BIT 410: Manipulation of Recombinant DNA
BLAST Searching Tips
About BLAST | Search
BLASTn | Look at Results | GenBank
Records
What BLAST Does
- You can use BLAST to compare an unknown sequence to published
sequences in databases such as GenBank. BLAST will make pairwise
comparisons between a sequence and the sequences in these databases.
- There are many types of BLAST searches that compare nucleotides,
amino acids, genomic, and translated nucleotide sequences to each
other. This link describes the different types of BLAST searches:
http://www.ncbi.nlm.nih.gov/BLAST/
- The rest of this tip sheet describes how to do a BLASTn search
to compare nucleotide sequences with each other.
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Searching BLASTn
Go to the BLAST home page: http://www.ncbi.nlm.nih.gov/BLAST/
Under nucleotides, select nucleotide-nucleotide BLAST (BLASTn)
Search Steps:
- Copy and paste your sequence into the search box. You can also
type or paste in a sequence accession number (e.g., from GenBank,
etc.), and your sequence will be entered automatically. (Click on
the Search link next to the box for information about acceptable
sequence Formats.)
- Choose database: leave this on the default, “nr” for
this project (“nr” is a combination of GenBank and several
other databases).
- Click the “BLAST” button to start the search. Next
you will get a page with a Request ID.
- Click on the “Format!” button to obtain your results.
If the results aren’t ready, you will get an interim “waiting”
page that will show your results when they are ready. (BLAST searches
may take a long time; you can always copy the Request ID and come
back later to get the results.)
Tip: If you want to know what the other information
on the page is, just click on the hyperlinked term and you will get
more information.
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Understanding the Results of your BLASTn Search
The results page has 4 sections.
- The first section presents references and background information
about the development of the BLAST program, etc. Don’t worry
about this for now.
- Graphical Overview of Sequence Comparisons (Distribution of Hits)
a. The score of each alignment is color coded, and divided into
5 groups, displayed in decreasing order of similarity.
b. Mousing over a sequence bar displays the definition and score
of the sequence in the window (part of Section 3).
c. Clicking on the sequence bar will take you to the detailed pairwise
alignment between the sequences (part of Section 4).
- The list of Sequences with significant alignments to your query
a. These are ranked by score, better (more significant) scores are
listed first.
b. Each line includes:
i. A series of numbers and letters—these are mostly database
acquisition numbers. The gi number is assigned by GenBank; the second
number indicates the sources of the sequence (e.g, emb=EMBL, gb=GenBank;
the third number identifies another accession number or the locus.
These numbers can be used as a search term to find a sequence in
selected databases.
ii. If you click anywhere in this block of numbers you will link
directly to the GenBank, or other database, record for that sequence.
iii. The rest of the text is a description of the sequence.
iv. For scores, the “e” value indicates significance,
lower is better.
- Details of alignments for each sequence pair
a. The results show a base-by-base comparison between your search
sequence and each sequence listed in the results.
b. Lines between bases indicate exact matches. Spaces indicate a
non-matching pair of bases or a small gap.
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Interpreting a GenBank Record
As described above, clicking on the gi or other accession numbers
in the results list will open up the database record for that sequence.
Detailed information about individual sequences is contained in GenBank
records.
For more detailed information see this Sample GenBank Record: http://www.ncbi.nih.gov/Sitemap/samplerecord.html.
This document includes detailed descriptions of each of the record
components.
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