl
│ │ ├── genes_speciesA-genes_speciesC.txt.homologos
│ │ ├── guia.ctl
│ │ ├── output
│ │ │ ├── nad4_Echinococcus_multilocularis_AB018440-nad4_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ │ ├── nad4_Echinococcus_multilocularis_AB018440-nad4_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ │ ├── nad4L_Echinococcus_multilocularis_AB018440-nad4L_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ │ ├── nad4L_Echinococcus_multilocularis_AB018440-nad4L_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ │ ├── nad5_Echinococcus_multilocularis_AB018440-nad5_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ │ ├── nad5_Echinococcus_multilocularis_AB018440-nad5_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ │ ├── nad6_Echinococcus_multilocularis_AB018440-nad6_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ │ └── nad6_Echinococcus_multilocularis_AB018440-nad6_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ ├── Results.genes_speciesA-genes_speciesC.txt.vic
│ │ └── Results.genes_speciesA-genes_speciesC.txt.vic2
│ └── genes_speciesB-genes_speciesC.txt.carpeta
│ ├── codeml.ctl
│ ├── genes_speciesB-genes_speciesC.txt.homologos
│ ├── guia.ctl
│ ├── output
│ │ ├── nad4_Eudiplozoon_nipponicum_MW704020-nad4_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ ├── nad4_Eudiplozoon_nipponicum_MW704020-nad4_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ ├── nad4L_Eudiplozoon_nipponicum_MW704020-nad4L_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ ├── nad4L_Eudiplozoon_nipponicum_MW704020-nad4L_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ ├── nad5_Eudiplozoon_nipponicum_MW704020-nad5_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ ├── nad5_Eudiplozoon_nipponicum_MW704020-nad5_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ │ ├── nad6_Eudiplozoon_nipponicum_MW704020-nad6_Fasciola_hepatica_AF216697.cds_aln.paml
│ │ └── nad6_Eudiplozoon_nipponicum_MW704020-nad6_Fasciola_hepatica_AF216697.cds_aln.paml.mlc
│ ├── Results.genes_speciesB-genes_speciesC.txt.vic
│ └── Results.genes_speciesB-genes_speciesC.txt.vic2
└── All_results_single-copy
Please cite the dependencies used:
PAML: Yang, Z. (2007). PAML 4: Phylogenetic Analysis by Maximum Likelihood. Molecular Biology and Evolution, 24(8), 1586-1591.
ParaAT: Zhang, Z., Xiao, J., Wu, J., Zhang, H., Liu, G., Wang, X., & Dai, L. (2012). ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochemical and Biophysical Research Communications, 419, 779-781.
Please also cite the dependencies if used:
MAFFT: Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30, 772-780.
MUSCLE: Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792-1797.
T-Coffee: Notredame, C., Higgins, D. G., & Heringa, J. (2000). T-Coffee: A novel method for fast and accurate multiple sequence alignment. Journal of Molecular Biology, 302, 205-217.
Clustal W: Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948.
The example files are located in Example_pipeline.zip
This example have information about orthologous group obtained from OMA software.
To run execute
-bash-4.4$ ~/path/PANAS.sh -p protein_directory/ -n nucleotide_directory/ -o Orthologs/ -z 4 -a mafft -g 1 -t OMA -s single-copy
This example have information about orthologous group obtained from a tab-delimited text file generated manually.
To run execute
-bash-4.4$ ~/path/PANAS.sh -p protein_directory/ -n nucleotide_directory/ -o Orthologs/ -z 4 -a mafft -g 1 -t other -s single-copy