INPUT DATA

In this section, the user must define the input for the prediction server following these steps:

1) Specify the desired input data type (FASTA or PEPTIDE) using the drop down menu.

2) Provide the input data by means of pasting the data into the blank field, uploading it using the "Choose File" button or by loading sample data using the "Load Data" button. All the input sequences must be in one-letter amino acid code. The alphabet is as follows (case sensitive):

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

Any other symbol will be converted to X before processing. At most 5000 sequences are allowed per submission; each sequence must be not more than 20,000 amino acids long and not less than 8 amino acids long.

3) Specify the input format (and peptide length(s) if required):

Sample input data is given after each of the listed options.

a. If FASTA was selected as input type, the user must choose the peptide length(s) the prediction server is going to work with. The input FASTA sequence is digested in overlapping peptides of the provided length(s) and the program predicts binding against all of them. By default input proteins are digested into 9-mer peptides.

Also, the user must decide between these 3 input format options:

1. Lookup TPM values per peptide
   
   The digested peptides are searched against the HPA database. Gene expression value annotation for a given peptide is defined as the sum of the TPM values of all protein-coding transcripts containing the exact queried peptide. If a peptide is not found, the program will assign TPM=0.
   
   
2. Lookup TPM values per protein (using PROT_ID)
   
   The PROT_ID is searched against the HPA database (or the user-specified expression database). Gene expression value annotation is the same for all digested peptides and is equal to the TPM value associated with the queried PROT_ID. If the PROT_ID is not found, the program will assign TPM=0.
   
   Sample input data for options 1. & 2. :
   
   >ENSP00000253039.4
   MAGGEAGVTLGQPHLSRQDLTTLDVTKLTPLSHEVISRQATI...
   
   
3. TPM values given in the input file
   
   The digested peptides are assigned the TPM value annotated in the input file. This number should be a positive rational number. If this is not satisfied, the program will assign TPM=0.
   
   >ENSP00000253039.4 TPM=80.3919983
   MAGGEAGVTLGQPHLSRQDLTTLDVTKLTPLSHEVISRQATI...

b. If PEPTIDE was selected as input type, the user must select one of the following 3 input format options:

In all cases, the input format can contain an additional 2^nd column that has numerical values. This column can be used to store scores (or target values) that can be futher on employed to compute evaluation metrics.

1. [PEP] ([Score])
   
   Peptides are searched against the HPA database. Gene expression value annotation works the same way as described above in a.1.
   
   AAADIVNFL
   AAADSIKIW
   AAAHFYFEL
   AAAPQLLIV
   ...
   
   
2. [PEP] ([Score]) [PROT_ID]
   
   PROT_ID(s) are searched against the HPA database (or the user-specified expression database). Gene expression value annotation is performed by assigning to each peptide its corresponding PROT_ID's TPM value. If the PROT_ID is not found, the program will assign TPM=0. For a list of the Ensembl protein IDs present in the HPA database (genome assembly GRCh38.p12, GENCODE v.28), click HERE.
   
   AAADIVNFL ENSP00000404403.1
   AAADSIKIW ENSP00000308179.4
   AAAHFYFEL ENSP00000415612.1
   AAAPQLLIV ENSP00000252593.6
   ...
   
   
3. [PEP] ([Score]) [TPM]
   
   The input is already in the required format for prediction.
   
   AAADIVNFL 0.497551
   AAADSIKIW 3.67981
   AAAHFYFEL 8.54361
   AAAPQLLIV 108.95
   ...
   
   NOTE: If PEPTIDE was chosen as input type, peptide length selection is unnecessary and thus the corresponding selection box will directly not appear in the interface.

MHC SELECTION

4) The user must define which MHC(s) molecule(s) the input data is going to be predicted against:

a. Select the HLA/MHC supertype family. After choosing the MHC family, you will be able to select a single or multiple MHC molecules from the updated "Select Allele(s)" list.

b. Type the MHC allele names in the provided blank field (separated by commas and without blank spaces). If this is the case, there will be no need to select an MHC supertype familiy from the drop-down menu.

Click here for a list of MHC molecule names. Please note that a maximum of 20 MHC types is allowed per submission.

c. Paste a full MHC protein sequence in the blank box, or

d. directly upload it by clicking the "Choose file" button. Such sequence must be in FASTA format.

ADDITIONAL CONFIGURATION

In this section, the user may define additional parameters to further customize the run:

5) & 6) Specify thresholds for strong and weak binders. They are expressed in terms of %Rank, that is percentile of the predicted binding affinity compared to the distribution of affinities calculated on set of random natural peptides. The peptide will be identified as a strong binder if it is found among the top x% predicted peptides, where x% is the specified threshold for strong binders (default: 0.5%). The peptide will be identified as a weak binder if the % Rank is above the threshold of the strong binders but below the specified threshold for the weak binders (default: 2%).

7) Specify a %Rank threshold to filter out predictions. Only sequences with a predicted %Rank value less than the specified threshold will be printed. To print all predictions, leave this value set to -99.

8) Tick this box to have the output sorted by descending prediction score.

9) Tick this box to exclude expression values from the prediction. The method will ignore expression values in the input file, if present.

10) Specify the maximum TPM value to map input TPM values to the [0,1] interval. If the values already fall within this unit range, set this option to -1.

11) Load a file with a reference protein expression database. The file must contain two columns: [PROT_ID] [TPM]. This file will be used perform automatic TPM value annotation of input peptides or proteins (FASTA format). Be aware that the input peptides (or proteins) must have PROT_ID(s) that match the ones present in the provided database.

12) Enable this option to export the prediction output to .XLS format (readable by most spreadsheet softwares, like Microsoft Excel).

SUBMISSION

After the user has finished the "INPUT DATA", "MHC SELECTION" and "ADDITIONAL CONFIGURATION" steps, the submission can now be done. To do so, the user can click on "Submit" to submit the job to the processing server, or click on "Clear fields" to clear the page and start over.

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.

After the server has finished running the corresponding predictions, an output page will be delivered to the user. A description of the output format can be found at output format HERE.

At any time during the wait you may enter your e-mail address and simply leave the window. Your job will continue; when it terminates you will be notified by e-mail with a URL to your results. They will be stored on the server for 24 hours.