### Mastering BLAST: A Comprehensive Tutorial
#### Introduction to BLAST
BLAST, which stands for Basic Local Alignment Search Tool, is a powerful
bioinformatics program used for comparing an input sequence (query) against a
database of sequences. It identifies regions of local similarity, helping researchers
infer functional and evolutionary relationships between sequences and identify
members of gene families. Developed by Altschul et al. in 1990, BLAST has
become one of the most widely used tools in molecular biology and genetics.
#### Types of BLAST Programs
BLAST comes in several flavors, each tailored for specific types of sequence
comparisons:
1. **BLASTN**: Compares a nucleotide query sequence against a nucleotide
database.
2. **BLASTP**: Compares an amino acid query sequence against a protein
database.
3. **BLASTX**: Compares a nucleotide query sequence, translated in all six
reading frames, against a protein database.
4. **TBLASTN**: Compares a protein query sequence against a nucleotide
database translated in all six reading frames.
5. **TBLASTX**: Compares the six-frame translations of a nucleotide query
sequence against the six-frame translations of a nucleotide database.
Each program serves specific research needs, from identifying homologous genes
to studying evolutionary patterns.
#### Preparing for a BLAST Search
Before running a BLAST search, it's crucial to prepare your query sequence and
choose the appropriate database and parameters. Here are the key steps:
1. **Query Sequence**: Obtain the sequence you want to analyze. Ensure it is in
FASTA format, a standard text-based format for representing nucleotide or protein
sequences.
2. **Database Selection**: Choose the database against which you want to
compare your query. Common databases include:
- **nr**: Non-redundant database containing a diverse collection of sequences.
- **refseq**: Curated collection of reference sequences.
- **swissprot**: Manually annotated and reviewed protein sequences.
- **wgs**: Whole genome shotgun sequences.
3. **BLAST Program**: Select the appropriate BLAST program based on the type
of your query and the database.
4. **Parameters**: Customize search parameters to refine your search results:
- **Expect Value (E-value)**: Threshold for reporting matches. Lower E-values
indicate more significant matches.
- **Word Size**: Size of the initial matching segment. Smaller word sizes can
increase sensitivity but decrease speed.
- **Scoring Matrices**: For protein searches, choose an appropriate scoring
matrix (e.g., BLOSUM62 for general purposes).
#### Running a BLAST Search
Most BLAST searches are conducted online through the NCBI BLAST web
interface. Here’s a step-by-step guide to running a BLAST search:
1. **Access the NCBI BLAST Website**: Navigate to the NCBI BLAST
homepage (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
2. **Select the BLAST Program**: Choose the appropriate BLAST program
(BLASTN, BLASTP, BLASTX, TBLASTN, or TBLASTX) based on your query
sequence.
3. **Input the Query Sequence**: Paste your sequence into the query box or
upload a file in FASTA format.
4. **Select the Database**: Choose the database you want to search against.
5. **Set Parameters**: Customize parameters such as the E-value threshold, word
size, and scoring matrix.
6. **Submit the Search**: Click the “BLAST” button to initiate the search.
7. **Review Results**: Once the search is complete, results will be displayed in a
format that includes:
- **Summary Table**: Lists significant alignments with scores, E-values, and
descriptions.
- **Graphical Overview**: Visual representation of alignments along the query
sequence.
- **Alignments**: Detailed alignments showing the query and subject sequences
with matching regions highlighted.
#### Interpreting BLAST Results
Understanding BLAST results is crucial for drawing meaningful conclusions from
your search. Key elements of the BLAST output include:
1. **Score and Bit Score**: Indicate the quality of the alignment. Higher scores
suggest better alignments.
2. **E-value**: Indicates the number of matches expected by chance. Lower E-
values denote more significant matches. An E-value of 0 means the match is highly
significant.
3. **Identity**: Percentage of identical matches between the query and subject
sequences.
4. **Query Coverage**: Percentage of the query sequence that is aligned with the
subject sequence.
5. **Alignment**: Shows the actual alignment between the query and subject
sequences, highlighting matches, mismatches, and gaps.
#### Advanced BLAST Features
BLAST offers several advanced features to enhance your searches:
1. **Filtering Low Complexity Regions**: Masks regions of the query sequence
that have low complexity to prevent them from dominating the alignment scores.
2. **Composition-Based Statistics**: Adjusts scores based on the composition of
sequences to improve the statistical significance of the results.
3. **Gapped vs. Ungapped BLAST**: Gapped BLAST allows for insertions and
deletions in alignments, whereas ungapped BLAST does not. Gapped BLAST is
more realistic for biological sequences.
4. **PSI-BLAST (Position-Specific Iterative BLAST)**: An iterative version of
BLASTP that builds a position-specific scoring matrix (PSSM) from the first round
of hits and uses it for subsequent searches. Useful for finding distant homologs.
5. **MegaBLAST**: Optimized for aligning very similar sequences and is much
faster than the standard BLASTN. Ideal for large-scale nucleotide searches.
#### Practical Applications of BLAST
BLAST has numerous applications in biological research:
1. **Gene Identification**: Identifying unknown genes by comparing them with
known sequences in databases.
2. **Evolutionary Studies**: Analyzing the evolutionary relationships between
sequences from different organisms.
3. **Functional Annotation**: Inferring the function of a gene or protein based on
its similarity to known sequences.
4. **Genome Assembly**: Assisting in the assembly and annotation of genomes
by comparing sequencing reads to reference genomes.
5. **Medical Research**: Identifying potential drug targets and understanding the
genetic basis of diseases by comparing pathogenic sequences with known
sequences.
#### Conclusion
BLAST is an indispensable tool in bioinformatics, offering robust and versatile
options for sequence comparison. By understanding how to effectively use
BLAST, from selecting the appropriate program to interpreting the results,
researchers can gain deep insights into the genetic and functional landscape of
biological sequences. Whether you are identifying a new gene, exploring
evolutionary relationships, or annotating a genome, mastering BLAST is a key
skill in the modern biologist’s toolkit.
