Wikiomics:BLAST tutorial

Blast most popular DNA/protein sequence search algorithm tool. See also Wikipedia:BLAST.

This tutorial covers previous version of blast (blastall). The newest BLAST+ tools use NCBI C++ Toolkit and offer new functionality not covered here. For detailed help on command line blastall switches type:

blastall --help

Download blast searches results from here

BLAST input

• sequence as FASTA

>MIMI_L4 complement(6238..7602)
MPQKTSKSKSSRTRYIEDSDDETRGRSRNRSIEKSRSRSLDKSQKKSRDK
SLTRSRSKSPEKSKSRSKSLTRSRSKSPKKCITGNRKNSKHTKKDNEYTT
EESDEESDDESDGETNEESDEELDNKSDGESDEEISEESDEEISEESDED
VPEEEYDDNDIRNIIIENINNEFARGKFGDFNVIIMKDNGFINATKLCKN

• accession number (web only)

proteins:

AAA68881.1

• gi number

129295
gi|129295

More about input formats is @NCBI

Databases

Nucleotide

• nr - All Non-redundant GenBank+EMBL+DDBJ+PDB sequences (no EST, STS, GSS)
• month - All new GenBank+EMBL+DDBJ+PDB sequences released in the last 30 days
• est - Non-redundant Database of GenBank+EMBL+DDBJ EST Divisions
• est_human - human EST sequences
• est_mouse - mouse EST sequences

and many more..

Protein

• nr - All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF
• month - GenBank CDS translation+PDB+SP+PIR+PRF released in the last 30 days.
• swissprot - the last major release of the SWISS-PROT protein sequence database

+++

BLAST algorithms

regular BLAST

blastn

• nucleotide query vs nucleotide database

>DNA_seq1
CGGGAGGCGGCAGCGGCTGCAGCGTTGGTAGCATCAGCATCAGCATCAGCGGCAGCGGCAGCGGCCTCGG
GCGGGGCCGGCCGGACGGACAGGCGGACAGAAGGCGCCAGGGGCGCGCGTCCCGCCCGGGCCGGCCATGG
AGGGCGCCTCCTTCGGCGCGGGCCGCGCAGGGGCCGCCCTGGACCCCGTGAGCTTTGCGCGGCGGCCCCA

• select Somewhat similar sequences (blastn)
• use nr/nt database

blastp

• protein query vs protein DB

>MIMI_L4 complement(6238..7602)
MPQKTSKSKSSRTRYIEDSDDETRGRSRNRSIEKSRSRSLDKSQKKSRDK
SLTRSRSKSPEKSKSRSKSLTRSRSKSPKKCITGNRKNSKHTKKDNEYTT

• search nr protein database

blastx

• translated in 6 frames nucleotide query vs protein DB

>human_genomic_seq
TGGACTCTGCTTCCCAGACAGTACCCCTGACAGTGACAGAACTGCCACTCTCCCCACCTG
ACCCTGTTAGGAAGGTACAACCTATGAAGAAAAAGCCAGAATACAGGGGACATGTGAGCC
ACAGACAACACAAGTGTGCACAACACCTCTGAGCTGAGCTTTTCTTGATTCAAGGGCTAG
TGAGAACGCCCCGCCAGAGATTTACCTCTGGTCTTCTGAGGTTGAGGGCTCGTTCTCTCT
TCCTGAATGTAAAGGTCAAGATGCTGGGCCTCAGTTTCCTCTTACATACTCACCAAAAGG
CTCTCCTGATCAGAGAAGCAGGATGCTGCACTTGTCCTCCTGTCGATGCTCTTGGCTATG
ACAAAATCTGAGCTTACCTTCTCTTGCCCACCTCTAAACCCCATAAGGGCTTCGTTCTGT
GTCTCTTGAGAATGTCCCTATCTCCAACTCTGTCATACGGGGGAGAGCGAGTGGGAAGGA
TCCAGGGCAGGGCTCAGACCCCGGCGCATGGACCTAGTCGGGGGCGCTGGCTCAGCCCCG
CCCCGCGCGCCCCCGTCGCAGCCGACGCGCGCTCCCGGGAGGCGGCGGCAGAGGCAGCAT
CCACAGCATCAGCAGCCTCAGCTTCATCCCCGGGCGGTCTCCGGCGGGGAAGGCCGGTGG
GACAAACGGACAGAAGGCAAAGTGCCCGCAATGGAGGGAGCATCCTTTGGCGCGGGCCGT
GCGGGAGCTGCCTTTGATCCCGTGAGCTTTGCGCGGCGGCCCCAGACCCTGTTGCGGGTC
GTGTCCTGG

• search human proteins database:
  • BLAST@NCBI ->
  • Human ->
  • select:
    • Database: RefSeq protein
    • Program: BLASTX

tblastn

• protein query vs translated in 6 frames nucleotide database

>Q9BDJ6|GHRL_BOVIN Appetite-reg.hormone precursor
MPAPWTICSLLLLSVLCMDLAMAGSSFLSPEHQKLQRKEAKKPSGRLKPRTLEGQFDPEV
GSQAEGAEDELEIRFNAPFNIGIKLAGAQSLQHGQTLGKFLQDILWEEAEETLANE

• search non-human, non-mouse ESTs

tblastx

• translated in 6 frames nucleotide query vs translated in 6 frames nucleotide database
• nowadays seldom used except ESTs/genomic sequences of more exotic genomes

>gi|50539273|gb|CO636075.1 Gregarina niphandrodes 
GCCATTACGGCCGGGGGAAAGAAGTACTACCAACACAATGGTGGCTGATGACAAGATAATCACCACGAAG
GTTGTTTCTAAGAAGGTGGTTTCCACCAAGGTGGTCACCGGCGGCGACAAGAAGAAGAAGCATGTTGTTG
CCGAATAATTGATCTACATTATTAACTTGTTCATCAACATCCTCAACGACCTCATCATCATCTCCTACAC
GATGATCATCGTACACAACTTCTTACTCGACAACTTCTCCTCAATCTTCTATGATGCCACCAACCTCTGC
TGCCGCACCACCACTAATACAATTGTTGCTCGTCAAATCCTCAACGCCGTCCGTCTTATCATCCTCGCCG
AACTGTTGACCCACGCTACCACCAACGGGGCCACCGCCGCCACCCACTACTACAGACCCGCCTACCAACG
TTGTACATACATATTCTACATCATCAACGACTGATGCCACCGAGCTTCTTTCCACGCCTGATCTACTTGC
CCACCCAACAACCCCCCTAAAACAAAATCTGTGTCCCCCGTCTCGGCCCGCGTATCCAATATTTCTAGAG
CCGCCCCCACCGGTGCGGATCCCCCTCTTGTGCCCCCCTTCACGGGGGTTTATTCCCGCCCGTGGGTGTA
CCACAGTGCCCCACACCTGGTCCCCTGGTGCACAAACGTGCTTACCCCCTCTACAC

• try blastx first (no sensible hits) then
• tblastx vith non-human, non-mouse ESTs (some hits to other ESTs)

megaBLAST

• very fast search for highly similar DNA sequences (95% or more )
• good for:
  • cDNA vs genome (same or very close species)
  • splice variants of the same gene

PSI-BLAST Position-Specific Iterative BLAST

• starts with a regular blastp search (protein query vs protein database)
• uses best hits above a threshold (E -10^5) to create alignment and compute protein profile
• uses protein profile to perform next iterations of search, adding new best hits to the profile
• very good at picking distant relationships between proteins

>X.trop_syngr1_NP_001016195.1
MEGGAYGAGKAGGAFDPQTFVRQPHTVLRMVSWVFSIVVFGCIINEGYINASTEAEEHCIFNRNSSACAY
GVTVGVLAFLTCLLYLAVDVYFPQISSVKDRKKTVISDIAVSGLWAFFWFVGFCFLANQWQVSNPNDNPM
NEGADAARAAIAFSFFSIFTWAGQAVLAYQRYRLGSDSALFSQDYMDPSQDQGPPYPPYASNEDLDPSAG
YQQPPSDAYDAGSQGYQTQDY

• run search with regular blastp, observe hits
• repeat serch with PSI-BLAST, run 3 iterations, note novel hits

Command line BLAST

check first if $BLASTDB and $BLASTMAT are set (it should display these names sans $ signs & pathways):

env | grep BLAST

If you get a blank and these are not set in your '~/.ncbirc' file (tcsh):

setenv $BLASTDB /path/to/directory/with/blast/databases/
setenv $BLASTMAT /path/to/your/director/blast/data

or follow instructions for creating '~/.ncbirc' from here: [1]

to create a database out of your fasta file:

formatdb -i your_multiple_protein_file.fa -p T -V
formatdb -i your_multiple_DNAseq_file.fa  -p F -V

For more options type 'formatdb -h'

• • nucleotide search

blastall -p blastn -d ecoli_nn -i my_nn_fasta_file.fa -o my_nn_fasta_file_vs_ecolidb.blastn_out

• • protein search

blastall -p blastp -d ecoli_aa -i my_nn_fasta_file.fa -o my_nn_fasta_file_vs_ecolidb.blastp_out

Non-command line BLAST

BLAST can be setup to be queried using an internet browser instead of a command line, using NCBI's wwwblast software. The command line must be used during the setup of wwwblast, but not when running the actual BLAST queries.

Advanced topics

Substitution Matrices

For calculating scores of protein query vs putative protein hit BLAST uses so called substitution matrices. These are calculated from multiple protein alignments by counting amino acid frequences in a given columns. You can find more info @NCBI.

By default blastp uses BLOSUM62 ( a matrix constructed from local alignment of proteins with similarity 62% and better). One can use BLOSUM45 for more divergent sequences and BLOSUM80 for closer related ones.

For very short protein queries (15 aa and less) use PAM30 [1].

See also: [2]

1. Jonathan Pevsner, Bioinformatics and Functional Genomics (John Wiley and Sons, 2009), p110.

Word size

BLAST uses 'chunks' of a given length as an initial matches between query sequence and putative match in database. There is a trade off (sensitivity vs speed):

• shorter the word size, more sensitive the search.
• longer word sizes, faster the search

Typical word sizes:

• blastp: 3 (default), 2
• blastn: 28 (default), up to to 64

More @NCBI

Filters and Masking

Input query sequence may contain:

• repetitive sequences
  • simple i.e CACACA, proline-rich etc. (filtered by default by DUST/SEG)
  • complex i.e. Alu/LINE1 sequences (filtered by using "Species-specific repeats" and picking the correct organism, if available)
  • CAVEAT: transmembrane and coiled-coil regions are not masked by NCBI BLAST tools and therefore will give a spurious, non-homologous hits. You have to detect them and mask them yourself.

• custom filtering (lower case masking)

By providing query sequence i.e. genomic sequence with EXONS as capitals and introns in small letters we can search exonic sequences only.

Filtering blast output using Entrez query

One can be interested only in blast hits from i.e. specific organism. On a command line:

blastall -u "[Arabidopsis thaliana[organism]" -p blastp -d nr -i my_protein_input.fas -o my_protein_input_vs_nr_Athaliana.blastout.txt 

This can also be written as:

blastall -u "txid3702[ORGN]" -p blastp -d nr -i my_protein_input.fas -o my_protein_input_vs_nr_Athaliana.blastout.txt 

BLAST implementations

Read:

• Cha, I.E., and E.C. Rouchka. “Comparison of Current BLAST Software on Nucleotide Sequences.” In Parallel and Distributed Processing Symposium, 2005. Proceedings. 19th IEEE International, 8 pp., 2005.

Most popular are WU-BLAST and a distant second MPI-BLAST. Things to watch: ScalaBLAST, Grid-based implementation on one side and FPGA/CUDA hardware.

WU-BLAST / AB-BLAST

Often faster implementation of BLAST algorithm from Washington University.

• http://blast.wustl.edu/

Program was licensed to Advanced Biocomputing and becoming AB-BLAST. Previous licenses are still valid, but WU-BLAST versions except an obsolete 2.0a19 from 1998 are unavailable. Latest AB-BLAST version is 2009-10-30. Licensing is here (1k$ /year/lab, free for personal use on your own computer).

MPI-BLAST

BLAST implementation suitable for clusters, under active development.

• http://www.mpiblast.org/

Latest version: mpiBLAST-1.6.0 from 2010-07-13.

ScalaBLAST

Novel implementation for clusters, requires MPI.

• http://hpc.pnl.gov/projects/scalablast/

Latest version: 1.0.640 from 2010-04-05

CS-BLAST

New protein search program claimed to be more (twice) as sensitive as blastp.

• http://toolkit.lmb.uni-muenchen.de/cs_blast

Biegert, A. and Soding, J. (2009) Sequence context-specific profiles for homology searching. Proc Natl Acad Sci USA, 106 (10), 3770-3775

Latest version: 2.1.0 from 2009-08-17

FSA-BLAST

Fast but not tested/popular implementation by Michael Cameron (ver. 1.05 Released: 8 March 2006)

• http://fsa-blast.sourceforge.net/

Hardware speedups

There are several BLAST implementations running on FPGA processors. There are also projects porting BLAST to fast but not mainstream hardware, starting from Cell to GPUs.

DeCypher

from TimeLogic http://www.timelogic.com/decypher_intro.html

BioBoost

from Progeniq http://www.progeniq.com/

Other efforts

• Mercury BLASTP
• CAAD BLASTP

External tutorials/courses

• Tutorial_1 @NCBI
• PSI-BLAST tutorial @NCBI
• Altschul @NCBI The Statistics of Sequence Similarity Scores
• BLAST 'Rules of Thumb' etc @NCBI
• lecture @Knowledge Systems Institute good graphics
• tutorial @Geospiza