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Workshop OUTLINEPart 1:
• Introduction and motivation
• How does BLAST work?
Part 2:
• BLAST programs
• Sequence databases
• Work Steps
• Extract and analyze results
BLAST programs
2
• All types of searches are possibleQuery: DNA Protein
Database: DNA Protein
blastn – nuc vs. nucblastp – prot vs. protblastx – translated query vs. protein databasetblastn – protein vs. translated nuc. DBtblastx – translated query vs. translated database
Amino acid sequence – most suitable for homology search
• The database and the query can be either nucleotides or amino acids!
• We prefer amino acid sequence:-amino acid sequence is more conserved-20 letter alphabet. Two random hits share 5% identity in average (comparing to 25% in DNA seq).-protein comparison matrices are more sensitive .- protein databases are smaller – less random hits.- we want to conclude about the structure- proteins are much more relevant.
BLAST programs
• Where? (to find homologues)
• Structural templates- search against the PDB
• Sequence homologues- search against SwissProt or Uniprot (recommended!)
• How many?
• As many as possible, as long as the MSA looks good (next week…)
General Issues
• How long? (length of homologues)
• Fragments- short homologues (less than 50,60% the query’s length) = bad alignment
• Ensure your sequences exhibit the wanted domain(s)
• N/C terminal tend to vary in length between homologues
• How close? (distance from query sequence)
• All too close- no information
• Too many too far- bad alignment
• Ensure that you have a balanced collection!
General Issues
• From who? (which species the sequence belongs to)
• Don’t care, all homologues are welcome
• Orthologues/paralogues may be helpful
• Sequences from distant/close species provide different types of information
• Which method? (BLAST/PSI-BLAST)
• Depends on the protein, available homologues, the goal in mind…
General Issues
Sequence databases
Where do we want to search?DNA sequences
• ESTs- no annotated coding sequence pool. the largest pool of sequence data for many organisms (NCBI)
• NR- All GenBank + EMBL + DDBJ + PDB sequences. No longer "non-redundant" due to computational cost.
• Genomes a specific organisms
• RefSeq- mRna or genomic- an annotated collection from NCBI Reference Sequence Project.
• EMBL- Europe's primary nucleotide sequence resource (EBI)• ….
Sequence databases
Where do we want to search?Protein databases:
• PDB- the sequences of proteins for which structures are available
• NR (non-redundant)- Non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PRF, excluding those in env_nr
• RefSeq- sequences from NCBI Reference Sequence project.
• Proteins of a specific organisms
• Uniprot –swissprot or trembl
• ….
Sequence databases
Where do we want to search?
UniProt• UniProt is a collaboration between the
European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
• In 2002, the three institutes decided to pool their resources and expertise and formed the UniProt Consortium.
Sequence databases
Where do we want to search?
UniProt• The world's most comprehensive catalog of
information on proteins- Sequence, function & more…
• Comprised mainly of the databases:
– SwissProt – 366226 last year, 412525 protein entries now –high quality annotation, non-redundant & cross-referenced to many other databases.
– TrEMBL - 5708298 last year, 7341751 protein entries now – computer translation of the genetic information from the EMBL Nucleotide Sequence Database many proteins are poorly annotated since only automatic annotation is generated
Overall work steps
1.Run the search- 1. Select database2. E-value threshold3. BLAST or PSI-BLAST- how many rounds?
2.Take out sequences1. HSP or full sequences2. Can (should!) filter out redundant and sequences
that are too short (fragments)
3. Usually- align sequences- choose alignment program
4.View alignment with BioEdi tor another program
5.Calculate trees, conservatino scores (conseq) etc…
Multiple Sequence Alignment (MSA)
Overall work steps
• Perform alignment of a large collection of sequences
• Many algorithms, leading ones:
1. ClustalW2. MUSCLE3. T-COFFEE
Examining BaliBase 2005…
Edgar, R.C., 2004
MUSCLE is superior!
Overall work steps
BLAST NCBI
BLAST NCBI
• All program types
• Many databases to chose from, both nucleotide and protein
• 12 genome-specific databases
• Can also look for conserved domain, SNPs and more…
The well-known serverhttp://blast.ncbi.nlm.nih.gov/Blast.cgi
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
BLASTp
BLAST NCBI
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
QuerySequenc
e
Database
Run
BLAST NCBI
BLASTp
As many as possible
Matrix
BLAST NCBI
Evalue
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
Mark all
Mark onlywanted
BLAST NCBI
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
BLAST NCBI
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
BLAST NCBI
BLAST EBI
http://www.ebi.ac.uk/blastall/index.html
Many databases,including UniProt
Insert sequenc
e
RUN
Get maximum number of alignments!
BLAST EBI
http://www.ebi.ac.uk/blastall/index.html
Send sequences
to ClustalW
Mark all or wanted
Get sequences
BLAST EBI
PSI-BLAST
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
QuerySequenc
e
Database
Run
PSI-BLAST NCBI
PSI-BLAST
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
Pre-calculated PSSM
PSI-BLAST NCBI
Threshold for inclusionin PSSM
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
PSI-BLAST NCBI
Run next round
Include sequence in the PSSM
Not found inprevious round
http://www.ebi.ac.uk/blastpgp/
QuerySequenc
e
Database
Run
PSI-BLAST EBI
Number of iterations
(PSI-)BLAST on ConSeq, extract sequence & align
PSI-BLAST on ConSeq
The ConSeq webserver
• Calculates evolutionary conservation scores that are than displayed on the sequence.
• Requires a Multiple Sequence Alignment (MSA)- if nor provided, can create one automatically
• Runs (PSI-)BLAST, extracts hits from the BLAST results, filters according to e-value and aligns the sequences.
PSI-BLAST on ConSeq
The ConSeq webserver-http://conseq.tau.ac.il/
PSI-BLAST on ConSeq
The ConSeq webserver-http://conseq.tau.ac.il/
Query sequence
PSI-BLAST on ConSeq
The ConSeq webserver-http://conseq.tau.ac.il/
Alignment
algorithmDatabase
- swissprot or uniprot
No. of homologue
sIterations
E-value
PSI-BLAST on ConSeq
The ConSeq webserver-http://conseq.tau.ac.il/
PSI-BLAST on ConSeq
The ConSeq webserver-http://conseq.tau.ac.il/
All BLAST hits
MSA
Summary of web servers:
1. PSI-BLAST at NCBI-- Can control PSSM, included sequences & threshold- All types of BLAST programs- Not against UniProt- SwissProt or NR- Against RefSeq and NT- Full sequences downloaded like BLAST- Number of sequences up to 2000
NCBI vs. EBI vs. ConSeq
Summary of web servers:
2. BLAST at EBI – - Against UniProt or EMBL, not NR or specific genomes- Can’t control PSSM- just get last round
- Download and align only full sequences - The number of presented sequences is limited to 500- blastN, blastP, tblastN, tblastX
NCBI vs. EBI vs. ConSeq
Summary of web servers:
3. BLAST at ConSeq – • Get HSPs, not entire sequences!!!• Only blastP• Search uniprot/swissprot• Still, can’t control all options… such as redundancy and
minimal length of HSP
NCBI vs. EBI vs. ConSeq
(PSI-)BLAST via Max-Planck
(PSI-)BLAST via Max-PlanckRun (PSI-) BLAST
Send HSP or full sequences to an
alignment program
Forward HSP to filtrationvia “BLAMMER”
Download filtered sequences
Align the sequences via program of
choice
(PSI-)BLAST via Max-Planck
BLAST at Max-Planchttp://toolkit.tuebingen.mpg.de/sections/search
• Databases- swissprot, tremble, NR, env, pdb or any combination for proteins, but only NT for DNA.
• All BLAST programs
• Main advantage- you can easily extract and filter the HSPs, on top of full sequences.
The Query Protein
Name: Dihydrodipicolinate reductase
Enzyme reaction:
Molecular process: Lysine biosynthesis (early stages)
Organism: E. coli
Sequence length: 273 aa
Query:DAPB_ECOLI
<DAPB_ECOLIMHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIEIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNL
The Query Protein
(PSI-)BLAST via Max-Planckhttp://toolkit.tuebingen.mpg.de/psi_blast/
Choose database or databases
(selecting a few using CTRL)
Upload sequenceor MSA
(PSI-)BLAST via Max-Planc
Save PSi-BLAST result
(PSI-)BLAST via Max-Planck
E-value threshold can be assessed using the distribution
Filter Results via Max-Planck
Forward results to BLAMMER
BLAMMER
• Suppose to create MSAs from BLAST results, we will use it
just to filter the results and then align them via MUSCLE or
another known MSA program.
• Filter according to:• E-value• Min. coverage- min. percent of the query protein• Max. redundancy- extract similar sequences• Max. number of homolgoues- if wanted
Filter Results via Max-Planck
http://toolkit.tuebingen.mpg.de/blammer/
Filter Results via Max-Planckhttp://toolkit.tuebingen.mpg.de/blammer
Forwarded PSI-BLAST
result
Filtering parameters
Filter Results via Max-Planck
Save & thenre-align!
Align the BLAST sequences
Align via Max-Planck
http://toolkit.tuebingen.mpg.de/sections/alignment
1.Forward BLAST to MUSCLE, MAFFT etc...
Choose program
Use hits or full sequences
Align via Max-Planck
Align via Max-Planck
2. Filter via BLAMMER and then ALIGN:
Upload the results of the BLAMMER – downloaded
file
Align via Max-Planck
Alignment results:
Save the alignment
Alignmen viewing & editingBioEdit
• http://www.mbio.ncsu.edu/BioEdit/BioEdit.html
• Easy-to-use sequence alignment editor
• View and manipulate alignments up to 20,000 sequences. •Four modes of manual alignment: select and slide, dynamic grab and drag, gap insert and delete by mouse click, and on-screen typing which behaves like a text editor.
•Reads and writes Genbank, Fasta, Phylip 3.2, Phylip 4, and NBRF/PIR formats. Also reads GCG and Clustal formats
Easiest Using Bioedit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
Alignmen viewing & editing
Easiest Using Bioedit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
• Find a specific sequence: “Edit-> search -> in titles”
• Erase\add sequences: “Edit-> cut\paste\delete sequence”
• “Sequence Identity matrix” under “Alignment”- useful for a rough evaluation of distances within the alignment.
• After taking out sequences, “Minimize Alignment” under “Alignment” takes out unessential gaps.
• Can save an image using: “File -> Graphic View” & then “Edit -> Copy page as BITMAP”
Alignmen viewing & editing
Each sequence is a different story
adjust parameters:
• BLAST- E-value, substitution matrix, gap penalties, database, minimum length, redundancy level, fragment overlap…
• PSI-BLAST- BLAST parameters + PSSM inclusion threshold (or chose manually), number of rounds…
• Try using HSP or full sequences, different MSA programs…
No “Miracle solution”
THANKS
Some slides were taken from previous presentations by members of the Pupko lab and Prof. Beni Chor