BLAST

BLAST (Basic Local Alignment Search Tool) is the most frequently used sequence homology search tool. Given a query sequence (nucleotide or protein), BLAST compares it to a sequence database and picks out sequences with significant similarity to the probe sequence. BLAST uses a heuristic search protocol, which makes search very fast compared to non-heuristic methods. The heuristics used may however cause BLAST to fail to find all significant hits.

The command line version of NCBI-BLAST allows a user to modify all parameters of BLAST, to use special methods like PSI-BLAST and PHI-BLAST, and to analyze large data sets.

In Puhti you can use pb(Parallel Blast) command for large sets of query sequences. The pb program splits a large search jobs into several subjobs, that are executed simultaneously (more below).

The most commonly used BLAST commands are:

• blastn search hits for a nucleotide sequence from nucleotide database
• blastp search hits for a protein sequence from protein database
• blastx search hits for a nucleotide sequence from protein database
• psiblast do iterative search for a protein sequence from protein database
• rpsblast search hits for a protein sequence from protein profile database
• rpstblastn search hits for a nucleotide sequence from protein profile database
• tblastn search hits for a protein sequence from nucleotide database
• tblastx search hits for a nucleotide sequence from nucleotide database by using the protein translations of both query and database sequences.

Other blast commands

• blastdbcmd retrieve a sequence or a set of sequences form BLAST databases
• makeblastdb create a new BLAST database
• blast_formatter reformat a BLAST archive formatted BLAST result file. [TOC]

License

Free to use and open source under GNU LGPLv2.1.

Available

• Puhti: 2.12.0
• FGCI: 2.6.0
• Chipster graphical user interface

Usage

At CSC, BLAST searches can be executed in several ways:

• using the Chipster platform
• with normal BLAST commands in interctive batch jobs (sinteractive -i)
• as batch jobs with pb command in Puhti

Interactive usage in Puhti

To use the latest BLAST version in Puhti first give set up command :

module load biokit

Then launch an interactive bactch job session with command:

sinteractive -i

Reserve 8 GiB of memory, for your interactive session.

After that you can start using the BLAST commands listed above. For example following command would search for sequence homologs from UniProt database for a protein sequence.

blastp -query proteinseq.fasta -db uniprot -out result.txt

You can use -help option to see, what command line options are available for a certain BLAST command. For example

blastp -help

For example, command:

blastp -query proteinseq.fasta -evalue 0.001 -db uniprot -outfmt 7 -out result.table

Would run the same search as described above, except that the e-value threshold would be set to 0.001(-evalue 0.001) and the out put is printed out to a table (-outfmt 7).

Usage of pb (Parallel BLAST) at CSC

If your query sequence set contains less than 20 sequences then interactive batch job is probably the most effective way top do the search. However, if your query set contains hundreds or thousands of sequences then utilizing the parallel computing capacity of Puhti is more effective. For this kind of massive blast searches you can utilize the pb command.

pb (Parallel BLAST) is designed for situations, where the query file includes large amount of sequences. It splits the query task into several subjobs, that can be run simultaneously using the resources of the server very effectively. For large sets of query sequences, pb can speed up the search up to 50 fold. Two sample pb commands for puhti.csc.fi:

module load biokit

pb blastn -db nt -query 100_ests.fasta -out results.out

pb psiblast -db swiss -query protseqs.fasta -num_iterations 3 -out results.out

pb blast commands can be executed interactively in the login nodes of Puhti. You don't need to create any batch job file yourself. In stead pb command creates and submits a batch job automatically. Once BLAST job is started pb starts a process that monitor the progress of the blast job. As running a large BLAST jobs may take a long time you may need close the monitoring. You can do that by pressing: Ctrl-c. After that you can start other tasks or log out from Puhti. The BLAST jobs will still continue running in the batch job system.

To reconnect to your pb blast job, go to your scratch directory and run command:

blast_clusterrun

This lists the temporary directories of your unfinished pb blast jobs. You can check the job number of your blast job from the directory name. Use this number with -jobid option to define the pb blast job you wish to reconnect to.

blast_clusterrun -jobid some-number

Using own BLAST databases with pb

The pb program also allows users to do BLAST searches against their own fasta formatted sequence sets. This is done by replacing the -db option with option -dbnuc (for nucleotides) or -dbprot (for proteins). Example:

pb blastn -dbnuc my_seq_set.fasta -query querys.fasta -out results.out

If your database is big, building the BLAST indexes may require more that 1 GB of memory ( that is the job specific memory limit in Puhti login nodes). In those cases you can subhmit the job from an interactive batch job (with e.g. 8 GB of memory).

Using taxonomy lists to focus the search

Since BLAST version 2.10.0, the BLAST database format has changed to version 5. This version supports using a single taxonomy ID number or list of taxonomies, to focus the search only to an organism based subset from the search database.

The BLAST tools include a command get_species_taxids.sh that can be used to generate taxidlists. First you have to find the the higher lever TaxID number your wish to use. For example, the TaxID of Betacoronavirus genius can be found with command:

get_species_taxids.sh -n Betacoronavirus 

Then the TaxIDs of the spcies that belong to this genius (TaxID: 694002) can be retrieved with command:

get_species_taxids.sh -t 694002 > b-coronaviruses.txt

The command above produces a file containing TaxID numbers of Betacoronaviruses. This file can the be used with the -taxidlist to define BLAST to do the search only against the sequences originating form the defined species. For example:

pb blastp -db nr -query queryset.fasta -taxidlist b-coronaviruses.txt -out corona_results 

Note that -taxidlist can be used only with databases that include species information.

Using genome data from ensembl with pb

pb command can also automatically retrieve a species specific dataset from the Ensembl or Ensembl genomes servers and use the dataset as the search database. This is done by replacing the -db option with option -ensembl_dna (retrieves the genomic DNA), -ensenmbl_cdna (retrieves the cDNA sequences) or -ensembl_prot (retrieves the protein sequences). The latin name of a species or taxonomy index number is given as an argument for the ensembl options. You should use underscore (_) in stead of space in the species name.

For example to compare a set of nucleotide sequences against the human genome, you could use a command like:

pb blastn -query dna_fargments.fasta -ensembl_dna homo_sapiens -out  human_hits.txt

To compare the same dna fragments against the protein sequences, predicted from the chicken genome, you could use command:

pb tblastn -query dna_fargments.fasta -ensembl_prot gallus_gallus -out  chicken_hits.txt

You can see the list of species, available at Ensembl and Ensembl genomes databases with command:

ensemblfetch.sh -names

Below is a list of BLAST databases maintained at the servers of CSC.

**Name	Database	Source file**
Nucleotides
nt	NCBI non-redundant nucleotide database	ftp://ftp.ncbi.nih.gov/blast/db/FASTA/
refseq	NCBI RefSeq RNA database	ftp://ftp.ncbi.nih.gov/refseq/release/complete/
refseq_con	NCBI RefSeq human contigs	ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/H_sapiens/
Proteins
nr	NCBI non-redundant protein database	ftp://ftp.ncbi.nih.gov/blast/db/FASTA/
pdb_v5	PDB protein structure database	ftp://ftp.rcsb.org/pub/pdb/derived_data/
swiss	Uniprot/Swiss database	ftp://ftp.ebi.ac.uk/pub/databases/uniprot/knowledgebase/
trembl	Uniprot/TrEMBL database	ftp://ftp.ebi.ac.uk/pub/databases/uniprot/knowledgebase/
uniprot	Uniprot Swiss and TrEMBL
uniref100	Uniref100 database	ftp://ftp.ebi.ac.uk/pub/databases/uniprot/uniref/uniref100/
uniref90	UniRef90 database	ftp://ftp.ebi.ac.uk/pub/databases/uniprot/uniref/uniref90/
uniref50	UniRef50 database	ftp://ftp.ebi.ac.uk/pub/databases/uniprot/uniref/uniref50/
Ensembl genomes	Select one of the species with pb options: -ensembl_dna, -ensembl_cdna or -ensembl_pep	ftp://ftp.ensembl.org/

Support

servicedesk@csc.fi

Manual

More information on Blast can be found from the BLAST page of NCBI

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