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There is a statistical section at the very end of the BLAST report. BLAST query sequence windows will be lined up with similar regions in the database, then the algorithm will try to extend the alignment with the database sequence in both directions along the query sequence. What is the number of successful extensions for the first BLAST report?
What is, by far, the most common protein shown as a hit in the list of scores?
At the end of the list of scores, note the section entitled "Sequences with E-values WORSE than threshold". Why is it that PSI-BLAST includes this section, butBLAST does not?
Do any members from the Actin-like ATPase domain superfamily, other than actin (or actin-like proteins), show up as hits in this section? If so, name one.
There are several buttons within the results document entitled "Run PSI-BLAST iteration 2"; if they are difficult to locate, there is one at the end of the listof descriptions and scores; click on one of these buttons to run a second iteration of PSI-BLAST.Look at the results window and above the graphical display, it should say "Results of PSI-Blast iteration 2". Keep track of what the next iterationnumber should be, and make sure it matches the iteration number that PSI-BLAST displays to avoid getting lost within the PSI-Blast search. When the results appear,look at the legend under the color alignment graph. It says that a yellow starburst with the word "NEW" inside it indicates a new sequence that wasidentified as a result of the most recent iteration, and a green dot indicates a sequence that was already present prior to the most recent iteration.
What is the number of successful extensions for the second BLAST iteration?
Did the second iteration locate any new sequences within (meaning less than or equal to) the threshold E-value? If so, how many?
Are there new members of the Actin-like ATPase domain superfamily in the section entitled "Sequences with E-value WORSE than threshold"? If so, name one.
How many new sequences are identified as a result of the third iteration?
Are there new sequences returned that are within the threshold E-value that are NOT actin or actin-like proteins? If so, name one.
Finally, perform the first iteration of the PSI-BLAST search with the same query sequence as above, but with the database set to "nr".
What is the number of successful extensions for the first BLAST report when searching the nr database?
The "swissprot" database is a curated database, and all the descriptions onthe return list were informative. However, searching against the large "nr" database yields a list of returns that should contain some proteins describedas "unknown", or "unnamed". The advantage of "nr" is that usually many more hits are returned. Sometimes this is desirable, and sometimes it is not, but it is one thingto consider when designing a PSI-BLAST search strategy.
A PSI-BLAST search can be forced to include more sequences in the profile at a given iteration by altering the threshold. The default value is to accept anyresults with E values less than 0.005. It is common to change this threshold to 0.05 if the PSI-BLAST converges too quickly at 0.005. We did not take oursearch to convergence, that would require continuing iterations until the last iteration did not return any new hits. As this tutorialillustrates, PSI-BLAST is a useful tool, but it often requires putting some thought into the search strategy before it will produce meaningful results.
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