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$./phylophlan.py -u mygenomes when finished, the resulting tree will appear in the output/mygenomes folder. Example 1: Corynebacterium 'de novo' phylogenetic tree building You can try out this operation ( -u) using an example included in the PhyloPhlAn package you downloaded called examplecorynebacteria and stored in the input folder. In contains a protein multifasta file for each of the 30 genomes available for the as February 2012 plus two genomes as a meaningful outgroup. As mentioned above, the command for obtaining the phylogenetic tree is. $./phylophlan.py -u examplecorynebacteria -nproc 4 Using 4 threads (specified with -nproc 4) this operation should take no more than 4-5 minutes, but even using one processor only (default) should give you the results in 10 minutes or so. In the output/examplecorynebacteria/ folder you'll find a file of the resulting tree as provided by, and a file containing the same tree rerooted with a procedure which tries to maximize the distance from the root to any leaf.
The two files are available for download (, and can be inspected with and drawn with. Figure 3B in the reports and discuss this example. Also the full three of life reported above has been originally generated in this way. Notice that the concatenated alignment used to generate the tree with FastTree is stored in data/examplecorynebacteria/aln.fna and can be used as input for other phylogenetic reconstruction software such as or among. Inserting new genomes to the tree of life PhyloPhlAn let you insert a genome (or a set of genomes) into the already built microbial tree of life (containing 3,000 genomes, see figure and tree files above). Also in this case you need to create a dedicated folder (e.g.
Mygenomestoinsert) in the input folder to store the protein multifasta files of interest. The command is. $./phylophlan.py -i mygenomestoinsert -nproc 16 If possible, we would recommend to use as many threads as possible ( -nproc) because this operation is quite computationally demanding as it requires the alignments with other 3,000 genomes to be updated and the full tree of life to be rebuilt. The resulting tree file output/mygenomestoinsert/mygenomestoinsert.tree.int.nwk can be inspected with tree visualization software to check where the new genomes are rooted and their relations with already well characterized strains. Example 2: inserting Lactobacillus and Sulfolobus genomes into the tree of life As an example of insertion, we included in the input folder contained in the PhyloPhlAn package, three genomes recently sequenced and not yet included into the PhyloPhlAn tree and repository.
These are two and one genomes available in IMG (accessions, and respectively). $./phylophlan.py -i exampleinsertion -nproc 16 The resulting file exampleinsertion.tree.int.nwk now contains the thousands of genomes in the PhyloPhlAn repository as well as the three 'new' genomes. Imputing taxonomic labels for newly integrated genomes You can also ask PhyloPhlAn to try to automatically assign a taxonomic labels to the genomes integrated into the tree of life ( -i option introduced above). This is possible simply adding the -t flag (for taxonomic analysis) to the same command line. $./phylophlan.py -i -t mygenomestoinsert -nproc 16 In addition to the output/mygenomestoinsert/mygenomestoinsert.tree.int.nwk file, you will obtain tab-separated text files with the most confident taxonomic predictions for your genomes in the output/mygenomestoinsert/ folder.
Example 3: predicting the taxonomic labels of three 'new' genomes Suppose you don't know the taxonomic labels of the Lactobacillus and Sulfolobus genomes used as examples above, possibly because of insufficient phenotipic characterization or because you obtained them with metagenomic assembly. You can call the PhyloPhlAn taxonomic imputation pipeline as. $./phylophlan.py -i -t exampleinsertion -nproc 16 And check the predictions in the file that we report below: SulfolobusacidocaldariusN8 dArchaea.pCrenarchaeota.cThermoprotei.oSulfolobales.fSulfolobaceae.gSulfolobus.s?t? LactobacillusrhamnosusKATCC8530 dBacteria.pFirmicutes.cBacilli.oLactobacillales.fLactobacillaceae.gLactobacillus.srhamnosus.t? LactobacillusrhamnosusLRHMDP3 dBacteria.pFirmicutes.cBacilli.oLactobacillales.fLactobacillaceae.gLactobacillus.srhamnosus.t?
As expected, the all three genomes are assigned to the right genera. The two lactobacilli could also be assigned to the right species ( srhamnosus) whereas PhyloPhlAn does not find enough support to assign the Sulfolobus genome to the 'acidocaldarius' species.
All command line options and parameters $./phylophlan.py -h usage: phylophlan.py -h -i -u -t -taxtest TAXTEST -c -cleanall -nproc N -v PROJECT NAME NAME AND VERSION: PhyloPhlAn version 0.99 (8 May 2013) AUTHORS: Nicola Segata ([email protected]) and Curtis Huttenhower ([email protected]) DESCRIPTION PhyloPhlAn is a computational pipeline for reconstructing highly accurate and resolved phylogenetic trees based on whole-genome sequence information. The pipeline is scalable to thousands of genomes and uses the most conserved 400 proteins for extracting the phylogenetic signal. PhyloPhlAn also implements taxonomic curation, estimation, and insertion operations. Positional arguments: PROJECT NAME The basename of the project corresponding to the name of the input data folder inside input/. The input data consist of a collection of multifasta files (extension.faa) containing the proteins in each genome. If the project already exists, the already executed steps are not re-ran.
The results will be stored in a folder with the project basename in output/ Multiple project can be generated and they safetely coexists. Optional arguments: -h, -help show this help message and exit -i, -integrate Integrate user genomes into the PhyloPhlAn tree -u, -usertree Build a phylogenetic tree using user genomes only -t, -taxonomicanalysis Check taxonomic inconsistencies and refine/correct taxonomic labels -taxtest TAXTEST nerrors:type:taxl:tmin:tex:name (alpha version, experimental!) -c, -clean Clean the final and partial data produced for the specified project. (use -cleanall for removing general installation and database files) -cleanall Remove all instalation and database file leaving untouched the initial compressed data that is automatically extracted and formatted at the first pipeline run. Projects are not remove (specify a project and use -c for removing projects).nproc N The number of CPUs to use for parallelizing the blasting default 1, i.e.
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