Express Phylogenetic Inference based on Multiple Markers
EPhIMM is a shell script for express phylogenetic analysis of prokaryotic genomes.
- hmmer http://hmmer.org/ for marker inference using profile HMM
- NCBI BLAST+ https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ for marker inference using FASTA sequences of marker genes
- esl-sfetch from Easel for https://github.com/EddyRivasLab/easel for aequence extraction
- mafft https://mafft.cbrc.jp/alignment/software/ for multiple sequence alignment
- fasttree http://www.microbesonline.org/fasttree/ for tree inference
- prodigal https://github.com/hyattpd/Prodigal for protein prediction
- Java 8 or newer (11) for autoedit of alignment (typically installed in Linux ditributions)
You may install them with conda:
conda install mafft fasttree easel hmmer prodigal blast
Extract HMM profiles for marker genes from markers.tar.gz using tar xzf markers.tar.gz.
Run EPhIMM.sh in folder containing files with protein sequences. E.g. if You downloaded EPhiMM to /home/user/software/EPhiMM/ and data located in /home/user/data/mygenomes:
user@pc:~/ cd /home/user/data/mygenomes
user@pc:/home/user/data/mygenomes$ /home/user/software/EPhiMM/EPhiMM.sh
Default output folder is output-DATE, where DATE is current date. You can provide different output folder using flag -o, e.g. EPhiMM.sh -o MyOutput.
If You work with nucleotide genomic sequences use flag -p to predict CDS with Prodigal.
After marker genes search. fetching and alignment You will be asked for manual or automatic alignment editing. In case of automatic columns containing more than 50% (You may modify the value with -g option) gaps will be removed from the alignment using BioKotlin (compilled binary included in distribution, source code is available on GitHub: https://github.com/laxeye/BioKotlin). In case of manual editing open file all.aligned.faa in the output folder, make changes and save it. You will be prompted for relative path to edited file, e.g. if You saved file as edited.version.faa in results folder type results/edited.version.faa.
You can check genomes.stats.txt and marker.stats.txt for missing and duplicated genes. Resulting alignment and tree will be stored in output folder.
-o|--output - name of folder to store results
-p|--predict-proteins - predict proteins from genomic sequences
-a|--auto - perform autoedit of alignment (default: off)
-g|--max-gap-fraction <0-100> - remove columns having more than N% gaps from the alignment (implies --auto)
-m|--markers - folder with hmm profiles of target genes (by default EPhIMM uses 41 universal prokaryotic genes)
-s|--sequences - folder with FASTA sequences of marker genes. One sequence per file.
-e|--evalue - e-value for HMMER/BLAST
-t|--threads - number of threads for HMMER
-h|--help - show help message
Marker genes should be provided as HMM profiles.
Initial set containing 41 single copy marker genes was taken from CheckM software (Parks et al., 2015), 2 markers from the original set (TIGR00344 and TIGR00422) were excluded due to multiple hits.
If You like to use Your own set of markers put .hmm files in a folder, e.g. /home/user/MyMarkersHMM, and run the tool providing path to the markers with option -m:
EPhIMM.sh -m /home/user/MyMarkersHMM/
You may use FASTA-formated aminoacid sequences of marker genes putting them together in a folder and providing path to the folder with option -s:
EPhIMM.sh -s /home/user/MyMarkersFASTA/
Default e-value for hmmsearch is 1e-6, You can set it with option -e, e.g.:
EPhIMM.sh -e 1e-10.
If You find EPhIMM usefull please cite it as:
Korzhenkov A. (2020) EPhIMM: computational workflow for fast phlyogenetic inference based on multiple alignment of prokaryotic single copy marker genes. Presented at: BGRS/SB-2020, Novosibirsk, Russia. DOI: 10.18699/BGRS/SB-2020-027
Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P., & Tyson, G. W. (2015). CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome research, 25(7), 1043-1055.
Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution, 30(4), 772-780.
Price, M.N., Dehal, P.S., and Arkin, A.P. (2010) FastTree 2 -- Approximately Maximum-Likelihood Trees for Large Alignments. PLoS ONE, 5(3):e9490. doi:10.1371/journal.pone.0009490.
Eddy, S. R. (2011). Accelerated profile HMM searches. PLoS computational biology, 7(10).
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research, 25(17), 3389-3402.