This tutorial assumes that local copies of the GenBank and GenPept databases are installed.
This tutorial will introduce the several
types of sequence database searches, using the antifreeze protein from the sea raven, Hemitripterus
americanus. The cDNA sequence for this gene is found in SRAAFP.gen.
Create a directory called 'db' and save
this sequence to the db directory.
To read this sequence into bldna, choose 'File --> Open', and select SRAAFP.gen.
Since this tutorial will demonstrate database searches
using both DNA and protein sequences as query sequences,
we need to generate the amino acid sequence. This is done
in two steps. First, we need to extract the protein coding
sequence from the cDNA. (Remember that mRNAs contain both
5' and 3' untranslated sequences. If you look at SRAAFP.gen,
you'll see that the CDS (protein coding sequence) runs
from positions 10 to 597. We will use the FEATURES program
to extract the CDS, and then translate the CDS using
Click on 'Run' with the above settings. The CDS will appear in a
new bldna window.
Note that the CDS begins with a start codon, indicating
that this cDNA contains the entire protein coding region.
Next translate the CDS into protein. Select SRAAFP:CDS1, and choose DNARNA --> Ribosome. By default, Ribosome sends the output to a new blprotein window.
|First, choose a database. All GenBank divisions are listed, along with a choice to search ALL divisions, or a User-created file in Pearson/FASTA format. Most of the time, it is best to search the taxonomic group from which your query sequence is derived. This is especially true for DNA/DNA searches, since only closely-related DNA sequences are likely to give statistically-significant matches. If you do need to search all of GenBank, this can take more than an hour, depending on the length of the sequence and the K-tuple value (each K-tuple speeds up the search by a factor of 4, but decreases sensitivity).|
from long-running searches to a file.
bldna runs all database searches in the background, ie. as independent jobs. If you quit bldna or even logout, the job will run to completion. For long-running searches, it is usually best to send output directly to a file. Simply click on 'Output file', and type an Output file name. In the example above, output will be directed to two files: SRAAFP.gbvrt.fasta, which contains the FASTA report, and SRAAFP.gbvrt.fasta.nam, which contain the names of the hits. This namefile could be used as input to FETCH to retrieve the hits. Notice that biolegato automatically appends the .nam file extension.
For very closely-related sequences, speed up the
search with K-tuple = 5 or 6.
In some cases, you are specifically looking for sequences that share very high similarity with your query. Suppose you had a cDNA and wanted to find the corresponding genomic clone. In that case, the similarity is likely to be greater than 90%. A K-tuple value of 5 or 6 will give a very fast search at low sensitivity, which is all you need.
For DNA searches a very simple scoring scheme is used, in which +1 is added to the score for a match, and -1 is added to the score for a mismatch. The greater diversity of amino acids lends itself to more complex scoring schemes. The menu shows a variety of protein scoring matrices. The Blosum matrices were constructed using distantly-related sequences. If you need a highly sensitive search, use a low-numbered Blosum matrix. The PAM matrices were constructed from a set of closely-related sequences. For a high-sensitivity search, use PAM250, or PAM250 Gonnet (based on a more recent dataset.). For closely-related sequences, use PAM120.
More information of scoring matrices can be found in
Hugh B. Nicholas Jr., David W. Deerfield II., and Alexander J. Ropelewski, (1998) A Tutorial on Searching Sequence Databases and Sequence Scoring Methods Developed by the Biomedical Supercomputing Initiative of the Pittsburgh Supercomputing Center.[http://www.nrbsc.org/education/tutorials/sequence/db/].
The results of this protein search are found in SRAAFP.genpept.fasta and SRAAFP.genpept.fasta.nam.
| Hint: Oligopeptides need
logarithmic score weighting
For very short query sequences such as oligopeptides, unweighted scores would not exceed the minimum cutoff values needed to appear in the output. The FASTA programs allow you to specify a logarithmic weighting ratio that gives short query sequences higher matches. The score is weighted by the natural log of the query length divided by the natural log of the database size.
Two versions of TFASTA are available. TFASTX allows 3-base insertions (ie. 1 codon), while TFASTY allows 1 or 2 base insertions. TFASTY is therefore the most sensitive program, but it is also slower. TFASTY would be especially good at detecting similarities between a query protein and ESTs, since ESTs typically have more frameshifts than most sequences.
The results of this protein search are found in SRAAFP.gbvrt.tfasta and SRAAFP.gbvrt.tfasta.nam.
on automatic translation:
Programs that translate sequences on the fly (eg. TFASTX, TBLASTN, TBLASTX) have no knowledge whatsoever about gene structure (ie. exons, introns, 5' UTR, 3'UTR). All these programs do is take every group of 3 nucleotides and assign a codon to it. Even stop codons are represented by an asterisk (*). Consequently, non-coding sequences and non-coding open reading frames are translated into meaningless amino acid sequences.
FASTX and FASTY are the converse of TFASTX and TFASTY, in that they translate a DNA query sequence, allowing either codon-sized gaps (TFASTX) or 1 or 2 base gaps (TFASTY). TFASTY is therefore the slower, but more sensitive. These programs are particularly well-suited for comparing a sequence that may have frameshifts (eg. an EST) with a protein database.
The result of this protein search is found in SRAAFP.genpept.fasty.
|Click on Send to and fill in the
When you click on Create File, a file chooser will pop up.
|The default name will be something like
"sequence.gb", but it is good practice to give it a name
consistent with the rest of the files in this exercise. A
good name would be
NOTE: Remember to save this file in the current working directory! Your file chooser will show a different location from the one shown a right.