NetMHC-3.0 predicts the binding affinity of either a list of peptides with a defined length (8–11 residues) or all possible sub-peptides hosted within full-length proteins. The input must be in the FASTA format, or as peptides all of equal length, one peptide pr. line. The server will accept a maximum of 5000 sequences per submission; each sequence not more than 20 000 amino acids with a minimum length corresponding to the selected length of prediction (see subsequently). Input data can be pasted into a text field or uploaded from a local file on the user's computer.
If the input is in peptide, format the corresponding tick-box must be selected. The input must not exceed 5000 sequences and with a maximum of 20 000 amino acids in each sequence. One or more MHCs must be selected, as well as the desired peptide length. Only one prediction length at a time can be used. The output can optionally be sorted according to the predicted affinity by selecting a tick-box. The predictions start by clicking the Submit button. An example input in FASTA format is shown in .
The output is displayed as raw text with a header indicating the server name, the type of prediction (PSSM, ANN or ANN-approximation) the first selected allele and the date () followed by the prediction output in a column format. The columns are named in the first line of the prediction output. The first column [pos] is the position of the first amino acid of the predicted peptide within the possibly longer sequence, numbering starting with 0. Column (peptide) is the primary sequence of the (sub-)peptide. Column (logscore) is the raw prediction output, which for ANNs is 1-log50000 to the affinity in nanomolar units. For PSSM predictions the raw prediction score is a log-odds likelihood score. Additionally a column is included for ANN predictions, [affinity (nM)], which is the predicted affinity presented in nanomolar units. Column (Bind Level) indicates if the peptide is predicted to bind stronger than a certain threshold [for ANN predictions stronger than 50 nM (SB) or stronger than 500 nM (WB); for PSSM high-binding peptides (SB) have a prediction score greater than the 0.1% percentile score value of 1 000 000 random natural peptides, and weak binding (WB) peptides a score value above the 1% percentile score of 1 000 000 random natural peptides predictions]. Predicted affinities weaker than 500 nM or lower than the 1% percentile score have no indications. Column (Protein Name) gives the name of the predicted protein. If peptide input was used, the name will always be ‘Sequence’. Column (Allele) gives the name of the MHC allele chosen. The output contains all the sub-peptides for each protein for a given allele either in the order they appear in the sequence or sorted by predicted affinity within each protein (if chosen). If more than one protein sequence were entered, a dashed line will separate the peptides from each protein. If more than one allele were chosen, the output will show a header similar to the first immediately after the first predictions, all in the same web output page.
Raw text output using the input in and selecting the alleles HLA-A0201 and HLA-A0301. 10-mer peptide predictions were chosen. Affinity sorting was chosen.
In each header, there is a link to a file with the output in tab as separated format, where the filename ends on.xls making it easily imported into spreadsheet programs. This file always contains the predicted peptides in the order they appeared in the input file. The output data for each peptide will be displayed on a single line with predictions for each of the selected alleles in different columns ().
Downloaded output sheet opened in Microsoft® Excel and adjusted with of column. The output was generated using input in and selecting the alleles HLA-A0201 and HLA-A0301. 10mer peptide predictions were chosen.