BLAST Tutorial

Overview of BLAST

BLAST, which stands for Basic Local Alignment Search Tool, is a cornerstone of modern bioinformatics. It's a suite of programs designed to identify regions of similarity between biological sequences. The tool can compare nucleotide or protein sequences to databases and calculate the statistical significance of the matches. This capability is crucial for a variety of scientific inquiries, including the identification of gene families, inference of functional and evolutionary relationships, and annotation of sequences.

BLAST was developed by the National Center for Biotechnology Information (NCBI) and has become an indispensable resource for researchers. It operates by taking a query sequence—either nucleotide or protein—and searching it against a database to find matching sequences. The original BLAST program focused on protein queries, but it soon expanded to include nucleotide queries and cross-comparisons between nucleotide and protein sequences through an intermediate translation step.

The tool is available in both standalone and web versions, with the latter offering access to complete genomes of various organisms, including humans, mice, fruit flies, and plants like Arabidopsis thaliana. This allows users to view BLAST alignments within the full genomic context, providing a more comprehensive understanding of the sequence relationships.

BLAST works by indexing strings of a certain length within the query sequence and then scanning the database for matches. The length of these strings, known as the "wordsize," is configurable and varies depending on the type of BLAST program used. For protein-to-protein searches, the typical wordsize is 3, while for nucleotide-to-nucleotide searches, it's usually 11. When a match is found, BLAST attempts to extend the alignment in both directions, continuing as long as the score increases or until it drops by a critical amount, known as the "dropoff."

The results of a BLAST search are presented in various alignment views, allowing users to see the query sequence aligned with one or multiple database sequences. These views can highlight conserved regions, gaps, and single nucleotide polymorphisms (SNPs), providing valuable insights into the sequence data.

In summary, BLAST is a powerful and versatile tool that has revolutionized the field of bioinformatics by enabling rapid and accurate comparisons of biological sequences.

Installation

To install BLAST, you would typically visit the NCBI website and download the appropriate version for your operating system. The installation process involves extracting the downloaded files and, for the standalone version, adding the BLAST program directory to your system's PATH environment variable. This allows you to run BLAST from any directory on your system.

For detailed installation instructions, it's best to refer to the official NCBI BLAST documentation, which provides step-by-step guidance for different platforms. It's important to ensure that your system meets the necessary requirements and that you download the correct version of BLAST for your needs.

Quick Start

Getting started with BLAST is straightforward, especially if you're using the web version. For a quick start, you can navigate to the BLAST web interface on the NCBI website, where you'll find options to input your query sequence and select the type of BLAST search you want to perform. You can choose from various databases and adjust search parameters to tailor the results to your specific research question.

The BLAST Quick Start mini-course available on the NCBI website is an excellent resource for beginners. It provides example-driven tutorials that guide you through the process of performing different types of BLAST searches, interpreting the results, and understanding the underlying theory that influences the choice of program, parameters, and database.

Code Examples Of Popular Commands

Here are five popular BLAST commands that you can use in the standalone version of the tool:

  1. blastn: This command is used for nucleotide-to-nucleotide BLAST searches. It compares a nucleotide query sequence against a nucleotide sequence database.
blastn -query my_query.fasta -db nt -out results.out
  1. blastp: This command is for protein-to-protein BLAST searches. It compares an amino acid query sequence against a protein sequence database.
blastp -query my_protein.fasta -db nr -out results.out
  1. blastx: This command is used to compare a nucleotide query sequence translated in all reading frames against a protein database.
blastx -query my_query.fasta -db nr -out results.out
  1. tblastn: This command is used to search protein queries against a nucleotide sequence database dynamically translated in all reading frames.
tblastn -query my_protein.fasta -db nt -out results.out
  1. tblastx: This command compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
tblastx -query my_query.fasta -db nt -out results.out

Each of these commands requires specifying a query file (-query), a database (-db), and an output file (-out). Additional parameters can be added to customize the search, such as limiting the search to specific organisms or adjusting the word size.

By mastering these commands and understanding how to interpret the results, researchers can leverage the full power of BLAST to advance their scientific inquiries.