The following section will take
you on a stepwise tour discussing how to use this server ?
To run prediction, follow these
1: Type the following URL address in your web browser
2: The user is required to fill the sequence submission
form. A brief description of each of the field is as follows :
of Antigen: This is an optional field.
your sequence below: Paste your antigen sequence in one of the
standard format (FASTA, EMBL, PIR etc.) or amino acid sequence only in single
submit from file: The user can also upload the antigen sequence
directly from a file.
NOTE: Care should be taken that the server accepts input from
either of two options, not both.
sequence format: The user has to select the appropriate format
according to the input sequence.
- The following parameters allow
users to perform a customizable MHC Class-II binding peptide prediction.
The threshold is an important parameter which is
defined as the 'percentage of best scoring natural peptides'. For example,
a threshold of 1% would predict peptides in any given protein sequence which
belong to the 1% best scoring natural peptides.
- The % threshold parameter allows
the user to select for different stringency levels, in order to modulate the
prediction results: a lower threshold corresponds to a high stringency prediction,
i.e. to a lower rate of false positives and to a higher rate of false negatives.
In contrast, a higher threshold value (low stringency) corresponds to a higher
rate of false positives and a lower rate of false negatives. In short, from
the same protein sequence input, a threshold setting of 1% will predict a
lower number of peptide sequences and for a lower number of HLA-II alleles,
compared to 2% or higher thresholds; however, this will also ensure a higher
likelihood of positive downstream experimental results. Normally, at least
for a first round of screening, threshold values higher than 3% are not
desirable, since the rate of false positives can increase the size of the
predicted repertoire to an amount unacceptable for later experimental testing.
top scorer: Value in this field represent the number of top scorer
in query antigen, to be displayed in tabular format. The peptide score of
each nanomer in an antigen is calculated using quantitative matrices. The
higher the score of any peptide frame the greater is the probability of it's
binding to given MHC molecule. Default value is 5 % of the total number of
nanomeric frames in query antigen.
The user can select single/multiple allele form a list of 51 HLA-DR alleles.
Multiple allele option is helpful in locating promiscuous binding regions
Display Format: The server offers different result display formats
to ease the identification of promiscuous binders. The server display the
binding peptides in sequence for selected alleles. User can select from the
view I: Predicted binders are displayed as region underlined with "
* " . This display is handy in locating overlapping binding regions
in terms of their extend of overlap.
view II: Predicted binders are displayed as blue colored region, with
P1 anchor or the starting residue of each predicted binding frame as red colored.
This display is useful in locating promiscuous binding regions.
View : The server allows to present the following parameters in graphical
Score distribution profile
Best scoring subsequence profile (only during
Step 3: Finally click on "Submit" button.
Each of the graphical
output generated by server upon click provide description of corresponding