?? BLAST Beyond the Browser: Unlocking the Power of Local Sequence Analysis

When it comes to comparing biological sequences, BLAST (Basic Local Alignment Search Tool) is one of the most powerful tools in a bioinformatician’s toolkit. Typically, we use the online BLAST tool hosted by NCBI, which allows us to compare a sequence against huge public databases. But did you know that you can run BLAST offline on your own computer? Welcome to the world of local offline BLAST, where you take control of your own sequence searches without relying on internet access!

In this article, we’ll explore what local BLAST is, why it’s worth setting up, and how to use it. We'll also cover building custom databases, the different types of BLAST searches, and step-by-step commands to get started with local BLAST.

What is BLAST?

BLAST (Basic Local Alignment Search Tool) is a family of algorithms used to identify regions of similarity between biological sequences (DNA, RNA, or protein). It works by comparing an input sequence, or query, against a database of known sequences to find matches based on local alignment. Instead of aligning entire sequences globally, BLAST identifies short, similar regions, which makes it much faster than many other alignment tools.

Why Choose Local Offline BLAST?

Using BLAST locally has many advantages that make it essential for bioinformaticians:

1. Speed: Running BLAST searches locally can be much faster for large datasets. No waiting for uploads!
2. Privacy: Your data stays on your computer, which is especially helpful when working with unpublished or sensitive sequences.
3. Customization: Local BLAST allows you to use custom databases with genomes of your interest, fine-tune parameters, and even create entirely new databases tailored to your research.
4. Batch Processing: Local BLAST allows you to run multiple sequences together in one file, which is incredibly useful when analyzing large datasets or multiple samples simultaneously.

Setting Up Local BLAST

To get started, you’ll need to install BLAST+ on your system. BLAST+ is the command-line version of BLAST, available for download from NCBI. You can also install it directly on some systems using package managers.

Option 1: Install with sudo apt install

On Debian-based systems (such as Ubuntu), you can quickly install BLAST+ using this command:

$ sudo apt install ncbi-blast+

This command will download and install the BLAST+ package from the official repositories, which includes tools like blastn, blastp, and makeblastdb. However, this version might not always be the latest. If you need the most recent features or updates, downloading directly from NCBI is recommended.

Option 2: Download and Install the Latest BLAST+ from NCBI

Visit the NCBI BLAST+ download page and download the appropriate version for your operating system. To make it easier to use, add the BLAST installation directory to your system’s PATH in bashrc file. This allows you to run BLAST commands directly from any terminal window.

Creating Your Own BLAST Database

One of the biggest advantages of local BLAST is the ability to build custom databases. This is particularly useful if you’re working with a specific set of sequences, like those from your own research or a unique dataset.

Step 1: Format Your Data

First, make sure your sequences are in FASTA format. Each sequence should have a header starting with a “>” symbol, followed by a unique identifier. For example:

>Sequence_1 AGCTGACTGAGCTA...

>Sequence_2 CGTAGCTAGGCTGA...

Step 2: Build the Database

With your FASTA file ready, use the makeblastdb command to build a BLAST-compatible database:

$ makeblastdb -in your_sequences.fasta -dbtype nucl -out my_custom_db

• -in specifies the input file.
• -dbtype specifies the type of database (nucl for nucleotide, prot for protein).
• -out specifies the name of the database.

Now, you have a database that’s ready to be queried locally with BLAST!

Common Output Files from makeblastdb

1. *.nhr – Binary file for the nucleotide database or header file for protein databases.
2. *.nin – Binary file for the nucleotide database index or index file for protein databases.
3. *.nsq – Contains nucleotide sequence data or sequence file for protein databases.

For protein databases, these files will have extensions like *.phr, *.pin, and *.psq, following the same structure as above.

#NCBI even provides pre-built databases that you can download and use offline. Some popular ones are nt (nucleotide), nr (non-redundant protein), and SwissProt (curated protein sequences).

Running BLAST Searches Locally

Now that your database is set up, it’s time to run some searches. Here are some common types of BLAST searches and example commands.

1. Nucleotide BLAST (blastn)

To search a nucleotide query against a nucleotide database:

$ blastn -query your_query.fasta -db my_custom_db -out results.txt -evalue 0.01 -outfmt 6

• -query is the file containing the query sequence.
• -db is the database to search against.
• -out specifies the output file.
• -evalue sets the E-value threshold (0.01 here).
• -outfmt sets the output format (6 gives tabular output).

2. Protein BLAST (blastp)

If you’re working with protein sequences, use blastp to compare your protein query to a protein database:

$ blastp -query your_protein.fasta -db swissprot -out results_protein.txt -evalue 1e-5 -outfmt 6

The options here are similar to blastn, but be aware that E-values tend to be stricter in protein searches (e.g., 1e-5).

Translating Nucleotide BLAST (tblastx)

For comparing translated nucleotide sequences to other translated sequences, use tblastx:

$ tblastx -query dna_sequence.fasta -db nt -out results_tblastx.txt -evalue 1e-3 -outfmt 7

This is helpful for finding homologous sequences even if the DNA sequences are not similar but encode similar proteins.

Common BLAST Output Formats:

1. 0 (Pairwise) Default format. It shows alignments in a pairwise view with detailed descriptions of matching sequences and alignment sections.
2. 5 (XML) Output in XML format. Useful for downstream processing with scripts or when working with software that can parse XML, such as bioinformatics pipelines or databases.
3. 6 (Tabular) Produces tab-separated values without headers, making it easy to read into analysis tools (like Excel, R, or Python). This format provides one line per hit with specific fields (default is 12 fields), which include:
4. 7 (Tabular with Comment Lines) Similar to format 6 but includes comment lines (headers) describing each field, which can be useful for understanding the output at a glance.
5. 10 (Comma-separated values - CSV) Similar to 6, but uses commas instead of tabs. This format is also easy to import into spreadsheets for further analysis.
6. 11 (BLAST Archive Format) A binary format designed to store BLAST results compactly. It can be read back into BLAST+ tools for further analysis, making it useful for saving and sharing complete BLAST results.
7. 15 (JSON) Outputs in JSON format, which is structured and lightweight, making it easy to integrate with web applications or other bioinformatics tools that can parse JSON.

Choosing the Right BLAST Tool

Each BLAST tool is optimized for specific types of comparisons:

• BLASTN: Nucleotide-to-nucleotide comparisons.
• BLASTP: Protein-to-protein searches.
• TBLASTN: Compares a protein to a nucleotide database (translates the nucleotide).
• TBLASTX: Translates both the query and database, useful for detecting distant homologs.

Bringing It All Together: Efficient and Flexible Sequence Analysis

Local offline BLAST is a powerful option for biologists and bioinformaticians alike. It combines the flexibility of customizable searches with the efficiency of working offline. Whether you’re working on sensitive data, building a unique database, or aiming for quicker searches, local BLAST is a valuable tool that lets you take full control of your sequence analysis.

Happy BLASTing!!!!