# Looking for sequences in databases

## 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

## What is the best threshold?

We want big scores

How big is big enough?

We need to make several hypothesis

The most common hypothesis is statistical

## 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

## 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
• can also send jobs in NCBI servers with NCBI databases

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

## NCBI protein databases

nr
Non-redundant protein sequences
refseq_protein
Reference proteins
refseq_select
Reference Select 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

## NCBI protein databases

landmark
Model Organisms
swissprot
UniProtKB/Swiss-Prot
pat_aa
Patented protein sequences

## NCBI protein databases

pdb
Protein Data Bank proteins
env_nr
Metagenomic proteins
tsa_nr
Transcriptome Shotgun Assembly proteins

## NCBI nucleotide databases

Human G+T
Human genomic plus transcript
Mouse G+T
Mouse genomic plus transcript
nr/nt
Nucleotide collection

## NCBI nucleotide databases

Bacteria and Archaea
16S ribosomal RNA sequences
refseq_select
Reference Select sequences
refseq_rna
Reference RNA sequences

## NCBI nucleotide databases

refseq_representative_genomes
RefSeq Representative genomes
refseq_genomes
RefSeq Genome Database

SRA
TSA
Transcriptome Shotgun Assembly
HTGS
High throughput genomic sequences

## NCBI nucleotide databases

pat
Patent sequences
pdb
nucleotides in Protein Data Bank
RefSeq_Gene
Human RefSeqGene sequences

## BlastN variants

megablast
Highly similar sequences
discontiguous megablast
More dissimilar sequences
blastn
Somewhat similar sequences

## 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.

## Let’s test it

Let’s look for Terje Steinum’s data

This is a 16S gene amplified by PCR

(see the course’s Homepage)

## Let’s go to the NCBI website

https://blast.ncbi.nlm.nih.gov/