AMPHORA2 User Manual
Contents
1. AMPHORAZ User Manual An Automated Phylogenomic Inference Pipeline for Bacterial and Archaeal Sequences COPYRIGHT 2011 by Martin Wu AMPHORA2 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 AMPHORA2 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 AMPHORAZ please cite Wu M Scott AJ Phylogenomic analysis of bacterial and archaeal sequences with AMPHORAZ Bioinformatics 2012 28 7 1033 1034 DEPENDENCY AMPHORAZ2 depends on several external programs 1 HMMERS3 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 required only if you analyze DNA sequences using AMPHORA2 Make s2. igh the alignment columns with the RAxML s a option for untrimmed alignment only perl MarkerAlignTrim pl 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 Default home foo AMPHORA2 Marker 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 OutputFormat phylip If AMPHORAZ 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 31 ecoli marker proteins 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 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 perl Phylotyping p Options Method use maximum likeliho
3. l MarkerScanner pl sequence file Options DNA input sequences are DNA Default no Evalue HMMER evalue cutoff Default 1e 3 Bacteria input sequences are Bacterial sequences Archaea input sequences are Archaeal sequences ReferenceDirectory the file directory that contain the reference alignments hmms and masks Default home foo AMPHORA2 Marker Help print help Example 1a Identify phylogenetic markers from the E coli proteome Perl MarkerScanner pl Bacteria TestData ecoli pep 1b Identify phylogenetic markers from the E coli genome assembly perl MarkerScanner pl Bacteria DNA TestData ecoli fasta If AMPHORAZ has been installed correctly at the end of the run for example 1a or 1b you should see 31 marker protein sequences pep in your working directory 1c If you want to identify phylogenetic markers from metagenomic sequence reads e g 454 reads of a mixed bacterial and archaeal population perl MarkerScanner pl DNA metagenomic fasta However if you know your input sequences only contain bacterial or archaeal sequences then use the Bacterial or Archaeal option This makes the AMPHORA2 run faster and the results will be more accurate 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 rpoB aln rpsJ aln and their corresponding alignment masks The alignment masks can be used to we
4. o AMPHORAZ2 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 AMPHORAZ see the marker list file in the Marker folder 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 bacterial and arachaeal genome trees in newick format The genome trees are RAxML maximum likelihood trees made from concatenated protein sequences 5 TestData This folder contains the E coli genome assembly ecoli fasta and proteome sequences ecoli pep for testing AMPHORAZ RUNNING AMPHORA2 1 Marker identification Use MarkerScanner pl to identify bacterial and or archaeal 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 rpoB pep rpsJ pep When DNA input sequences are used this program first identifies ORFs longer than 100 bp in all six reading frames then scans the translated peptide sequences for the phylogenetic markers per
5. od 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 Example assign phylotypes using the maximum likelihood method perl Phylotyping pl CPUs 6 gt phylotype result Again if AMPHORA2Z is installed correctly you should see something like this as the output Query Marker Superkingdom Phylum Class Order Family Genus Species NP_417776 NC_000913 rplB Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 50 Escherichia coli 0 48 NP_417097 NC_000913 rplS_ Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 80 Escherichia coli 0 80 NP_414711 NC_000913 rpsB Bacteria 0 96 Proteobacteria 0 96 Gammaproteobacteria 0 96 Enterobacteriales 0 96 Enterobacteriaceae 0 96 Escherichia 0 80 Escherichia coli 0 78 The phylotyping results are tab delimited The numbers within the parentheses are the confidence scores of the assignment KNOWN ISSUES Bioperl AMPHORAZ has been tested on Bioperl 1 6 1 People have reported problems running AMPHORAZ on Bioper 1 6 9 For example the following error has been re
6. ported when running Phylotyping pl EXCEPTION Bio Root Exception MSG parse error expected or or STACK Error throw STACK Bio Root Root throw usr local share perl 5 14 2 Bio Root Root pm 472 STACK Bio TreelO NewickParser parse_newick usr local share perl 5 14 2 Bio TreelO NewickParser pm 195 STACK Bio TreelO newick next_tree usr local share perl 5 14 2 Bio TreelO newick pm 143 STACK main assign_phylotype usr local Bioinf AMPHORA2 Scripts Phylotyping pl 154 STACK usr local Bioinf AMPHORA2 Scripts Phylotyping pl 70 Downgrading Bioperl from 1 6 9 to 1 6 1 solves the problem NCBI Taxonomy 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
7. ure 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 AMPHORAZ 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 AMPHORA2 2 Unpack AMPHORA2 tar zxvf AMPHORA2 tar gz 3 Install dependencies if they have not been installed cd AMPHORA2 sudo perl preinstall pl 4 Setup AMPHORA2 You need to set up the environment variable AMPHORA2_home so the AMPHORA2 scripts know where to look for the phylogenetic marker database and the NCBI taxonomy information Let s suppose your unpacked AMPHORA folder is at home foo AMPHORAZ2 If you are using a bash shell you can add the following lines to the end of the file bashrc export AMPHORA2_home home foo AMPHORA2 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 AMPHORAZ_home home foo AMPHORA2 Then in the terminal issue this command source tcshrc 5 Make the AMPHORA2Z scripts executable chmod x home fo
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