Local sequence similarity search can be performed for any DNA or protein sequence. These include PiMS targets' protein and nucleotide sequences, expressed and final proteins, primers and constructs.
Search results are displayed in a summary table containing links to the individual 'Hits' and sequence alignments.
PIMS employs Smith-Waterman algorithm for local sequence similarity search which is slightly more sensitive than BLAST. The inclusion of gaps is controlled by the two penalties known as gapopen and gapextend. Given a novel protein sequence, this may be the choice to detect distantly related proteins. These proteins may align with long gapped regions, possibly in loops on protein surfaces that may not contain critical functional residues. If you want to detect weak candidate alignments it may be necessary to repeat the searches with a variety of gap penalties.

First you need to supply PIMS with a sequence to searched against PIMS local database To do that click on the Target->Similarity Search on the menubar

Sequence

You can cut and paste or type a protein sequence into the large text window. A free text (raw) sequence is simply a block of characters representing a protein or a nucleotide sequence. Partially formatted sequences will not be accepted. If you paste a sequence in FASTA format, it's name will be used in a hit list and alignments. Please note that sequence name will be chopped to 13 characters to preserve alignment formatting.

Matrix (scoring function)

This is the scoring matrix used when comparing sequences. By default it PIMS uses 'BLOSUM62' for both proteins and nucleotide sequences. However, you are welcome to change this according to the task.

Gap

Default in PIMS 10.0 for any sequence. The gap open penalty is the score taken away when a gap is created. The best value depends on the choice of comparison matrix. (Valid number from 0 to 100.0)

Gap extend

The PIMS default is 0.5 for any sequence. The gap extension penalty is added to the standard gap penalty for each base or residue in the gap. This is how long gaps are penalized. Usually you will expect a few long gaps rather than many short gaps, so the gap extension penalty should be lower than the gap penalty. An exception is where one or both sequences are single reads with possible sequencing errors in which case you would expect many single base gaps. You can get this result by setting the gap open penalty to zero (or very low) and using the gap extension penalty to control gap scoring. (Number from 0 to 10.0)

Search results comprising information describing any entries in PIMS database (Hits) which match the search parameters. At the top of a search results page a hit table is displayed. It is sorted by the score. Example screenshots


Hit table columns form left to right:

No - (Result Number)

This is the result number, results are ranked in descending order of score. By clicking on this number you will be forwarded to the details of the alignment further down the page.

Hit id

The name of the molecule which sequence matches query sequence. By clicking on this name you can view other information related to it.

Hit type

Either Target, Construct or Primer sequence. By howevering a mouse other the type you can see its name, click on it and you'll be forwarded to the details.

Length

Alignment sequence length.

Score

Alignment score, representing the likelihood that the described alignment is not random, providing an indication of its validity. It is calculated by totaling the scores for each matched pair of residues at each position in the alignment, plus unmatched residues are given the gap penalty, and the gap extension penalty.

Identity

The number of residues (either an amino acid or a nucleotide) which are identical between the query sequence and the Hit, over the region of the match.

Similarity

The number of identical or related residues over the region of the match.

Gaps

The number of gaps in the alignment

For each Hit, PiMS displays the alignment between the Query and the Hit sequences in a table. The alignment tables are located at the end of the Hit list table. You can view them by scrolling to the bottom of the Hit table or by clicking the "View Alignments" link at the top of it. There is also a numbered link to each individual alignment table in the first column of the hit list. PIMS adopt Intelligenetics format which uses `|' to show identities and `:' to show conservative replacements and places these indicators between the two aligned sequences. An example alignment is shown below.

• The lengths of the Target and Hit sequences are shown, along with the length and score for the alignment. Also shown are the number of identical amino acids, the number of identical and similar amino acids and the number of gaps in the region of the alignment.
• Below this are values for the % of identical residues in the Hit, alignment and Target and the length of the Hit as a % of the Target length.
• The sequence alignment is displayed in three rows. The top and bottom rows are labelled Query (or sequence name if the sequence was in FASTA format) and hit name and these are separated by a pattern in red.
• Markup Line is placed between a pairwise alignment it shows where sequences are mismatched, gapped, identical or similar.
  In general the markup line uses a space for a mismatch or a gap, "." for any small positive score, ":" for a similarity which scores more than 1.0, and "|" for an identity where both sequences have the same residue regardless of its score ("W" matching "W" scores much more than "L" matching "L" because a conserved tryptophan is more significant than a conserved leucine).
• Any two residues or bases are defined as similar when they have positive comparisons (as defined by the comparison matrix being used in the alignment algorithm).
• Note that the sum of identical and similar positions is greater than 100%. This is because the count of similar positions includes the count of identical positions; if residues are identical, they must also be similar.
• Score used to determine which is the best possible alignment to report. Smith-Waterman score of an alignment is equal to the sum of the matches taken from the scoring matrix.