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.ncbi.nlm.nih.gov"> NCBI. As of Jan 1998, the NCBI 'experimental' servers (Cruncher and Muncher), which used to process blast jobs, have been replaced. The new system now sends blast jobs to the NCBI web server. If the server is overloaded or the network is slow, you may see the following error message:


helix% blast x89712.genpept -default

 Sending query...
 Awaiting results...

 ** Server did not say "OK".

Your options are to try again later, use the Blast email server (add '-mail' to the command line), or use Blast via the web.

The old 'blast -batch' command doesn't work anymore; to run blast in batch mode see How do I run blast searches in batch mode

Why have my alignments changed in Version 9?

The following information is taken from the GCG Newsletter, July 1997.

Protein sequence alignment programs in the Wisconsin Package use scoring matrices to make comparisons between pairs of amino acids. In Version 9.0, released in December 1996, changes were made to the scoring matrices which may affect the alignments you create. If the sequences you aligned in previous versions of the Package were very similar, you may not have noticed much difference to those same alignments produced using Version 9.0. However, with more distantly related sequences the alignments produced may be substantially different.

Version 9 Changes to Scoring Matrices

There are two main changes to protein scoring matrices in Version 9.0.

BLOSUM62¹, the new default protein scoring matrix, is the primary cause for the difference in alignments created in Version 9.0. Versions of the Wisconsin Package prior to 9.0 used a scoring matrix based on the PAM250 table from M. Dayhoff², where all perfect matches were set to a single value. BLOSUM62, in contrast, assigns scores ranging from 4 to 11 to perfect matches (identity). Scores for a non-perfect but similar matches range from 0 to 3. Scores for mismatches (non-similar matches) are assigned vales less than 0. Knowing the range of these values is helpful for setting display thresholds for similar amino acids.
Scoring matrix values are now integers. Matrices which previously held floating point (real number) values have had their values multiplied by 10 so that they now hold integer values. In addition, the values for gap creation and extension penalties also must be entered as integer values.

The modified PAM250 matrix which was used in previous software versions is still available in Version 9.0. The matrix has been renamed oldpep.cmp, and the original floating point values were changed to integer values. However, we do not recommend using this matrix. If you are trying to align distantly related sequences and are not achieving expected results with BLOSUM62, use the BLOSUM30 matrix. You can specify this alternative matrix with the -MATRix=blosum30.cmp (UNIX) or /MATRix=blosum30.cmp (OpenVMS) command-line parameter when you run a sequence comparison program.

How Does This Affect Me?

Two common questions you may have about the alignments created in Version 9.0 and their answers are detailed below.

Q: The global alignment displayed by PileUp or Gap shows one sequence completely to the right of the others, with only a few end bases shown as matched when run with BLOSUM62 (see examples below). But with the old matrix the sequences appeared to align together. Why?

A: The old matrix (PAM250) was overly permissive of mismatches and allowed you to align unrelated sequences. The BLOSUM62 matrix is more restrictive and will not routinely align unrelated sequences along their entire lengths. Thus, you may find that the displaced sequence(s) may be unrelated to the rest of your alignment. If you have reason to believe that the sequences are globally but very distantly related, then you might want to align them with a matrix based on an evolutionary model that assumes greater divergence time, like BLOSUM30. (A complete series of BLOSUM matrices is provided in the GenMoreData directory.)

(UNIX) prompt> namels genmoredata:blosum*

(OpenVMS) prompt> dir genmoredata:blosom*

51 100 Calm_Human RHVMTNLGEK LTDEEVDEMI READIDGDGQ VNYEEFVQMM TAK~~~~~~~ Calm_Drome RHVMTNLGEK LTDEEVDEMI READIDGDGQ VNYEEFVTMM TSK~~~~~~~ Calm_Wheat RHVMTNLGEK LTDEEVDEMI READVDGDGQ INYEEFVKVM MAK~~~~~~~ Hsp2_Mouse ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~MV RYRMRSPSEG 101 147 Calm_Human ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ Calm_Drome ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ Calm_Wheat ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ Hsp2_Mouse PHQGPGQDHE REEQGQGQGL SPERVEDYGR THRGHHHHRH RRCSRKR

Global alignments created by PileUp in Version 9.

51 RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMTSK....... 93 |. : 1 ......................................MVRYRMRSPSEG 12
Global alignment created by Gap in Version 9.

Pileup doesn't find conserved motifs in my alignment in Version 9

The following information is taken from the GCG Newsletter, July 1997.

PileUp was always intended as a global alignment tool, which aligns sequences along their entire lengths. Because of this, PileUp is not ideally suited to finding the best local region of similarity (such as a shared motif) among all of the sequences. Global alignment algorithms tend to overlook areas of local similarity in favor of aligning the entirety of all sequences.

If you know where the areas of conserved motifs lie, use the SeqLab Editor to manually edit the resulting global alignme