NARWHAL

NARWHAL — Neoantigens Recognition Website and HLA Genotyping Tool

Start Neoantigen Start HLA typing

Neoantigen identification

Input data types

In this step, please select input data types. You can select DNA-seq, RNA-seq, or both of them.

DNA-seq RNA-seq

Basic Information

please provide your basic information.

Email address:

Please re-enter your Email for comfirmation.

Re-enter Email address:

Step 1-1. DNA Reads Upload (Tumor sample)

Select a method for uploading files:
From browser From URLs

Please upload your tumor DNA-seq files R1 and R2 here. Only allow .fastq.gz format. Maximum size for each file: 20 GB. Please note that uploading your files may take some time, so please refrain from refreshing the page while the data is being uploaded.

Start upload

Please provide the Google Drive file IDs (e.g., https://drive.google.com/file/d/Google Drive file ID /view) of your sequencing files in R1 and R2 fields. Using a share link from Google Drive is available. Only allow .fastq/.fq and .gz format.

R1:

R2:

Confirm URLs

Step 1-2. DNA Reads Upload (Normal sample)

Select a method for uploading files:
From browser From URLs

Please upload your normal DNA-seq files R1 and R2 here. Only allow .fastq.gz format. Maximum size for each file: 20 GB. Please note that uploading your files may take some time, so please refrain from refreshing the page while the data is being uploaded.

Start upload

R1:

R2:

Confirm URLs

Step 2. HLA genotype selection

Before binding affinity prediction, please select specific HLA genotypes. Personal HLA types will be predicted during analysis if select "Personal HLA types". Manual selection is also provided to select specific HLA types.

Personal HLA types Manual selection

Personal HLA types will be predicted during analysis, and the step of HLA binding prediction will be conducted based on the predicted HLA types.

Based on HLA major allele group frequencies.

Maximum number of HLA types: 8

Step 3. Peptide-HLA binding affinity

In this step, we provide pvactools to predict binding affinity scores between peptides and specific HLA(s). The default standard of ic50 is as followed, and the standard can be adjusted in customized mode. Noted that stronger binding affinity has lower ic50 number!

No binding (ic50 > 500) | Weak binding (500 ≥ ic50 > 250) | Intermediate (250 ≥ ic50 > 50) | Strong binding (50 ≥ ic50)
Default Settings Customized

Quast settings	Values
Maximum ic50 for Strong binding	[default: 50]
Maximum ic50 for Intermediate binding	[default: 250]
Maximum ic50 for Weak binding	[default: 500]

Step 4. LC-MS/MS peptides Upload (optional)

Once an incorrect or missing LC-MS/MS file upload, we will employ the IEDB database as a viable alternative to generate aeTSA.

Select a method for uploading files:
From browser From URLs

Please upload your FASTA files consisting of peptide sequences from LC-MS/MS here. Only allow .fasta.gz format.

Maximum size for each file: 1 GB.

Please provide the Google Drive file IDs (e.g., https://drive.google.com/file/d/Google Drive file ID /view) of your peptide sequencing files in fasta format. Using a share link from Google Drive is available. Only allow .fasta.gz format.

Peptide:

Confirm the URL

Step 5. Privacy Statement

NARWHAL NEVER shares users' uploaded data and results with others. All data and metadata of one task will be removed automatically from the server without any copies after 7 days of the report generated. To comply with ethical data handling principles, we strongly recommend that users obtain explicit patient consent prior to uploading any personal or genomic data to the platform. If such consent cannot be secured, users are advised to run the pipeline via a locally installed version to maintain full data control.

I have read the Privacy Statement of NARWHAL and accept NARWHAL to process my upload data.

Step 6. Data submission

Basic Information

please provide your basic information.

Email address:

Please re-enter your Email for comfirmation.

Re-enter Email address:

Step 1-1. RNA Reads Upload (Tumor sample)

Select a method for uploading files:
From browser From URLs

Please upload your tumor RNA-seq files R1 and R2 here. Only allow .fastq.gz format. Maximum size for each file: 20 GB. Please note that uploading your files may take some time, so please refrain from refreshing the page while the data is being uploaded.

Start upload

R1:

R2:

Confirm URLs

Step 1-2. RNA Reads Upload (Normal sample)

Select a method for uploading files:
From browser From URLs

Please upload your normal RNA-seq files R1 and R2 here. Only allow .fastq.gz format. Maximum size for each file: 20 GB. Please note that uploading your files may take some time, so please refrain from refreshing the page while the data is being uploaded.

Start upload

R1:

R2:

Confirm URLs

Step 2. Variant calling

In this step, we provide Varscan to call somatic mutation from RNA sequencing.
Default Settings Customized

Quast settings	Values
minimum coverage in normal & tumor to call variant (>=0)	[default: 8]
minimum coverage in normal to call somatic (>=0)	[default: 8]
minimum coverage in tumor to call somatic (>=0)	[default: 8]

Step 3. RNA expression level filtering

In this step, we perform RNA expression level filtering to filtering out variants with low expression levels.
Default Settings Customized

Quast settings	Values
minimal threshold of tumor RNA expression level (unit: tpm)	[default: 1.0]
minimal ratio of tumor RNA expression level to normal RNA expression level	[default: 0.0 (none)]

Step 4. HLA genotype selection

Personal HLA types Manual selection

Personal HLA types will be predicted during analysis, and the step of HLA binding prediction will be conducted based on the predicted HLA types.

Based on HLA major allele group frequencies, default HLA types are HLA-A*02:01, A*11:01, A*24:02, and A*33:03.

Maximum number of HLA types: 20

Step 5. Peptide-HLA binding affinity

Quast settings	Values
Maximum ic50 for Strong binding	[default: 50]
Maximum ic50 for Intermediate binding	[default: 250]
Maximum ic50 for Weak binding	[default: 500]

Step 6. LC-MS/MS peptides Upload (optional)

Once an incorrect or missing LC-MS/MS file upload, we will employ the IEDB database as a viable alternative to generate aeTSA.

Select a method for uploading files:
From browser From URLs

Please upload your FASTA files consisting of peptide sequences from LC-MS/MS here. Only allow .fasta.gz format.

Maximum size for each file: 1 GB.

Peptide:

Confirm the URL

Step 7. Privacy Statement

Confirm URLs

Step 3. RNA expression level filtering

In this step, we perform RNA expression level filtering to filtering out variants with low expression levels.
Default Settings Customized

Quast settings	Values
minimal threshold of tumor RNA expression level (unit: tpm)	[default: 1.0]
minimal ratio of tumor RNA expression level to normal RNA expression level	[default: 0.0 (none)]

Step 4. HLA genotype selection

Personal HLA types Manual selection

Personal HLA types will be predicted during analysis, and the step of HLA binding prediction will be conducted based on the predicted HLA types.

Based on HLA major allele group frequencies, default HLA types are HLA-A*02:01, A*11:01, A*24:02, and A*33:03.

Maximum number of HLA types: 20

Step 5. Peptide-HLA binding affinity

Quast settings	Values
Maximum ic50 for Strong binding	[default: 50]
Maximum ic50 for Intermediate binding	[default: 250]
Maximum ic50 for Weak binding	[default: 500]

Step 6. LC-MS/MS peptides Upload (optional)

Once an incorrect or missing LC-MS/MS file upload, we will employ the IEDB database as a viable alternative to generate aeTSA.

Select a method for uploading files:
From browser From URLs

Please upload your FASTA files consisting of peptide sequences from LC-MS/MS here. Only allow .fasta.gz format.

Maximum size for each file: 1 GB.

Peptide:

Confirm the URL

Step 7. Privacy Statement

I have read the Privacy Statement of NARWHAL and accept NARWHAL to process my upload data.