School B&I TCD Bioinformatics

Database homology searching

May 2010

Why search a Database

• To find homologous sequences to your unknown to determine function

• To find other related sequences to do evolutionary studies (trees) or to make specialised database (nematode 16sRNA)

• To find the mouse or E.coli homolog of your gene of interest

• To find genes in a newly sequenced genome• To predict 3-D structure (blast vs PDB)

BLAST

• For many molecular biologists

• a Blast search – with a DNA sequence – at NCBI – accepting default parameters

• IS bioinformatics– 70% of searches at NCBI at blastN

BLAST

• Basic local alignment search tool• More or less unchanged for 20 years• Extraordinarily quick:

– Submit seq via satellite to Bethesda MD– Crunch thro 400 GB of sequence– Return results via satellite– ALL in 30 seconds

• Available at other sites (EBI, ExPaSy etc.)• with better response times, fewer conditions

NCBI penalties

• 1st request: current time• 2nd request: current time + 60 seconds• 3rd request: current time + 120 seconds• 4th request: current time + 180 seconds• 5th request: current time + 240 second

• DON’T submit multiple simultaneous queries

Reliable servers

• http://www.ch.embnet.org/software/bBLAST.html • http://www.ch.embnet.org/software/aBLAST.html

– Basic and advanced SwissBlast

• http://www.ebi.ac.uk/blast2/index.html – EBI (Cambridge, UK) NCBI Blast

• http://www.ncbi.nlm.nih.gov/blast/– Not the only Blast server!

BLAST varieties• blastn: searches a DNA sequence against a DNA database like

EMBL, Genbank, or dbEST. also megablast for exact matches• blastp: searches a protein sequence against a protein database

such as UniProt, or "nr" a non-redundant database which ideally contains one copy of every available sequence.

Then you have:• blastx: searches a DNA sequence (translated in all six reading

frames) against a protein database.• tblastn: searches a protein sequence against a DNA database

(translated in all six reading frames) – essential for searching EST databases.

and in the interests of completeness there is:• tblastx: searches a DNA sequence (translated in all six reading

frames) against a DNA database (translated in all six reading frames).

finally• Psi-blast an iterative process using position specific subst matrix

PSSM to find distantly related sequences

Algorithm

• Five steps– Break the query seq into short “words” – typically 3

consecutive residues for protein– Search the database for (nearly) exact matches to

these words– Extend match “hits” out on either side until score

stops going up – these are HSPs (high scoring segment pairs)

– Sort the HSPs by some “optimum” criterion– Significant hits are then formally scored, aligned and

displayed

Alternatives to BLAST

• FASTA– A little slower than Blast– More sensitive (so recommended) for DNA to

DNA searches

• Smith-Waterman (Blitz)– Much slower than Blast (20x slower)– Much more sensitive

• But Blast is standard because it gives a “good enough” answer quickly.

Blast Blast at NCBI

Parallel search in Conserved domain DB

Paste your query sequence here

Input sequence parameters

Optional parameters to change include• Parameters that alter the hits found

– Database searched– Word size– Substitution matrix– Gap penalties– Low complexity masking

• Parameters that alter the results delivered– Expectation cut-off– Limit by organism or taxonomic group– Number of hits reported– Number of alignments shown

Database

• If query is a coding gene: translate and search protein database

• Search PDB if you want a 3-D structure• Search NR if you want any hit• Search UniProt to know what the hits are• Search dbEST to know if your sequence is expressed

• UniProt90: no seq is more than 90% ident to any other (for an uncluttered tree) also UniProt50

Word size

• Default at NCBI Blast– 11 for DNA; option of 7 and 5– 3 for protein; option of 2

• Increasing wordsize will speed search

… but will lose sensitivity– It will miss some useful but distant hits

• Decreasing wordsize will be more sensitive … but take longer

Substitution matrix

• By default BLAST uses BLOSUM62

• Can change this– Blosum90 mirrors changes in closely related

sequences– Blosum30 changes in distant sequences

• Should run 3 blast searches with different substitution matrices.

Substitution matricesTop left part of a BLOSUM 90 matrix A R N D C Q E G H I LA 5 -2 -2 -3 -1 -1 -1 0 -2 -2 -2R -2 6 -1 -3 -5 1 -1 -3 0 -4 -3N -2 -1 7 1 -4 0 -1 -1 0 -4 -4D -3 -3 1 7 -5 -1 1 -2 -2 -5 -5C -1 -5 -4 -5 9 -4 -6 -4 -5 -2 -2Q -1 1 0 -1 -4 7 2 -3 1 -4 -3E -1 -1 -1 1 -6 2 6 -3 -1 -4 -4G 0 -3 -1 -2 -4 -3 -3 6 -3 -5 -5H -2 0 0 -2 -5 1 -1 -3 8 -4 -4I -2 -4 -4 -5 -2 -4 -4 -5 -4 5 1L -2 -3 -4 -5 -2 -3 -4 -5 -4 1 5

Changes 30-62-90 not linear

Positive off-diagonals aresimilar

Reality of matrix change

• Query: GHDEICI• GH + C • Sbjct: GHACNCG

– Scores 39 with Blosum30; 5 with Blosum90

• Query: HEQCRLEN• +E LEN• Sbjct: QENAHLEN

– Scores 19 with Blosum30; 24 with Blosum90

• So GHDEICIHEQCRLEN will find different hits

Matrix families

• PAM – point accepted mutation– Margaret Dayhoff 1970s (before Genbank)

• BLOSUM – based on aligned blocks– Henikoff and Henikoff 1992– Blosum 62 default (based on aligned seqs

62% identical – don’t ask why 62: it works)

• Low PAM = High Blosum– PAM 250 == Blosum 30– PAM 30 == Blosum 90

Gap penalty

• Original Blast was gap-free– Because gapped aligns much more CPU

• Now can insert “affine” gaps– Gap open 10; gap extend 1

• Raise gap penalty to discourage gaps– Preferentially gets closely related hits

• Lower gap penalty for sensitive search for distant relatives

Low Complexity Masking• Seg masks low-complexity regions

– “too many” serines, prolines etc.• What a masked sequence looks like:>P04729 Wheat gamma gliadinMKTFLVFALIAVVATSAIAQMETSCISGLERPWQQQPLPPQQSFSQQPPFSQQQQQPLPQQPSFSQQQPPFSQQQPILSQQPPFSQQQQPVLPQQSPFSQQQQLVLPPQQQQQQLVQQQIPIVQPSVLQQLNPCKVFLQQQCSPVAMPQRLARSQMWQQSSCHVMQQQCCQQLQQIPEQSRYEAIRAIIYSIILQEQQQGFVQPQQQQPQQSGQGVSQSQQQSQQQLGQCSFQQPQQQLGQQPQQQQQQQVLQGTFLQPHQIAHLEAVTSIALRTLPTMCSVNVPLYSATTSVPFGVGTGVGAY*and after low complexity masking:

>P04729 SEG low-complexity maskedMKTFLVFALIAVVATSAIAQMETSCISGLERPWXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXLNPCKVFLQQQCSPVAMPQRLARSQMWXXXXXXXXXXXXXXXXXXXXXXXRYEAIRAIIYSIIXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXHQIAHLEAVTSIALRTLPTMCSVNVPLYSATTSVPFGVGTGVGAY*

• Xnu masks repeats (from database of common repeats)

Masking• Check if masking on/off by default (differs at

sites)• Run 2 searches with masking on, masking off

ANNYway• Masking by hand:

– If you know about the DNA binding domain already• Which is really common and will occupy the top 100 hits

against any database– Replace the region with Xs– Re-run blast to find other motifs/domains/information– NCBI blast allows select subsequences

• DUST masks low info DNA sequences– Like polyA tails

Advanced

Expectation cut-off• E value

– Expected number of chance hits• Given the database searched• Query HSP length

• Related to probability• Default is 10: “expect to find 10 hits as

good as this by chance alone”• E values less than 10-4 unreliable • So different from p < 0.05 • For short seqs crank E up to 100 or 1000

Data deluge – hits delivered

• Default suits general purpose

• V = 50 num of “hits” one line descriptions

• B = 50 num of alignments between query and hit which are displayed

Swiss Blast

EBI blast

Limit by taxon/organism

Also search at www.ensembl.org for “genomed” organisms

Database choice

NR for getting aNNy hitswissprot or refseq for getting hits that have annotationMonth for recent hits

The output

• Is in five parts

1. Admin – date, size of database, id of query

2. Graphic display of query and hits

3. List of hits with links to database and to…

4. …alignments (may be > 1 HSP per hit)

5. More admin, including errors/warnings

Notes!

• Record the top admin stuff. Your search will be different – Next week– On a different server– With different DB– With different input parameters

• Mouse-over any graphic display– Shows domain structure– Shows if hit is global or local

• Read the bottom admin stuff

Blast outputsp|P06471|HOR3_HORVU B3-HORDEIN Length = 264 Score = 62.5 bits (149), Expect = 1e-09 Identities = 32/63 (50%), Positives = 38/63 (59%)Query: 131 LNPCARSQMWXXXXXXXXXXXXXXXXXXXXXXXRYEAIY LNPCARSQM R+EA+YSbjct: 111 LNPCARSQMLQQSSCHVLQQQCCQQLPQIPEQLRHEAVY Query: 191 SII 193 SI+Sbjct: 171 SIV 173

Low-complexity masked regionWhat sort of residues masked inThe query sequence??

May be more HSPs for same hitIf big deletion in either seq

General blast protocol1. Find server, choose DB, paste seq, GO

2. Rerun search with/out masking

3. Rerun search with two diff subs matrix

4. 2 x 3 for six searches

5. If top N hits all same family/domain then XXX this region and resubmit

6. LOOK at the results; esp strange ones

7. Limit results by organism

Blast notes

• Twilight zone <25% protein <70% NA• Because two sequences give high blast scores

to a third, doesn’t mean they are related– NNNNDOMAINANNNNDOMAINBNNN– NNNNDAMIANANNNNNNNNNNNNNN– NNNNNNNNNNNNNNNDIMIAMBNNN

• E-value vs % ID. >10-4 unreliable• Bit score <50 unreliable

PSI-BLAST

• Uses a PSSM rather than BloSum/PAM

• Iterative …– Can find very distant relatives– …so deep insight

• BUT can iterate off with the fairies