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Example of matching an input sequence

(Print this page so it can be consulted during the subsequent steps described below)

Input sequence
The following sequence is that of the mature protein of the allergen Zea m 14.0101 from maize pollen. As may be noticed, this sequence contains a one-letter code for each amino acid, while the complete sequence is made up of 93 letters or amino acids:

aiscgqvasaiapcisyargqgsgpsagccsgvrslnnaarttadrraacnclknaaagvsglnagnaasipskcgvsipytiststdcsrvn
While the original protein sequence in the UniProt database entry P19656 consisted of 120 amino acids, removal of the signal peptide comprising the first 27 amino acids has yielded this mature protein sequence containing 93 amino acids.

If users enter their own input sequences, numbers in this sequence should be removed, whereas spaces, paragraph- or line- returns, need not be removed. In addition, three-letter codes for amino acids, such as IleSerCys... (first 3 residues of Zea m 14) should be changed into one-letter codes, for example by using web-based conversion tools (for example, "Three-to-One").

Entering an input sequence and selecting the alignment of interest
Enter the input sequence, by typing or copy-pasting it, in the searchbox (below "Copy Paste your amino acid sequence here") of the Allermatchtm search page. With the cursor, select one of the following options:

  • "Do an 80-amino-acid sliding window alignment"
  • "Look for a small exact wordmatch"
  • "Do a full fasta alignment"
In case the 80-amino-acid sliding windows has been chosen, the default threshold value of 35% identity may be modified by the user in the box next to "Cut-off Percentage (only applicable to the 80-amino-acid sliding window)". The threshold is the lower limit for alignments that will be displayed in the following steps (alignments scoring below the threshold will therefore not be displayed).

If the option for a small exact wordmatch has been chosen, the default value 6 for the wordlength can be modified by the user in the box next to " Wordlength (only applicable to the exact wordmatch search)". The wordlength is the minimal number of amino acids in an exact match.

After having selected the options and thresholds (if applicable) of interest, click then the "Go" button. The results will appear in the new page that is created in the same window on the user's screen. The various outcomes are discussed below for each of the specific options.

80-amino-acid sliding window

Summary table

The new page that appears after starting the 80-amino-acid sliding window alignment on the input sequence provides a table with a summary of the "hits", which are alignments scoring above the cutoff value. Each specific allergenic protein whose database sequence scored hits is presented in a new line, while data on this allergenic protein and the alignment are presented under the following column headings:
  • "Hit No", the rank of the best hit (see third column) of the allergenic protein, such as 1, 2, or 3, while the rank for the highest best hit is 1.
  • "Db", database from which the allergen sequence has been retrieved.
  • "Description", the description for the allergenic protein as provided by UniProt/GenBank in the protein database accession.
  • "Best hit (identity)", highest number of identical amino acids in the hits, expressed as percentage of 80- or more- amino acids, for example 30% for 24 identical amino acids
  • "No of windows ident>....", the number of amino-acid subsequences (windows) of the input sequence that showed hits above the cut-off value with the database sequence of the allergenic protein 
  • "% of windows ident>.... ", the fraction (percentage, %) of the total number of analysed subsequences (windows) of the input sequence that showed hits above the cutoff value with the allergenic protein
  • "Full identity", identical amino acids in the FASTA alignment of the complete input sequence against the database sequence of the allergenic protein. The first number is the percentage of identical amino acids as part of the total length of the alignment, while the second number is the total length of this alignment expressed as number of amino acids (including non-identical amino acids).
  • "External link", the external accession id, which is clickable and provides a link to the original accession for the database sequence on the source database's website (the user's browser will exit the Allermatchtm website)
  • "Scientific name", Latin name of the organism from which the allergenic protein is derived
  • "Detailed information", the clickable "Go" button links to a page with specific details on the database sequence of the allergenic protein, as well as the complete FASTA alignment and the subsequences (windows) of the input sequence aligning to the database sequence. After having clicked on the "Go" button, a new page will appear in the same window on the user's screen.

Detailed information

This page provides the following information:
  • The input sequence (amino acid sequence).
  • Details on the database sequence of the allergenic protein, including allergen name, species name, external accession id, remarks (for example, signal-, pro-, or transit- peptides that have been removed from the sequence) and amino acid sequence.
  • The complete amino acid sequences of the input- and database- sequences are shown in this page. Below each one-letter code for amino acid residues in both of these sequences, a "#"-marking may be displayed. The residues marked with "#" were aligned with residues in the other sequence (database or input, respectively) in the 80-amino-acid window alignments that had 35% or more identical amino acids in the window. Please note that these "#" markings also include nonidentical residues in both the input- and database- sequences that were aligned to each other. The 35% cut-off value is fixed for these "#" markings and cannot be changed by the user.
  • Details of the full alignment between the complete input sequence (no 80-amino-acid windows) and the allergenic protein.
By clicking the "Show all alignments" button all the separate hits, i.e. alignments of those 80-amino-acid subsequences (windows) of the input sequence that scored equal to- or above- the cut-off value of 35% (fixed value, cannot be changed by the user), can be viewed. The new page that appears in the same window on the user's screen contains the same information as the previous page, in addition to the separate hits. After clicking "Hide all alignments", the previous page re-appears.

Example

For the input sequence Zea m 14 screened against the Allermatchtm database, for example, the summary table lists various database sequences of allergenic proteins that score hits if the cut-off value equals 35%. Since the Zea m 14 sequence contains 93 amino acids, 14 subsequences (windows) of 80 amino acids have been generated (1-80, 2-81, ...., 13-92, 14-93). The highest ranking database sequence in the table is Zea m 14 itself, because the same sequence has also been stored in the Allermatchtm database, which shows a best hit of 100%, while all of the 14 windows of the input sequence scored hits, as expected. One of the lower ranking sequences in the table is the allergenic protein Par j 2 derived from weed pollen from Parietaria judaica. The best hit for this sequence is 36.59% identity, while 4 of the 14 windows scored hits. The detailed information on the alignments with Par j 2 show that a large part of both the input and database sequence are part of the 80-amino-acid sliding window- and full- alignments. Interestingly, many of the sequences listed in the table are lipid transfer proteins, as mentioned in the original external accession to which the table provides links.

Exact hits of small stretches of identical amino acids

Summary table

The new page that appears after starting the alignment of small identical stretches using WordMatch provides a table summarising the "hits", which are the alignments equal to- or above- the wordlength, i.e. the minimal number of identical contiguous amino acids. Each of the database sequences of allergenic proteins that showed a hit with the input sequence is shown in a separate line of the table, while the data on the allergenic protein are shown under the following column headings:
  • "No", rank of the database sequence of the allergenic protein, while the sequence that scores the highest number of wordmatches ranks number 1.
  • "Db", database from which the allergen sequence has been retrieved.
  • "Description", the description of the allergenic protein as provided by UniProt/GenBank in the protein database accession.
  • "Number of exact wordmatches", the number of identical stretches of a given wordlength shared by the input- and database- sequences.
  • "% of exact wordmatches", the identical stretches of a given wordlength shared by the input- and database- sequences, expressed as percentage of the maximum number of stretches (nonidentical and identical) of the same wordlength that can be made from the input sequence.
  • "External db", the external accession number, which is clickable and provides a link to the original accession for the database sequence on the source database website (the user's browser will exit the Allermatchtm website)
  • "Scientific name", Latin name of the organism from which the allergenic protein is derived
  • "Detailed information", after the "Go" button has been clicked on, a new page is created in the same window on the user's screen that contains information on the allergenic protein, and the hits of short identical stretches.

Detailed information

This page provides the following information on the hits of the selected wordlength with a specific allergenic protein:

  • The input sequence (amino acid sequence)
  • Details on the database sequence of the allergenic protein, including allergen name, scientific name, external accession id and amino acid sequence.
  • The complete amino acid sequences of the input- and database- sequences, while the "#"-symbols mark the residues within these sequences that are part of the exact hits with the wordlength of 6 amino acids (fixed wordlength, which does not change to the wordlength entered by the user).
  • Matches that are shorter than 6 amino acids may be found in the output of the full alignment (see below).

Example

For the Zea m 14 test sequence, tested against the Allermatchtm database, the summary table mentions various database sequences of allergenic proteins, including Zea m 14 itself, if a wordlength of 6 is selected. Besides Zea m 14, the other database sequences include, among others, allergenic proteins that are classified as lipid transfer proteins. Among the low ranking database sequences are Pru av 3 and Pru ar 3 from cherry and apricot, respectively, each of which scored one hit. As can be inferred from the detailed information, the single identical stretch of 6 amino acids (acnclk) in Pru av 3 and Pru ar 3 is also present in some of the other listed database sequences.

Full alignment

The new page that appears after starting the full alignment contains the following information:
  • Bar diagram showing the number of hits for certain statistical scores (E, opt) of the FASTA alignments of the input sequence with the database sequences of allergenic proteins.
  • List of database sequences of allergenic proteins, ranked in descending order of best statistical scores for the alignment of these sequences with the input sequence.
  • Details of each specific alignment from the previous list, in the same order.

Example

If Zea m 14 has been entered as input sequence, the highest scoring database sequences are the same as for the 80-amino acids sliding window alignment, i.e. lipid transfer proteins.

Questions: Dr. Gijs Kleter