Class 7: BLAST

Bioinformatics

Andrés Aravena

October 21, 2022

Looking for sequences in databases

We know how to compare two sequences

GROUSE
M-OUSE
  • We 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

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

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
    • 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

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

NCBI nucleotide (reads)

SRA
Sequence Read Archive
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/