Package: automlsa2
Table of ContentsWho might want this software?InstallationDependenciesJust tell me how to run itOverviewUsageRunning TestsTODOContributingAuthor ContactAcknowledgmentsLicenseCopyrightsWho might want this software?The intended audience is scientific researchers, computational biologists, and
bioinformaticians who are interested in exploring phylogenetic and phylogenomic
relationships between genes and organisms. The general idea is to allow the
input of sequence data along with marker genes and output a robust phylogenetic
tree. I’ve implemented commands to help with the installation of external
dependencies, and I hope the software is easy to use.If you have feature requests or otherwise have ideas about how to make this
software better, please submit an issue with your ideas!PhD/Masters students and undergraduates are especially encouraged to submit
issues if they are having trouble using this software.Windows users, please install WSL to make use of this software. Using a Linux
distribution will make your life as a computational researcher significantly
easier.Installationautomlsa2 is distributed onPyPIas
a universal wheel and is available on Linux/macOS and Windows (untested) and
supports Python 3.7+ and PyPy.$python3-mpipinstall-Uautomlsa2While I will do my best to keep the git version usable, stick to a release
and/or pypi install for the most stable experience.git version install:$gitclonehttps://github.com/davised/automlsa2.git$cdautomlsa2$python3-mpipinstall-rrequirements.txt$python3-mpipinstall-U.for developers, clone as above, then:$python3-mpipinstall-e.--no-use-pep517DependenciesPython modules:pandasnumpybiopythonrichpackagingpsutilSeerequirements.txtfor more info.External programs:NCBI BLAST+ >= 2.10.0mafft >= 7.471IQ-TREE COVID-19 release >= 2.1.1You can install external programs using theautomlsa2--install_depscommand. These will be installed to${HOME}/.local/externalunless
otherwise specified.Just tell me how to run it$automlsa2--filesGenus_species_1.fnaGenus_species_2.fna...\Genus_species_N.fna--queryqueries.fasta-tTHREADS--runIDAlternatively:$automlsa2--dirpath/to/genomes--queryqueries.fasta-tTHREADS\--runIDOverviewautomlsa2 is a re-imagination ofautoMLSA.plThe entire codebase has been re-written in python. While the general algorithm
produces similar output, and several steps are shared, there are many
updates and differences between the two programs, which will be covered later.The general overview can be summarized here:Input is a set of marker genes as queries, and a set of target genome FASTA
files.BLAST databases are generated for each target genome, and each query gene
is extracted from the input query FASTA files.BLAST searches are done with the extracted sequences and genomes.Per genome hits are calculated pending the cut-offs, and genomes are
filtered from the analysis.Sequences are extracted from the BLAST results as unaligned multi-FASTAs.Unaligned sequences are aligned using mafft.A nexus file is generated pointing to all aligned sequences.A phylogenetic tree is generated using the nexus file as input.BLAST searches are threaded, or, optionally, written to a file to be submitted
to a compute cluster. mafft alignment commands can also be written to a file
for submission to a compute cluster.Input query files and genome directories are scanned for updates - if
sequences are added, removed, or changed, the analysis is re-done.Multiple queries targeting the same gene sequence can be used to improve
coverage of disparate gene sequences, e.g. attempting to cover an entire
phylum with multiple reference genomes being used.Usage$automlsa2-husage:automlsa2[-h][--queryQUERY[QUERY...]][--filesFILES[FILES...]][--dirDIR[DIR...]][-eEVALUE][-cCOVERAGE][-iIDENTITY][-p{blastn,tblastn}][--configCONFIG][--missing_check][-tTHREADS][--dups][--allow_missingALLOW_MISSING][--outgroupOUTGROUP][--protect][--checkpoint{validate,preblast,filtering,prealign,postalign,nexus,none}][--install_deps[INSTALL_DEPS]][--externalEXTERNAL][--mafftMAFFT][--iqtreeIQTREE][--debug][--version][--quiet]runidThisisarewriteofautoMLSA.pl.Generatesautomatedmulti-locussequenceanalyses.positionalarguments:runidNameoftherundirectory.optional arguments:-h,--helpshow this help message and exit--query<QUERY [QUERY …]>Path to file with input seq(s).--files<FILES [FILES …]>Path to the target genome FASTA files.--dir<DIR [DIR …]>Path to the target genome directory with FASTA files.-eEVALUE,--evalueEVALUEE-value cutoff for BLAST searches. [1e-5]-cCOVERAGE,--coverageCOVERAGESets the coverage cut-off threshold. [50]-iIDENTITY,--identityIDENTITYSets the identity cut-off threshold. [30]-pPROGRAM,--programPROGRAMWhich BLAST program to run. [tblastn] {tblastn, blastn}--configCONFIGPath to configuration json file to copy.--missing_checkUse this to confirm that settings have been
checked when genes are missing.-tTHREADS,--threadsTHREADSNumber of threads to use. [1]--dupsAllow for duplicate query names for more sequence
coverage across disparate organisms.--allow_missingALLOW_MISSINGAllow for N missing genes per genome. [0]--outgroupOUTGROUPName of outgroup file or strain to root on.--protectSave files from getting overwritten. By default, as input
files update, older alignments and trees are deleted.--checkpointCHECKPOINTName of stage to stop computing on. [none]
{validate,preblast,filtering,prealign,postalign,nexus,none}--install_deps<[INSTALL_DEPS]>Install dependencies into given directory. [~/.local/external]--externalEXTERNALPath to installed external programs. [~/.local/external]--mafftMAFFTmafft settings [–localpair –maxiterate 1000 –reorder]--iqtreeIQTREEiqtree2 settings [-m MFP -B 1000 -alrt 1000 –msub
nuclear –merge rclusterf]--debugTurn on debugging messages.--versionshow program’s version number and exit--quietTurn off progress messages.One or more input target genome FASTA files is required, either using--filesor--dir. Additionally, one or more query FASTA files
containing one or more query gene sequences is necessary for analysis.By default, protein queries are expected, and nucleotide FASTA sequence is
required for the target genomes.tblastnis used to target the genome
sequences using the amino acid queries.blastnis also available, targeting
the genome sequences using nucleotide queries.Threads will speed things up significantly. BLAST searches are threaded in
python; submitting multiple threads to the blast executable often does not
result in much speed up, so each BLAST search is run with one CPU given.Query marker genes often come from a well-studied representative of, at most,
the same genus. Intergenera phylogenies should have a representative sequence
from each genus. This can be accomplished by giving all examples of a
particular gene the same name in the reference FASTA file. e.g.>Gene1Refgenus1refspeciesABC<AAsequence>>Gene1Refgenus2refspeciesDEF<AAsequence>>Gene1Refgenus3refspeciesGHI<AAsequence>This ^ FASTA ^ file would have three representatives of Gene1 in the analysis.
The resulting alignments would have one copy of the gene, with the best hits
from each target genome included.Target genome files will be named based on the filename in the final output.
Generally, one will want to have Genus_species_strain.fasta or
G_species_strain.fasta as the filenames prior to analysis.Genomes can be downloaded using myget_assembliesprogram, here:https://pypi.org/project/get-assemblies/. Locally produced genomes can be
renamed as required.Running TestsThe test data are included in a separate repository -https://github.com/davised/automlsa2-examplesTo download this test data, you can rungit submodule update--init.Alternatively, you can run therun_tests.shscript and it will download
the git submodule for you and run the test command.The tests should take about 2-3 minutes with 4 CPUs.TODO☐ Write detailed list of intermediate files.☐ Compare functionality of this version to prior autoMLSA.pl version.☑ Check for version numbers for external programs.ContributingBug reports are encouraged! Submit a github issue and I’ll be happy to take
a look. Also, feel free to clone and submit merge requests.Author ContactEd DavisAcknowledgmentsSpecial thanks for helping me test the software and get the python code packaged:Alex WeisbergShawn O’NeilAlso, thanks to these groups for supporting me through my scientific career:OSU Chang LabCenter for Genome Research and Biocomputing @ OSULicenseautomlsa2 is distributed under the terms listed in theLICENSEfile. The
software is free for non-commercial use.CopyrightsCopyright (c) 2020 Oregon State UniversityAll Rights Reserved.