1. BLAST

2. What is BLAST?
“BLAST® (Basic Local Alignment Search Tool) is a set of similarity search programs
designed to explore all of the available sequence databases regardless of whether
the query is protein or DNA.
The BLAST programs have been designed for speed, with a minimal sacrifice of
sensitivity to distant sequence relationships.
The scores assigned in a BLAST search have a well-defined statistical interpretation,
making real matches easier to distinguish from random background hits.
BLAST uses a heuristic algorithm which seeks local as opposed to global alignments
and is therefore able to detect relationships among sequences which share only
isolated regions of similarity (Altschul et al., 1990).

3. Selecting BLAST programme
Compares an amino acid query sequence against a protein sequence database.
blastn
Compares a nucleotide query sequence against a nucleotide sequence database.
blastx
Compares a nucleotide query sequence translated in all reading frames against a protein sequence database. You could use this option to find potential translation products of an unknown nucleotide sequence.
tblastn
Compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
tblastx
Compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

4. Selecting the Database (protein)nr
All non-redundant GenBank CDS translations+PDB+SwissProt+PIR 
month
All new or revised GenBank CDS translation+PDB+SwissProt+PIR
reased in the last 30 days. 
swissprot
The last major release of the SWISS-PROT protein sequence
database (no updates). These are uploaded to our system when
they are received from EMBL.
patents
Protein sequences derived from the Patent division of GenBank.
yeast
Yeast (Saccharomyces cerevisiae) protein sequences. This database
is not to be confused with a listing of all Yeast protein sequences.
It is a database of the protein translations of the Yeast complete genome.
E. coli
E. coli (Escherichia coli) genomic CDS translations.
pdb
Sequences derived from the 3-dimensional structure Brookhaven
Protein Data Bank.
alu
Translations of select Alu repeats from REPBASE, suitable for
masking Alu repeats from query sequences.

5. Nucleotide Databases nr
All non-redundant GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or HTGS sequences).
month
All new or revised GenBank+EMBL+DDBJ+PDB sequences released in the last 30 days.
dbest
Non-redundant database of GenBank+EMBL+DDBJ EST Divisions.
dbsts
Non-redundant database of GenBank+EMBL+DDBJ STS Divisions.
mouse ests
The non-redundant Database of GenBank+EMBL+DDBJ EST Divisions limited to the organism mouse.
human ests
The Non-redundant Database of GenBank+EMBL+DDBJ EST Divisions limited to the organism human.
other ests
The non-redundant database of GenBank+EMBL+DDBJ EST Divisions all
organisms except mouse and human.
yeast
Yeast (Saccharomyces cerevisiae) genomic nucleotide sequences. Not a
collection of all Yeast nucelotides sequences, but the sequence fragments
from the Yeast complete genome.
E. coli
E. coli (Escherichia coli) genomic nucleotide sequences.

6. Entering your Sequence
The BLAST web pages accept input sequences in three formats;
FASTA sequence format, NCBI Accession numbers, or GIs.

7. Is the query sequence represented in the database?
Setting Up a Query
Is the query sequence represented in the database?
Choose a current nucleic acid database. Select from among organism-specific
(e.g.: yeast), inclusive (e.g., nonredundant), or specialized set (e.g., dbEST,
dbSTS, GSS, HTG) databases
blastn

8. Are there homologs or evolutionary relatives of the query sequence in the database? Are there proteins whose function is related to the query sequence?
Choose a protein database if the query is protein or DNA expected to encode
a protein because amino acid searches are more sensitive
blastp for amino acid queries; blastx for translated nucleic acid queries. Use
Tblastn or tblastx for comparisons of an amino acid or translated nucleic
acid query versus a translated nucleic acid database.

9. search parameters Default Special Cases Short Query
Default
Special Cases
Short Query
Large Sequence Family
Ungapped BLAST
Filter on
off
Scoring Matrix
BLOSUM62
PAM30 for 35 and under
Word Size 3
3, or reduce to 2
E value 10
1000 or more
Gap costs
11,1 4
Alignments 50
2000

10. Filter
The default setting will filter repetitive or low-complexity sequences from the
query using the SEG (protein) or DUST (nucleic acid) programs
If a low complexity region in the query is of interest, filtering will need to
be turned off.
If the number of hits returned is small when searching with
a short query, it may help to re-search with filtering turned off.
The Human repeat filter option human repeats such as LINEs and
SINEs and is especially useful for human sequences that may contain
these repeats.

11. which that substitution is known to occur within conserved blocks of
Scoring Matrices
BLOSUM62 is the default matrix. The BLOSUM matrix assigns a probability
score for each position in an alignment that is based on the frequency with
which that substitution is known to occur within conserved blocks of
related proteins.
BLOSUM62 has been empirically shown to be among the best for detecting
weak protein similarities
Other supported options include PAM30, PAM70, BLOSUM80, and BLOSUM45.
Short query sequences can only produce short alignments, and therefore
database searches with short queries should use an appropriately tailored matrix
. The BLOSUM series does not include any matrices with relative entropies
suitable for the shortest queries, so the older PAM matrices may be used instead.

12. Gap opening and gap extension penalties
No alternate scoring matrices are available for BLASTN
Gap opening and gap extension penalties
BLAST program
Default Gap Penalty (G)
Default Gap Extension Penalty (E)
Other supported (G) and (E) values
blastp
-11 -1
-10, -1;
-10, -2;
-11, -1;
-8, -2;
-9, -2
blastn -5 -2
none

13. E value threshold
The E value for an alignment score "S" represents the number of
hits with a score equal to or better than "S" that would be
"expected" by chance (the background noise) when searching
a database of a particular size.
The default E value for blastn, blastp, blastx and tblastn is 10. At
this setting, 10 hits with scores equal to or better than the
defined alignment score, S, are expected to occur by chance
(in a search of the database using a random
query with similar length).
Increase the E value to 1000 or more when searching with a short
query, since it is likely to be found many times by chance
in a given database.

14. Alignments
If the number of alignments requested (x) is fewer than those exceeding the significance threshold only the top (x) hits will be reported.
To detect low-similarity matches, the number of alignments to be
shown should be increased when searching with a member
of a large sequence family.

15. Analyzing the output
Step 1.  Examine the alignment scores and statistics
Scores for each position of an alignment are derived from a
substitution matrix
The raw score "S" of the alignment is usually calculated by
summing the scores for each letter-to-letter and letter-to-null
position in the alignment.
The bit score is calculated from the raw score by normalizing
with the statistical variables that define a given scoring system.
Therefore, bit scores from different alignments, even those
employing different scoring matrices can be compared.

16. The higher the score the better the alignment
There is no widely accepted theory for selecting gap costs.
It is rarely necessary to change gap opening or extension
Values from the default.

17. Statistics
Local alignments with no gaps are referred to as
High scoring pairs (HSPs).
For gapped alignments, the significance of a given alignment
with score S is represented by the E (Expect) value (shown in
the right-most column in the output), the expected number of
chance alignments with a score of S or better.
The E value decreases exponentially as the Score (S) that is
assigned to a match between two sequences increases.
A convenient way to create a significance threshold for reporting
hits is to alter the E value. When the Expect value threshold is
increased from the default value of 10, more hits can be reported.

18. Step 2. Examine the alignments
Descriptions
The highest scoring alignments are described by one line summaries
called "descriptions".
The description lines are sorted by increasing E value, thus the most significant
alignments (lowest E values) are at the top.

19. Graphic Representation

20. At the top is a linear map of the query. Each bar drawn
below the map represents a protein (or protein fragment)
that matches the query sequence. The position of each
bar relative to the linear map of the query allows the user
to see instantly the extent to which the database matches
align with a single or multiple regions of the query.
The most similar hits are shown at the top in red. Pink, green,
blue and black bars follow, representing proteins in
decreasing order of similarity.

21. PSI_BLAST
Position-Specific Iterative (PSI) BLAST is a program based on the BLAST 2.0
algorithm that is designed to detect weak relationships between the query and
members of the database not necessarily detectable by standard BLAST searches.
The added sensitivity of this program over regular BLAST comes from the use of a
profile that is constructed (automatically) from a multiple alignment of the highest
scoring hits in the initial BLAST search.
A highly conserved position will receive a high score and weakly conserved
positions receive scores near zero.
The profile is then used to perform additional BLAST searches (called iterations)
and the results of each iteration used to refine the profile.

22. PSI-BLAST analysis is useful both for identifying the distant members of a
protein family, whose relationship is not recognizable by straight sequence
comparison, and also for deducing the function of hypothetical proteins that
are unannotated in the database.
A PSI-BLAST query is identical to a BLAST query with added specification by the
use of the expectation (E) value cut-off for inclusion of a match in the first
and subsequent iterations.
The initial PSI-BLAST search uses the same matrix options available for
Gapped BLAST, since it is a Gapped BLAST search.
The user can continue to search iteratively until satisfied that no new matches will
be identified. The point at which no new hits are identified by additional searches
is known as "convergence".

23. Motif searching with PHI-BLAST
A new service called Pattern Hit Initiated BLAST (PHI-BLAST), that searches for
particular patterns in protein queries is now available in Version 2.0 of the
BLAST program suite.
PHI-BLAST expects as input a protein query sequence and a pattern contained
in that sequence. PHI-BLAST searches the specified database for other protein
sequences that also contain the input pattern and have significant similarity to the
query sequence in the vicinity of the pattern occurrences.
PHI-BLAST is integrated with Position-Specific Iterated BLAST (PSI-BLAST), so
that the results of a PHI-BLAST query can be used to initiate one or more
rounds of PSI-BLAST searching.