[Optional] Using Singularity to run BLAST+
Overview
Teaching: 30 min
Exercises: 30 minQuestions
How can I use Singularity to run bioinformatics workflows with BLAST+?
Objectives
Show example of using Singularity with a common bioinformatics tool.
We have now learned enough to be able to use Sigularity to deploy software without us needed to install the software itself on the host system.
In this section we will demonstrate the use of a Singularity container image that provides the BLAST+ software.
Source material
This example is based on the example from the official NCBI BLAST+ Docker container documentation Note: the
efetchparts of the step-by-step guide do not currently work using Singularity version of the image so we provide a dataset with the data already downloaded.(This is because the NCBI BLAST+ Docker container image has the
efetchtool installed in the/rootdirectory and this special location gets overwritten during the conversion to a Singularity container image.)
Download the required data
Download the blast_example.tar.gz.
Unpack the archive which contains the downloaded data required for the BLAST+ example:
tar -xvf blast_example.tar.gz
x blast/
x blast/blastdb/
x blast/queries/
x blast/fasta/
x blast/results/
x blast/blastdb_custom/
x blast/fasta/nurse-shark-proteins.fsa
x blast/queries/P01349.fsa
Finally, move into the newly created directory:
cd blast
ls
blastdb blastdb_custom fasta queries results
Create the Singularity container image
NCBI provide official Docker containers with the BLAST+ software hosted on Docker Hub. We can create a Singularity container image from the Docker container image with:
singularity pull ncbi-blast.sif docker://ncbi/blast
INFO: Converting OCI blobs to SIF format
INFO: Starting build...
Getting image source signatures
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Storing signatures
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INFO: Creating SIF file...
Now we have a container with the software in, we can use it.
Build and verify the BLAST database
Our example dataset has already downloaded the query and database sequences. We first
use these downloaded data to create a custom BLAST database by using a container to run
the command makeblastdb with the correct options.
singularity exec ncbi-blast.sif \
makeblastdb -in fasta/nurse-shark-proteins.fsa -dbtype prot \
-parse_seqids -out nurse-shark-proteins -title "Nurse shark proteins" \
-taxid 7801 -blastdb_version 5
Building a new DB, current time: 06/16/2023 14:35:07
New DB name: /home/auser/test/blast/blast/nurse-shark-proteins
New DB title: Nurse shark proteins
Sequence type: Protein
Keep MBits: T
Maximum file size: 3000000000B
Adding sequences from FASTA; added 7 sequences in 0.0199499 seconds.
To verify the newly created BLAST database above, you can run the
blastdbcmd -entry all -db nurse-shark-proteins -outfmt "%a %l %T" command to display
the accessions, sequence length, and common name of the sequences in the database.
singularity exec ncbi-blast.sif \
blastdbcmd -entry all -db nurse-shark-proteins -outfmt "%a %l %T"
Q90523.1 106 7801
P80049.1 132 7801
P83981.1 53 7801
P83977.1 95 7801
P83984.1 190 7801
P83985.1 195 7801
P27950.1 151 7801
Now we have our database we can run queries against it.
Run a query against the BLAST database
Lets execute a query on our database using the blastp command:
singularity exec ncbi-blast.sif \
blastp -query queries/P01349.fsa -db nurse-shark-proteins \
-out results/blastp.out
At this point, you should see the results of the query in the output file results/blastp.out.
To view the content of this output file, use the command less results/blastp.out.
less results/blastp.out
...output trimmed...
Query= sp|P01349.2|RELX_CARTA RecName: Full=Relaxin; Contains: RecName:
Full=Relaxin B chain; Contains: RecName: Full=Relaxin A chain
Length=44
Score E
Sequences producing significant alignments: (Bits) Value
P80049.1 RecName: Full=Fatty acid-binding protein, liver; AltName... 14.2 0.96
>P80049.1 RecName: Full=Fatty acid-binding protein, liver; AltName: Full=Liver-type
fatty acid-binding protein; Short=L-FABP
Length=132
...output trimmed...
With your query, BLAST identified the protein sequence P80049.1 as a match with a score of 14.2 and an E-value of 0.96.
Accessing online BLAST databases
As well as building your own local database to query, you can also access databases that are available online. For example, to see which databases are available online in the Google Compute Platform (GCP):
singularity exec ncbi-blast.sif update_blastdb.pl --showall pretty --source gcp
Connected to GCP
BLASTDB DESCRIPTION SIZE (GB) LAST_UPDATED
nr All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects 369.4824 2023-06-10
swissprot Non-redundant UniProtKB/SwissProt sequences 0.3576 2023-06-10
refseq_protein NCBI Protein Reference Sequences 146.5088 2023-06-12
landmark Landmark database for SmartBLAST 0.3817 2023-04-25
pdbaa PDB protein database 0.1967 2023-06-10
nt Nucleotide collection (nt) 319.5044 2023-06-11
pdbnt PDB nucleotide database 0.0145 2023-06-09
patnt Nucleotide sequences derived from the Patent division of GenBank 15.7342 2023-06-09
refseq_rna NCBI Transcript Reference Sequences 47.8721 2023-06-12
...output trimmed...
Similarly, for databases hosted at NCBI:
singularity exec ncbi-blast.sif update_blastdb.pl --showall pretty --source ncbi
Connected to NCBI
BLASTDB DESCRIPTION SIZE (GB) LAST_UPDATED
env_nr Proteins from WGS metagenomic projects (env_nr). 3.9459 2023-06-11
SSU_eukaryote_rRNA Small subunit ribosomal nucleic acid for Eukaryotes 0.0063 2022-12-05
LSU_prokaryote_rRNA Large subunit ribosomal nucleic acid for Prokaryotes 0.0041 2022-12-05
16S_ribosomal_RNA 16S ribosomal RNA (Bacteria and Archaea type strains) 0.0178 2023-06-16
env_nt environmental samples 48.8599 2023-06-08
LSU_eukaryote_rRNA Large subunit ribosomal nucleic acid for Eukaryotes 0.0053 2022-12-05
ITS_RefSeq_Fungi Internal transcribed spacer region (ITS) from Fungi type and reference material 0.0067 2022-10-28
Betacoronavirus Betacoronavirus 55.3705 2023-06-16
...output trimmed...
Notes
You have now completed a simple example of using a complex piece of bioinformatics software through Singularity containers. You may have noticed that some things just worked without you needing to set them up even though you were running using containers:
- We did not need to explicitly bind any files/directories in to the container. This worked because Singularity automatically binds the current directory into the running container, so any data in the current directory (or its subdirectories) will generally be available in running Singularity containers. (If you have used Docker containers, you will notice that this is different from the defalt behaviour there.)
- Access to the internet is automatically available within the running container in the same way as it is on the host system without us needed to specify any additional options.
- Files and data we create within the container have the right ownership and permissions for us to access outside the container.
In addtion, we were able to use the tools in the container image provided by NCBI without having to do any work to install the software irrespecetive of the computing platform that we are using. (In fact, the example this is based on runs the pipeline using Docker on a cloud computing platform rather than on your local systeam.)
Key Points
We can use containers to run software without having to install it
The commands we use are very similar to those we would use natively
Singularity handles a lot of complexity around data and internet access for us