Topic 4: Homology Search, FASTA, and BLAST

What is a homology search?

Searching a sequence database for sequences that may share common ancestry with a query.

What is a query sequence?

The sequence being searched against a database.

What is a subject sequence?

A database sequence compared with the query.

Why use heuristic homology search?

Exact local alignment of a query against every database sequence is too slow for large databases.

What is a heuristic?

A fast search shortcut that does not guarantee the optimal alignment or all true matches.

What is an exact homology search example?

Smith-Waterman applied against database sequences.

What are two heuristic search tools?

FASTA and BLAST.

What is the shared idea behind FASTA and BLAST?

Find short promising hits, then extend them into longer alignments.

How does FASTA begin?

It finds exact short-word matches (k-tuples).

How does BLAST begin?

It identifies words with similarity scores meeting a threshold T.

What is FASTA ktup?

Length of exact matching words used to seed alignments.

What happens if FASTA ktup increases?

Search is generally faster but less sensitive.

What is BLAST word length w?

Length of the query words used for seeding.

What is BLAST threshold T?

Minimum word-pair score required for a hit.

What is BLAST extension drop-off X?

How far the extension score can fall from its best value before extension stops.

What happens after FASTA finds promising regions?

It extends them and can refine the best alignments with dynamic programming.

What happens after BLAST finds promising hits?

It extends hits and evaluates their statistical significance.

What is an HSP?

High-Scoring Segment Pair: a high-scoring local alignment region.

What is a raw score S?

An alignment score based on substitutions and gaps.

What is a bit score?

A normalized alignment score; higher means a stronger alignment.

What is an E-value?

Expected number of chance alignments with an equal or better score.

How do you interpret a smaller E-value?

The alignment is more statistically significant.

What does an E-value displayed as zero mean?

Extremely small, rounded or displayed as zero; not literally impossible by chance.

What is the BLAST E-value formula?

E=Kmn×exp(−lambda×S).

What is the BLAST bit score formula?

S'=(lambda×S−ln K)/ln 2.

What is the relationship between E-value and p-value?

p=1−exp(−E).

What is sequence identity in search results?

Percentage of aligned positions with identical residues.

What is sequence similarity in protein results?

Resemblance including favorable nonidentical amino acid substitutions.

What is query coverage?

Fraction of the query sequence included in an alignment.

What is BLAST Max Score?

Highest bit score of an individual aligned segment.

What is BLAST Total Score?

Combined scores from aligned segments for a database sequence.

What is a false positive?

An unrelated sequence incorrectly identified as homologous.

What is a false negative?

A true homolog that is missed by the search.

What happens if the E-value cutoff is made smaller?

More stringent results; usually fewer false positives but potentially more false negatives.

Does a low E-value prove homology?

No; it provides statistical evidence supporting possible homology.

What makes a BLAST hit convincing?

Low E-value, high bit score, and meaningful identity and coverage together.