We know how to compare two sequences

GROUSE
M-OUSE

• We can calculate a distance between sequences
  • Smaller distances mean more similarity
  • It works for global and semi-global alignment
• We can calculate a score for each alignment
  • We can decide where to put gaps and substitutions to compare two sequences
  • Works for global, semi-global, and local alignment

Score can change

If mismatches and gaps have different cost, the score will change

Sometimes the optimal alignment changes

Therefore alignments are meaningless without knowing the scoring matrices

Later we will discuss how to choose the “best” scoring matrix for each case

Looking on a database

We have one sequence, called query

We compare our sequences with each sequence in a Database

(sequences in the database are called subjects)

We get the score of each alignment

We report all subjects with score over a threshold

Larger scores, less hits

A hit is a subject with score over a threshold

Larger score thresholds give less hits

We can estimate the number of hits in a given database, assuming randomness

That is called Expected value

Expected value as a threshold

In practice, we choose a small Expected value

(usually called E-value)

Something like 10^-5 or 10^-20

What we find is not random
and maybe it is biologically meaningful

E-value depends on the database

The formula for E-value depends on

• The substitution scoring matrix
• The query size
• The database size

Same alignments in different databases have different E-value
but the same score

BLAST

The most common tool for local alignment is BLAST

Basic Local Alignment Search Tool

BLAST is not Global Alignment

Using BLAST

There are two ways of using BLAST

• Going to NCBI’s website: https://blast.ncbi.nlm.nih.gov/
  • Runs in NCBI servers with NCBI databases
• Using a command line version
  • Runs in your server with your databases
  • can also send jobs in NCBI servers with NCBI databases
  • Download it from NCBI website

For today we can look at NCBI page

Types of BLAST

Depending on the alphabet of the query and subject

BlastN

Search nucleotides in nucleotide databases

BlastP

Search proteins in protein databases

BlastX

Search nucleotide in protein databases.

Each query is translated into 6 putative proteins

Types of BLAST

TBlastN

Search proteins in nucleotide databases.

Each subject is translated into 6 putative proteins

TblastX

Search nucleotides in nucleotide databases

Translate each query and each subject into 6 proteins

Compares all the resulting proteins

What is “Non-Redundant”?

These databases get data from several sources

Sometimes two people upload the same sequence but with different ID

For example, EMBL ID, GenBank ID, RefSeq ID, etc.

This database combines all identical entries into one, and keeps all the alternative IDs

BlastP variants

blastp

protein-protein BLAST.

PSI-BLAST

Position-Specific Iterated BLAST.

builds a position-specific scoring matrix.

PHI-BLAST

Pattern Hit Initiated BLAST.

limits alignments to those that match a pattern in the query.

BlastP variants

Quick BLASTP

Accelerated protein-protein BLAST.

very fast and works best if the target percent identity is 50% or more.

DELTA-BLAST

Domain Enhanced Lookup Time Accelerated BLAST.

builds a PSSM using a Conserved Domain Database search.

searches a sequence database.