1. Specify the input sequences

All the input sequences must be in one-letter amino acid code. The allowed alphabet (not case sensitive) is as follows:

A C D E F G H I K L M N P Q R S T V W Y and X (unknown)

All the other symbols will be converted to X before processing.

The server allows for input in either FASTA or PEPTIDE format.

Note that for Peptide input, all peptides MUST be of equal length.

The sequences can be input in the following two ways:

Both ways can be employed at the same time: all the specified sequences will be processed. However, there may be not more than 500 sequences in total in one submission. The sequences shorter than 15 or longer than 10000 amino acids will be ignored.

2. Customize your run

1. Select peptide lenght from the list. Multiple lengths can be chosen by using Ctrl key. If no lenght will be selected, the method will give predictions for 9mer peptides.

2. Select a method which you want to use to get predictions. NetMHCcons is a default method, which integrates all 3 methods and gives the best predictions for the query allele. But if needed NetMHC, NetMHCpan or PickPocket methods can be used separately.

3. Select the allele(s) you want to make predictions for from the scroll-down menu or type in the allele names separated by commas (with out blank spaces). Note that for NetMHC method not all the alleles are available. When you choose NetMHC method, the lists of possible alleles to choose will be updated. If you want to type in the alleles, check the list of alleles before choosing the query allele if you want to use NetMHC method.

4. You can also paste a single full length MHC protein sequence in FASTA format or submit a file containing a full length MHC protein sequence in FASTA format directly from your local disk.

5. Specify thresholds for strong and weak binders. Two different types of thresholds can be set: based on the binding affinity giving in nM IC50 values or based on % Rank obtained using the method on 200.000 random natural peptides. The peptide will be identified as a strong binder if the % Rank OR binding affinity (IC50) is below the specified threshold for the strong binders. The peptide will be identified as a weak binder if the % Rank OR binding affinity (IC50) is above the threshold of the strong binders but below the specified threshold for the weak binders.

6. Choose if you want to filter the output so that not all the predictions will be shown. If you choose Yes, you will be able to choose filtering thresholds for % Rank and IC50 values. Only predictions for peptides that will have the % Rank OR binding affinity (IC50) below the specified thresholds will be shown in the output.

7. Click the box Sort by affinity to have the output sorted by descending predicted binding affinity.

8. Click the box Save prediction to xls file if you want the output to be saved in xls file.

3. Submit the job

Click on the "Submit" button. The status of your job (either 'queued' or 'running') will be displayed and constantly updated until it terminates and the server output appears in the browser window.

At any time during the wait you may enter your e-mail address and simply leave the window. Your job will continue; you will be notified by e-mail when it has terminated. The e-mail message will contain the URL under which the results are stored; they will remain on the server for 24 hours for you to collect them.