Phyla_AMPHORA User Manual
Contents
1. Phyla_AMPHORA User Manual A Phylum specific Automated Phylogenomic Inference Pipeline for Bacterial Sequences COPYRIGHT 2012 by Martin Wu Phyla_AMPHORA is free software you may redistribute it and or modify its under the terms of the GNU General Public License as published by the Free Software Foundation either version 2 of the License or any later version Phyla_AMPHORA is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU General Public License for more details http www gnu org licenses For any other inquiries send an Email to Martin Wu mw4yv virginia edu CITATION When publishing work that is based on the results from Phyla AMPHORA please cite Wang Zand Wu M A Phylum level Bacterial Phylogenetic Marker Database Mol Biol Evol Advance Access publication March 21 2013 doi 10 1093 molbev mst059 DEPENDENCY Phyla_AMPHORA depends on several external programs 1 HMMER3 http hmmer janelia org Required for marker identification sequence alignment and trimming Earlier versions of HMMER will not work RAxML version 7 3 0 or later https github com stamatak standard RAxML downloads Required for phylotyping Bioperl 1 5 2 or later http www bioperl org wiki Getting_BioPerl EMBOSS http emboss sourceforge net download The getorf program of the EMBOSS package is required2. ed by other processes In this case you can run Phylotyping pl on 6 CPUs by using the CPUs 6 option Of course raxmlHPC PTHREADS needs to be installed Example Assign phylotypes using the maximum likelihood method perl Phylotyping pl CPUs 6 gt phylotype result Again if Phyla_ AMPHORA has been installed correctly you should see something like this as the output Query Marker Superkingdom Phylum Class Order Family Genus Species NP_414730 NC_000913 Gamma 134 Bacteria 1 00 Proteobacteria 1 00 Gammaproteobacteria 1 00 Enterobacteriales 1 00 Enterobacteriaceae 1 00 Escherichia 1 00 Escherichia coli 1 00 NP_417099 NC_000913 Gamma 167 Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 scherichia 0 70 Escherichia coli 0 70 NP_416616 NC_000913 Gamma 252 Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 74 Escherichia coli 0 74 NP_418155 NC_000913 Gamma 286 Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 84 Escherichia coli 0 84 igs NP_417422 NC_000913 Gamma 296 Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 76 Escherichia coli 0 76 NP_417226 NC_000913 Gamma 306 Bacteria 0 97 Proteobacteria 0 97 Gammaprote
3. er pl Phylum 3 DNA TestData ecoli fasta If Phyla_AMPHORA has been installed correctly at the end of the run in example 1a or 1b you should see 294 marker protein sequences pep in your working directory 1c If you want to identify phylogenetic markers of all the 20 phyla from a set of metagenomic sequence reads e g 454 reads perl MarkerScanner pl DNA Phylum 0 metagenomic fasta 2 Marker sequence alignment and trimming This program will align mask and trim the marker protein sequences Output will be aligned trimmed sequences For example Acidobacteria 102 aln Aquificae 33 aln and their corresponding alignment masks The alignment masks can be used to weigh the alignment columns with the RAxML s a option for untrimmed alignment only Usage perl MarkerAlignTrim pl lt options gt Options Trim trim the alignment using masks embedded with the marker database Default no Cutoff the Zorro masking confidence cutoff value 0 1 0 default 0 4 ReferenceDirectory the file directory that contain the reference alignments hmms and masks Directory the file directory where sequences to be aligned are located Default current directory OutputFormat output alignment format Default phylip Other supported formats include fasta stockholm selex clustal WithReference keep the reference sequences in the alignment Default no Help print help Example perl MarkerAlignTrim pl WithReference Outp
4. hyla_AMPHORA Then in the terminal issue this command source tcshrc 5 Make the Phyla AMPHORA scripts executable chmod x home foo Phyla_AMPHORA Scripts You should see five folders 1 Marker This folder contains a seed alignment file in Stockholm format stock an alignment mask file mask a profile HMM file HMM and a tree file in newick format tre for each marker gene For more information about the phylogenetic markers that are included in Phyla_AMPHORA see the marker list file in the Marker folder IMPORTANT Because the Marker folder exceeds the 1GB the size limit of github it is not included in the github package If you download Phyla_AMPHORA from github you should download the Marker database from http wolbachia biology virginia edu WuLab Software html and move it here 2 Scripts This folder contains the scripts for marker identification alignment trimming and phylotyping 3 Taxonomy This folder contains the NCBI taxonomy database that is used by the Phylotyping pl script for phylotyping 4 Tree This folder contains the genome trees for 20 bacterial phyla in newick format The genome trees are RAxML maximum likelihood trees made from concatenated protein sequences of the phylum specific markers 5 TestData This folder contains the E coli genome assembly ecoli fasta and proteome sequences ecoli pep for testing Phyla_AMPHORA RUNNING Phyla_AMPHORA We recommend that you all
5. obacteria 0 97 Enterobacteriales 0 97 Enterobacteriaceae 0 97 Escherichia 0 61 Escherichia coli 0 61 NP_417800 NC_000913 Gamma 44 Bacteria 0 95 Proteobacteria 0 95 Gammaproteobacteria 0 95 Enterobacteriales 0 95 Enterobacteriaceae 0 95 Escherichia 0 95 Escherichia coli 0 95 The phylotyping results are tab delimited The numbers within the parentheses are the confidence scores of the assignment If you see the following error message when you run Phylotyping pl you can delete the name2id file in the folder AMPHORA2 home Taxonomy and run the script again EXCEPTION Bio Root Exception MSG No such file or directory AMPHORA2_home Taxonomy names2id STACK Error throw STACK Bio Root Root throw lib site_perl 5 16 3 Bio Root Root pm 368 STACK Bio DB Taxonomy flatfile _db_connect lib site_perl 5 16 3 Bio DB Taxonomy flatfile pm 463 STACK Bio DB Taxonomy flatfile new lib site_perl 5 16 3 Bio DB Taxonomy flatfile pm 144 STACK Bio DB Taxonomy new lib site_perl 5 16 3 Bio DB Taxonomy pm 116 STACK AMPHORA2_home Scripts Phylotyping p1 58
6. ocate at least 4GB of memory to Phyla_AMPOHRA 1 Marker identification Use MarkerScanner pl to identify phylum specific bacterial marker sequences Given a sequence file this program will identify markers from the input sequences and generate a protein fasta file for each marker gene in your working directory For example Acidobacteria 102 pep Aquificae 33 pep When DNA sequences are used as input this program first identifies ORFs longer than 100 bp in all six reading frames then scans the translated peptide sequences for the phylogenetic markers Usage perl MarkerScanner pl lt options gt sequence file Options Phylum 0 All Default 1 Alphaproteobacteria 2 Betaproteobacteria 3 Gammaproteobacteria 4 Deltaproteobacteria 5 Epsilonproteobacteria 6 Acidobacteria 7 Actinobacteria 8 Aquificae 9 Bacteroidetes 10 Chlamydiae Verrucomicrobia 11 Chlorobi 12 Chloroflexi 13 Cyanobacteria 14 Deinococcus Thermus 15 Firmicutes 16 Fusobacteria 17 Planctomycetes 18 Spirochaetes 19 Tenericutes 20 Thermotogae DNA input sequences are DNA Default no Evalue HMMER evalue cutoff Default 1e 7 ReferenceDirectory the file directory that contain the reference alignments hmms and masks Help print help Examples 1a Identify phylogenetic markers from the E coli proteome perl MarkerScanner pl Phylum 3 TestData ecoli pep 1b Identify phylogenetic markers from the E coli genome assembly perl MarkerScann
7. only if you analyze DNA sequences using Phyla AMPHORA Make sure that these programs are installed and are in your system s executable search path To test in a terminal type raxmlHPC version raxmlHPC PTHREADS version hmmsearch h hmmalign h getorf help If you see version or help messages then these programs have been correctly installed It is important to make sure they are the correct versions A script named preinstall pl is also included with Phyla AMPHORA to check and install the dependencies automatically You need the privilege of the system administrator to run the script See below for instructions INSTALLATION 1 Download Phyla AMPHORA 2 Unpack Phyla AMPHORA tar zxvf Phyla_AMPHORA tar gz 3 Install dependencies if they have not been installed cd Phyla AMPHORA sudo perl preinstall pl 4 Setup Phyla AMPHORA You need to set up the environment variable Phyla_AMPHORA_home so the Phyla_AMPHORA scripts know where to look for the phylogenetic marker database and the NCBI taxonomy information Let s suppose your unpacked Phyla_AMPHORA folder is at home foo Phyla_AMPHORA If you are using a bash shell you can add the following lines to the end of the file bashrc export Phyla AMPHORA_home home foo Phyla_AMPHORA Then in the terminal issue this command source bashrc If you are using a C shell you can add the following lines to the end of the file tcshre setenv Phyla AMPHORA_home home foo P
8. utFormat phylip If Phyla_AMPHORA has been installed correctly at the end of the run you should see an alignment file aln and a mask file mask for each of the marker gene in your working directory 3 Phylotyping Use Phylotyping pl to assign phylotypes for each identified marker sequences This program will assign each identified marker sequence a phylotype using the parsimony method or the evolutionary placement algorithm of RAxML The marker sequences need to be aligned first with the reference sequences using MarkerAlignTrim pl see above The alignments should be in the phylip format Usage perl Phylotyping pl lt options gt Options Method use maximum likelihood ml or maximum parsimony mp for phylotyping Default ml CPUs turn on the multiple thread option and specify the number of CPUs cores to use Important Make sure raxmlHPC PTHREADs is installed If the number specified here is larger than the number of cores that are free and available it will actually slow down the script Help print help If your computer has multiple CPUs cores the phylotpying process can be sped up by running multiple threads of the RAxML However it is very important to check how many CPUs cores are free and available to Phylotyping pl If you specify a number that is larger than the number of CPUs cores that are actually available it will slow down the script For example your computer has 8 CPUs but 2 of them are us
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