MAdLandDB is a protein database comprising of a comprehensive collection of fully sequenced plant and algal genomes, with a particular emphasis on non-seed plants and streptophyte algae. Additionally, for comparative analysis, the database also includes genomes from various other organisms such as fungi, animals, the SAR group, bacteria, and archaea. The database is actively developed and maintained by the Rensing lab and released in the MAdLand setting. It employs a system of species abbreviation using a 5 letter code, which is constructed using the first three letters of the genus and the first two letters of the species name, for example, CHABR for Chara braunii. Furthermore, the database provides gene identification through the addition of gene ID’s and supplementary information such as the encoding source of the gene, whether it is plastome encoded (pt) or transcriptome-based (tr) in cases when a genome is not yet available. The key advantage of this database is its non-redundant nature, and the fact that all sequences are predominantly from genome projects, thereby increasing their reliability.

Agenda

In this tutorial, we will deal with:

1. Get data
2. Perform NCBI Blast+ on Galaxy
3. Blast output
4. More Similarity Search Tools on Galaxy

Get data

Hands-on: Data Upload

1. Create a new history for this tutorial and give it a proper name

   Tip: Creating a new history

   To create a new history simply click the new-history icon at the top of the history panel:

   

   Tip: Renaming a history

   1. Click on galaxy-pencil (Edit) next to the history name (which by default is “Unnamed history”)
   2. Type the new name
   3. Click on Save
   4. To cancel renaming, click the galaxy-undo “Cancel” button

   If you do not have the galaxy-pencil (Edit) next to the history name (which can be the case if you are using an older version of Galaxy) do the following:

   1. Click on Unnamed history (or the current name of the history) (Click to rename history) at the top of your history panel
   2. Type the new name
   3. Press Enter

2. Import the file query.faa from Zenodo

   https://zenodo.org/records/7524427/files/query.faa
   

   Tip: Importing via links

   • Copy the link location

   • Click galaxy-upload Upload Data at the top of the tool panel

   • Select galaxy-wf-edit Paste/Fetch Data

   • Paste the link(s) into the text field

   • Press Start

   • Close the window

We just imported a FASTA file into Galaxy. Now, the next would be to perfrom the BLAST analysis against MAdLandDB.

Perform NCBI Blast+ on Galaxy

Since MAdLandDB is the collection of protein sequences, You can perform BLASTp ( Galaxy version 2.10.1+galaxy2) and BLASTx ( Galaxy version 2.10.1+galaxy2) tools.

Hands-on: Similarity search against MAdLand Database

1. BLASTp ( Galaxy version 2.10.1+galaxy2) OR BLASTx ( Galaxy version 2.10.1+galaxy2) with the following parameters:
   • “Protein query sequence(s)”: Amino acid input sequence (In case of BLASTp) OR
   • “Translated nucleotide query sequence(s)”: Translated nucleotide input sequence (In case of BLASTx)
   • “Subject database/sequences”: Locally installed BLAST database
   • “Protein BLAST database”: MadLandDB (Genome zoo) plant and algal genomes with a focus on non-seed plants and streptophyte algae (22 Dec 2022)
   • “Set expectation value cutoff”: 0.001
   • “Output format”:
   • In “Output Options”: Tabular (extended 25 columns)

Blast output

tool The BLAST output will be in tabular format (you can select the desired output format from the drop down menu) and include the following fields :

The fields are separated by tabs, and each row represents a single hit. For more details for BLAST analysis and output, we recommand you to follow the Similarity-searches-blast tutorial.

Details: Further Reading about BLAST Tools in Galaxy

See Cock et al. 2015 and Cock et al. 2013

More Similarity Search Tools on Galaxy

• Diamond: Diamond ( Galaxy version 2.0.15+galaxy0) is a high-throughput program for alignment of large-scale data sets. It aligns sequences to the reference database using a compressed version of the reference sequences called a “database diamond” which is faster to read and can save computational time (~20,000 times the speed of Blastx, with high sensitivity).

Details: Diamond in depth

See Buchfink et al. 2014 for more discussion.

You've Finished the Tutorial

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Key points

• Blast tool searches a database of sequences for similar sequences to a query sequence.

• Diamond quickly aligns large-scale data sets using a compressed version of the reference sequences called a “database diamond”.

• MAdLand is a database of fully sequenced plant and algal genomes, with an emphasis on non-seed plants and streptophyte algae that can be use for sequence similarity search.

Frequently Asked Questions

Useful literature

Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.

References

1. Cock, P. J. A., B. A. Grüning, K. Paszkiewicz, and L. Pritchard, 2013 Galaxy tools and workflows for sequence analysis with applications in molecular plant pathology. PeerJ 1: e167. 10.7717/peerj.167
2. Buchfink, B., C. Xie, and D. H. Huson, 2014 Fast and sensitive protein alignment using DIAMOND. Nature Methods 12: 59–60. 10.1038/nmeth.3176
3. Cock, P. J. A., J. M. Chilton, B. Grüning, J. E. Johnson, and N. Soranzo, 2015 NCBI BLAST+ integrated into Galaxy. GigaScience 4: 10.1186/s13742-015-0080-7

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Citing this Tutorial

1. Deepti Varshney, NCBI BLAST+ against the MAdLand (Galaxy Training Materials). https://training.galaxyproject.org/training-material/topics/sequence-analysis/tutorials/ncbi-blast-against-the-madland/tutorial.html Online; accessed TODAY
2. Hiltemann, Saskia, Rasche, Helena et al., 2023 Galaxy Training: A Powerful Framework for Teaching! PLOS Computational Biology 10.1371/journal.pcbi.1010752
3. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012

BibTeX

@misc{sequence-analysis-ncbi-blast-against-the-madland,
author = "Deepti Varshney",
	title = "NCBI BLAST+ against the MAdLand (Galaxy Training Materials)",
	year = "",
	month = "",
	day = "",
	url = "\url{https://training.galaxyproject.org/training-material/topics/sequence-analysis/tutorials/ncbi-blast-against-the-madland/tutorial.html}",
	note = "[Online; accessed TODAY]"
}
@article{Hiltemann_2023,
	doi = {10.1371/journal.pcbi.1010752},
	url = {https://doi.org/10.1371%2Fjournal.pcbi.1010752},
	year = 2023,
	month = {jan},
	publisher = {Public Library of Science ({PLoS})},
	volume = {19},
	number = {1},
	pages = {e1010752},
	author = {Saskia Hiltemann and Helena Rasche and Simon Gladman and Hans-Rudolf Hotz and Delphine Larivi{\`{e}}re and Daniel Blankenberg and Pratik D. Jagtap and Thomas Wollmann and Anthony Bretaudeau and Nadia Gou{\'{e}} and Timothy J. Griffin and Coline Royaux and Yvan Le Bras and Subina Mehta and Anna Syme and Frederik Coppens and Bert Droesbeke and Nicola Soranzo and Wendi Bacon and Fotis Psomopoulos and Crist{\'{o}}bal Gallardo-Alba and John Davis and Melanie Christine Föll and Matthias Fahrner and Maria A. Doyle and Beatriz Serrano-Solano and Anne Claire Fouilloux and Peter van Heusden and Wolfgang Maier and Dave Clements and Florian Heyl and Björn Grüning and B{\'{e}}r{\'{e}}nice Batut and},
	editor = {Francis Ouellette},
	title = {Galaxy Training: A powerful framework for teaching!},
	journal = {PLoS Comput Biol}
}

                   

Funding

These individuals or organisations provided funding support for the development of this resource

MAdLand

DataPLANT

Galaxy Administrators: Install the missing tools

You can use Ephemeris's shed-tools install command to install the tools used in this tutorial.

shed-tools install [-g GALAXY] [-a API_KEY] -t <(curl https://training.galaxyproject.org/training-material/api/topics/sequence-analysis/tutorials/ncbi-blast-against-the-madland/tutorial.json | jq .admin_install_yaml -r)

Alternatively you can copy and paste the following YAML

---
install_tool_dependencies: true
install_repository_dependencies: true
install_resolver_dependencies: true
tools:
- name: diamond
  owner: bgruening
  revisions: e8ac2b53f262
  tool_panel_section_label: NCBI Blast
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: ncbi_blast_plus
  owner: devteam
  revisions: 0e3cf9594bb7
  tool_panel_section_label: NCBI Blast
  tool_shed_url: https://toolshed.g2.bx.psu.edu/
- name: ncbi_blast_plus
  owner: devteam
  revisions: 0e3cf9594bb7
  tool_panel_section_label: NCBI Blast
  tool_shed_url: https://toolshed.g2.bx.psu.edu/

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