# HG changeset patch # User peterjc # Date 1427730373 14400 # Node ID 2c8931827fa5b34e62914647051d13edeac6120b # Parent 3b5eecc9551e48f9aeb2502881af574dcd3f845d Uploaded with note about NR versioning diff -r 3b5eecc9551e -r 2c8931827fa5 N_abberans_piechart_mouseover.png Binary file N_abberans_piechart_mouseover.png has changed diff -r 3b5eecc9551e -r 2c8931827fa5 README.rst --- a/README.rst Fri Oct 25 10:22:35 2013 -0400 +++ b/README.rst Mon Mar 30 11:46:13 2015 -0400 @@ -1,99 +1,180 @@ -This is package is a Galaxy workflow for the identification of candidate -secreted proteins from a given protein FASTA file. +Introduction +============ + +Galaxy is a web-based platform for biological data analysis, supporting +extension with additional tools (often wrappers for existing command line +tools) and datatypes. See http://www.galaxyproject.org/ and the public +server at http://usegalaxy.org for an example. + +The NCBI BLAST suite is a widely used set of tools for biological sequence +comparison. It is available as standalone binaries for use at the command +line, and via the NCBI website for smaller searches. For more details see +http://blast.ncbi.nlm.nih.gov/Blast.cgi + +This is an example workflow using the Galaxy wrappers for NCBI BLAST+, +see https://github.com/peterjc/galaxy_blast + + +Galaxy workflow for counting species of top BLAST hits +====================================================== + +This Galaxy workflow (file ``blast_top_hit_species.ga``) is intended for an +initial assessment of a transcriptome assembly to give a crude indication of +any major contamination present based on the species of the top BLAST hit +of 1000 representative sequences. + +.. image:: https://raw.githubusercontent.com/peterjc/galaxy_blast/master/workflows/blast_top_hit_species/blast_top_hit_species.png + +In words, the workflow proceeds as follows: + +1. Upload/import your transcriptome assembly or any nucleotide FASTA file. +2. Samples 1000 representative sequences, selected uniformly/evenly though + the file. +3. Convert the sampled FASTA file into a three column tabular file. +4. Runs NCBI BLASTX of the sampled FASTA file against the latest NCBI ``nr`` + database (assuming this is already available setup on your local Galaxy + under the alias ``nr``), requesting tabular output including the taxonomy + fields, and at most one matching target sequence. +5. Remove any duplicate alignments (multiple HSPs for the same match). +6. Combine the filtered BLAST output with the tabular version of the 1000 + sequences to give a new tabular file with exactly 1000 lines, adding + ``None`` for sequences missing a BLAST hit. +7. Count the BLAST species names in this file. +8. Sort the counts. + +Finally we would suggest visualising the sorted tally table as a Pie Chart. + + +Sample Data +=========== + +As an example, you can upload the transcriptome assembly of the nematode +*Nacobbus abberans* from Eves van den Akker *et al.* (2015), +http://dx.doi.org/10.1093/gbe/evu171 using this URL: + +http://nematode.net/Data/nacobbus_aberrans_transcript_assembly/N.abberans_reference_no_contam.zip + +Running this workflow with a copy of the NCBI non-redundant ``nr`` database +from 16 Oct 2014 (which did **not** contain this *N. abberans* dataset) gave +the following results - note 609 out of the 1000 sequences gave no BLAST hit. -It runs SignalP v3.0 (Bendtsen et al. 2004) and selects only proteins with a -strong predicted signal peptide, and then runs TMHMM v2.0 (Krogh et al. 2001) -on those, and selects only proteins without a predicted trans-membrane helix. -This workflow was used in Kikuchi et al. (2011), and is a simplification of -the candidate effector protocol described in Jones et al. (2009). +===== ================== +Count Subject Blast Name +----- ------------------ + 609 None + 244 nematodes + 30 ascomycetes + 27 eukaryotes + 8 basidiomycetes + 6 aphids + 5 eudicots + 5 flies + ... ... +===== ================== + +As you might guess from the filename ``N.abberans_reference_no_contam.fasta``, +this transcriptome assembly has already had obvious contamination removed. + +At the time of writing, Galaxy's visualizations could not be included in +a workflow. You can generate a pie chart from the final count file using +the counts (c1) and labels (c2), like this: + +.. image:: https://raw.githubusercontent.com/peterjc/galaxy_blast/master/workflows/blast_top_hit_species/N_abberans_piechart_mouseover.png + +Note the nematode count in this image was shown as a mouse-over effect. + + +Disclaimer +========== + +Species assignment by top BLAST hit is not suitable for any in depth +analysis. It is particularly prone to false positives where contaminants +in public datasets are mislabelled. See for example Ed Yong (2015), +"There's No Plague on the NYC Subway. No Platypuses Either.": + +http://phenomena.nationalgeographic.com/2015/02/10/theres-no-plague-on-the-nyc-subway-no-platypuses-either/ + -See http://www.galaxyproject.org for information about the Galaxy Project. +Known Issues +============ + +Counts +------ + +This workflow uses the Galaxy "Count" tool, version 1.0.0, as shipped with +the current stable release (Galaxy v15.03, i.e. March 2015). + +The updated "Count" tool version 1.0.1 includes a fix not to remove spaces +in the fields being counted. In the example above, while the top hits are +not affected, minor entries like "cellular slime molds" are shown as +"cellularslimemolds" instead (look closely at the Pie Chart key).. + +The updated "Count" tool version 1.0.1 also adds a new option to sort the +output, which avoids the additional sorting step in the current version of +the workflow. + +A future update to this workflow will use the revised "Count" tool, once +this is included in the next stable Galaxy release - or migrated to the +Galaxy Tool Shed. + +NCBI nr database +---------------- + +The use of external datasets within Galaxy via the ``*.loc`` configuration +files undermines provenance tracking within Galaxy. This is exacerbated +by the lack of officially versioned BLAST database releases by the NCBI. + +This workflow assumes that you have an entry ``nr`` in your ``blastdb_p.loc`` +(the configuration file listing locally installed BLAST databases external +to Galaxy - consult the NCBI BLAST+ wrapper documentation for more details), +and that this points to a mirror of the latest NCBI "non-redundant" database +from ftp://ftp.ncbi.nlm.nih.gov/blast/db/ + +i.e. The workflow is intended to be used against the *latest* nr database, +and thus is not reproducible over the long term as the database changes. Availability ============ -This workflow is available to download and/or install from the main -Galaxy Tool Shed: +This workflow is available to download and/or install from the main Galaxy Tool Shed: -http://toolshed.g2.bx.psu.edu/view/peterjc/secreted_protein_workflow +http://toolshed.g2.bx.psu.edu/view/peterjc/blast_top_hit_species Test releases (which should not normally be used) are on the Test Tool Shed: -http://testtoolshed.g2.bx.psu.edu/view/peterjc/secreted_protein_workflow +http://testtoolshed.g2.bx.psu.edu/view/peterjc/blast_top_hit_species Development is being done on github here: -https://github.com/peterjc/pico_galaxy/tree/master/workflows/secreted_protein_workflow - - -Sample Data -=========== - -This workflow was developed and run on several nematode species. For example, -try the protein set for *Bursaphelenchus xylophilus* (Kikuchi et al. 2011): - -ftp://ftp.sanger.ac.uk/pub/pathogens/Bursaphelenchus/xylophilus/Assembly-v1.2/BUX.v1.2.genedb.protein.fa.gz - -You can upload this directly into Galaxy via this URL. Galaxy will handle -removing the gzip compression to give you the FASTA protein file which has -18,074 sequences. The expected result (selecting organism type Eukaryote) -is a FASTA protein file of 2,297 predicted secreted protein sequences. +https://github.com/peterjc/galaxy_blast/tree/master/workflows/blast_top_hit_species Citation ======== -If you use this workflow directly, or a derivative of it, in work leading -to a scientific publication, please cite: +Please cite the following paper (currently available as a preprint): -Cock, P.J.A. and Pritchard, L. (2014). Galaxy as a platform for identifying -candidate pathogen effectors. Chapter 1 in "Plant-Pathogen Interactions: -Methods and Protocols (Second Edition)"; P. Birch, J. Jones, and J.I. Bos, eds. -Methods in Molecular Biology. Humana Press, Springer. ISBN 978-1-62703-985-7. -http://www.springer.com/life+sciences/plant+sciences/book/978-1-62703-985-7 +NCBI BLAST+ integrated into Galaxy. +P.J.A. Cock, J.M. Chilton, B. Gruening, J.E. Johnson, N. Soranzo +bioRxiv DOI: http://dx.doi.org/10.1101/014043 (preprint) -Peter J.A. Cock, Björn A. Grüning, Konrad Paszkiewicz and Leighton Pritchard (2013). -Galaxy tools and workflows for sequence analysis with applications -in molecular plant pathology. PeerJ 1:e167 -http://dx.doi.org/10.7717/peerj.167 +You should also cite Galaxy, and the NCBI BLAST+ tools: -Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S. (2004) -Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–95. -http://dx.doi.org/10.1016/j.jmb.2004.05.028 - -Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. (2001) -Predicting transmembrane protein topology with a hidden Markov model: -application to complete genomes. J Mol Biol 305: 567- 580. -http://dx.doi.org/10.1006/jmbi.2000.4315 +BLAST+: architecture and applications. +C. Camacho et al. BMC Bioinformatics 2009, 10:421. +DOI: http://dx.doi.org/10.1186/1471-2105-10-421 -Additional References -===================== - -Kikuchi, T., Cotton, J.A., Dalzell, J.J., Hasegawa. K., et al. (2011) -Genomic insights into the origin of parasitism in the emerging plant -pathogen *Bursaphelenchus xylophilus*. PLoS Pathog 7: e1002219. -http://dx.doi.org/10.1371/journal.ppat.1002219 +Automated Installation +====================== -Jones, J.T., Kumar, A., Pylypenko, L.A., Thirugnanasambandam, A., et al. (2009) -Identification and functional characterization of effectors in expressed -sequence tags from various life cycle stages of the potato cyst nematode -*Globodera pallida*. Mol Plant Pathol 10: 815–28. -http://dx.doi.org/10.1111/j.1364-3703.2009.00585.x - +Installation via the Galaxy Tool Shed should take care of the dependencies +on Galaxy tools including the NCBI BLAST+ wrappers and associated binaries. -Dependencies -============ - -These dependencies should be resolved automatically via the Galaxy Tool Shed: - -* http://toolshed.g2.bx.psu.edu/view/peterjc/tmhmm_and_signalp -* http://toolshed.g2.bx.psu.edu/view/peterjc/seq_filter_by_id - -However, at the time of writing those Galaxy tools have their own -dependencies required for this workflow which require manual -installation (SignalP v3.0 and TMHMM v2.0). +However, this workflow requires a current version of the NCBI nr protein +BLAST database to be listed in ``blastdb_p.loc`` with the key ``nr`` (lower +case). History @@ -102,12 +183,7 @@ ======= ====================================================================== Version Changes ------- ---------------------------------------------------------------------- -v0.0.1 - Initial release to Tool Shed (May, 2013) - - Expanded README file to include example data -v0.0.2 - Updated versions of the tools used, inclulding core Galaxy Filter - tool to avoid warning about new ``header_lines`` parameter. - - Added link to Tool Shed in the workflow annotation explaining there - is a README file with sample data, and a requested citation. +v0.1.0 - Initial Tool Shed release, targetting NCBI BLAST+ 2.2.29 ======= ====================================================================== @@ -116,15 +192,39 @@ This workflow is under source code control here: -https://github.com/peterjc/pico_galaxy/tree/master/workflows/secreted_protein_workflow +https://github.com/peterjc/galaxy_blast/tree/master/workflows/blast_top_hit_species To prepare the tar-ball for uploading to the Tool Shed, I use this: - $ tar -cf secreted_protein_workflow.tar.gz README.rst repository_dependencies.xml secreted_protein_workflow.ga + $ tar -cf blast_top_hit_species.tar.gz README.rst repository_dependencies.xml blast_top_hit_species.ga blast_top_hit_species.png N_abberans_piechart_mouseover.png Check this, - $ tar -tzf secreted_protein_workflow.tar.gz + $ tar -tzf blast_top_hit_species.tar.gz README.rst repository_dependencies.xml - secreted_protein_workflow.ga + blast_top_hit_species.ga + blast_top_hit_species.png + N_abberans_piechart_mouseover.png + + +Licence (MIT) +============= + +Permission is hereby granted, free of charge, to any person obtaining a copy +of this software and associated documentation files (the "Software"), to deal +in the Software without restriction, including without limitation the rights +to use, copy, modify, merge, publish, distribute, sublicense, and/or sell +copies of the Software, and to permit persons to whom the Software is +furnished to do so, subject to the following conditions: + +The above copyright notice and this permission notice shall be included in +all copies or substantial portions of the Software. + +THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR +IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, +FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE +AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER +LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, +OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN +THE SOFTWARE. diff -r 3b5eecc9551e -r 2c8931827fa5 blast_top_hit_species.ga --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/blast_top_hit_species.ga Mon Mar 30 11:46:13 2015 -0400 @@ -0,0 +1,331 @@ +{ + "a_galaxy_workflow": "true", + "annotation": "", + "format-version": "0.1", + "name": "Species of top BLAST hits", + "steps": { + "0": { + "annotation": "", + "id": 0, + "input_connections": {}, + "inputs": [ + { + "description": "", + "name": "Transcriptome FASTA file" + } + ], + "label": null, + "name": "Input dataset", + "outputs": [], + "position": { + "left": 242, + "top": 119 + }, + "tool_errors": null, + "tool_id": null, + "tool_state": "{\"name\": \"Transcriptome FASTA file\"}", + "tool_version": null, + "type": "data_input", + "user_outputs": [], + "uuid": "e445b44b-02a7-4fd1-8944-cd680f967062" + }, + "1": { + "annotation": "This workflow is deliberately a simple/crude assessment, and there is no need to run BLASTX on all the sequences - a sample of 1000 should be enough.", + "id": 1, + "input_connections": { + "input_file": { + "id": 0, + "output_name": "output" + } + }, + "inputs": [], + "label": null, + "name": "Sub-sample sequences files", + "outputs": [ + { + "name": "output_file", + "type": "input" + } + ], + "position": { + "left": 435, + "top": 119 + }, + "post_job_actions": { + "RenameDatasetActionoutput_file": { + "action_arguments": { + "newname": "1000 sequences from #{input_file}" + }, + "action_type": "RenameDatasetAction", + "output_name": "output_file" + } + }, + "tool_errors": null, + "tool_id": "toolshed.g2.bx.psu.edu/repos/peterjc/sample_seqs/sample_seqs/0.2.1", + "tool_state": "{\"__page__\": 0, \"input_file\": \"null\", \"__rerun_remap_job_id__\": null, \"sampling\": \"{\\\"count\\\": \\\"1000\\\", \\\"type\\\": \\\"desired_count\\\", \\\"__current_case__\\\": 2}\", \"chromInfo\": \"\\\"/mnt/galaxy/galaxy-dist/tool-data/shared/ucsc/chrom/?.len\\\"\", \"interleaved\": \"\\\"False\\\"\"}", + "tool_version": "0.2.1", + "type": "tool", + "user_outputs": [], + "uuid": "87ce69ef-5fb0-41b0-9575-d3b96544f8be" + }, + "2": { + "annotation": "We only want one line per query, so limit this to the best scoring target sequence. 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@@ -1,288 +0,0 @@ -{ - "a_galaxy_workflow": "true", - "annotation": "Runs SignalP v3.0 and TMHMM v2.0 to look for secreted proteins.
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