FASTA Format in Bioinformatics: Definition, Examples, and Uses

After finding short word matches, FASTA extends them ungapped using an optimized offset to detect regions of local similarity.

The k-tuple parameter (or ktup) is the size of the short, consecutive word that the FASTA algorithm searches for as a first step. A smaller k makes the search more sensitive (able to find more distant relationships) but slower, while a larger k speeds up the search but can miss less similar sequences.

Converting a FASTQ file to a FASTA file involves removing the quality scores, which are typically less important for downstream analyses like sequence alignment (e.g., using BLAST) or database searching. This process makes the file smaller and easier to work with when quality information isn't needed.

A multi-FASTA file simply contains more than one sequence, each starting with a > header line. They can be from one or multiple species.

Substitution matrices like BLOSUM62 assign higher scores to likely substitutions and lower or negative scores to unlikely substitutions, guiding alignment scoring.

Smaller k makes the search more sensitive (detects weaker similarities), while larger k makes it faster but may miss weak similarities.

This is not a strict rule but a recommended convention for readability and database compatibility.

Every sequence in a FASTA file starts with > followed by a header/identifier line. The actual sequence is written in the lines below it.

FASTA uses substitution matrices and gap penalties to score alignments, evaluating similarity beyond exact matches.

FASTA is lightweight and simple, unlike GenBank, which contains detailed annotations and complex structure.

N represents any nucleotide (A, T, G, or C) and is commonly used when the exact base is unknown.

Protein sequences in FASTA are written as one-letter codes (e.g., M, A, L, K). Three-letter codes are not used in FASTA.

.faa indicates protein (amino acid) FASTA sequences. Nucleotide sequences usually use .fna or .fa.

The header may contain GI numbers, accession IDs, or descriptions. These don’t affect the sequence but help identify its source.

DNA = A, T, G, C (sometimes N), proteins = one-letter amino acid codes (A, R, N, etc.). Nothing encoded or compressed.

FASTA only stores sequence letters and does not include quality scores, which are crucial in NGS for assessing sequencing reliability. For quality scores, formats like FASTQ are used.

FASTA only stores sequences; FASTQ has both sequences and base quality scores (important for NGS data).

Clustal Omega is a multiple sequence alignment (MSA) tool, optimized for aligning several sequences at once. FASTA/BLAST are for pairwise similarity searches.

The line starting with > is called the defline (definition line), which describes the sequence.

A heuristic algorithm, like FASTA or BLAST, is designed to be computationally efficient. Instead of exhaustively comparing every possible alignment (which would be too slow for large databases), it uses a shortcut—like finding short exact matches first—to quickly identify likely regions of similarity. While fast, this approach means it might occasionally miss the best possible alignment.

FASTA starts with finding short exact word matches, then extends them to high-scoring regions, making it heuristic (faster than full dynamic programming).

BLAST is optimized for speed, while FASTA can detect weaker similarities but is slower.

Protein alignment uses substitution matrices (PAM, BLOSUM). GC content and transition/transversion ratios are for nucleotides, not proteins.