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.

• BLAST
  • License
  • Available
  • Usage
  • Interactive usage in Puhti
  • Usage of pb (Parallel BLAST) at CSC
  • Using own BLAST databases with pb
  • Using taxonomy to focus the search
  • Using genome data from Ensembl with pb
  • Support
  • More information

License

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

Available

• Puhti: 2.15.0
• Chipster graphical user interface

Usage

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

• using the Chipster platform
• with normal BLAST commands in interactive 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 command:

module load biokit

Then launch an interactive batch 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 output 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:

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. Instead, pb command creates and submits a batch job automatically. Once BLAST job is started, pb starts a process that monitors the progress of the blast job. As running a large BLAST job may take a long time, you may need to 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 than 1 GB of memory (that is the job specific memory limit in Puhti login nodes). In those cases you can submit the job from an interactive batch job (with e.g. 8 GB of memory).

Using taxonomy to focus the search

The general purpose NCBI BLAST databases like NT or NE have grown very large, which slows the searches in Puhti. Thus, if you can focus your search to only a subset of these databases it makes the search faster and prevents non-important hits to be reported.

Starting with BLAST+ 2.15.0, the BLAST+ command line applications support a new feature: they accept non-leaf taxIDs (i.e., those above an organism level, such as the one for primates). For example to focus the search to only bird sequences (Taxonomy ID: 8782) of NR database you could use command

pb blastp -db nr - query test.fasta -taxids 8782 -out test.res

If you know that you will use a specific subset of NR or NT databases several times, it is more effective to filter this part of sequences out once and then index your own database form the filtered sequences. You can use command blastdbcmd to filter out a specific taxonomic group from NR or NT databases.

For example all bird sequences can be retrieved from NR database with command:

blastdbcmd -taxids 8782 -db nr -dbtype prot -out nrbirds.fasta -target_only

The resulting fasta file can be indexed for BLAST searches with makeblastdb command.

makeblastdb -in nrbirds.fasta -dbtype prot

To use your own database, you must redefine environment variable BLASTDB so that it points to the directory where you have the index. For example if the indexes locate in directory my_blastdb in the scratch directory of project project_20012345, the variable would be set with command:

export BLASTDB=/scrartch/project_20012345/my_blastdb

Now you can use your own database in the BLAST search:

pb blastp -db nrbirds.fasta - queryquery.fasta -out bird-only-hits.res

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), -ensembl_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 (_) instead 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

Contact CSC Service Desk for technical support.

More information

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

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