GenomeProt Help Guide

Overview

GenomeProt is a comprehensive proteogenomic analysis tool used to identify:

  • Canonical proteins
  • Noncanonical proteins (uORFs, dORFs, noncoding RNA–derived proteins)
  • Variant peptides and proteoforms

The workflow consists of four steps:

  1. Database Generation
  2. Analyse MS Proteomics (External Step)
  3. Integrate Data
  4. Visualise Results

1. Database Generation

Users can choose between short-read and long-read options depending on their data type.

The Shiny web server supports database generation using:

  • Pre-aligned BAM files
  • GTF file of assembled transcripts from the dataset

Users can also generate a variant-aware proteome database using an optional multisample VCF file generated from variant calls derived from the same samples.

By default, the app loads a demo dataset (BAM file input) to demonstrate processing and output files.

Files used to run the demo dataset can be downloaded below:

Steps to generate a database using the demo dataset

  1. Navigate to the Generate Database tab.
  2. Select a checkbox Incoporate SNVs into protein sequences.
  3. Click Submit using default options.
  4. The app processes the BAM file.
Database Generation screenshot

Output files:

  • proteome_database.fasta: Multi-FASTA protein database
  • proteome_database_metadata.txt: TSV file containing annotations for candidate protein sequences
  • proteome_database_transcripts.gtf: GTF file with transcript coordinates used to generate the proteome database

Results from this step can be downloaded within the app. The test results file can be downloaded here.

Note: To generate a database directly from FASTQ files, or BAM files larger than 20 GB, use the command-line version.

Command-line instructions are available here.

2. Analyse MS Proteomics (External Step)

This step must be completed outside the Shiny application. Using the database file (.fasta) generated in Step 1, analyse the proteomics data with your preferred search algorithm to identify proteins and peptides in your dataset.

3. Integrate Data

This step maps peptides identified in Step 2 to spliced transcript coordinates.

  • Files required from step 1: proteome_database_metadata.txt and proteome_database_transcripts.gtf
  • Files required from step 2: peptide_data.tsv (must follow required format)

Steps to map peptides

  1. Download ZIP file containing: proteome_database_metadata.txt, proteome_database_transcripts.gtf
  2. Extract ZIP file
  3. Download demo peptide_data.tsv from here.
  4. Navigate to Integrate Data tab
  5. Upload required files
  6. Click Submit
  7. After successful mapping, the Download results (zip) button turns green.
  8. Click to download output zip file.
Integrate Data screenshot

Output directory contents:

  • summary_report.html: Summary report of mapped peptides, transcripts, and ORFs
  • report_images/: Folder containing PDF versions of graphs from the summary report
  • peptide_info.tsv: Detailed peptide mapping annotations
  • combined_annotations.gtf: GTF file with mapped peptides and transcript coordinates
  • peptides.bed12: BED12 file with mapped peptide coordinates for UCSC Genome Browser visualisation
  • transcripts.bed12: BED12 file with transcripts supported by peptide evidence for UCSC Genome Browser visualisation
  • ORFs.bed12: BED12 file with ORFs supported by peptide evidence for UCSC Genome Browser visualisation

The demo results files can be downloaded here.

4. Visualise Results

This step visualises peptides on transcript and gene coordinates using IsoVis.

  • Click 'Upload data' and select combined_annotations.gtf as the 'transcript data' file (max. 3 GB).
  • Optional: select raw transcript counts as the 'transcript counts' file.
  • Optional: select the peptide intensities file from MS analysis as the 'peptide intensities' file.
  • Click Apply.
  • Select your gene of interest. Start typing either its gene name or Ensembl ID into the search box, select it from the list of results displayed, then either press enter or click '>'.
  • Use the different visualization options provided in IsoVis. For example, parts of the visualization can be toggled, including protein domain labels in the protein diagram.
  • The entire visualization can be exported as a PNG, JPEG, PDF or SVG, and individual visualization components can be exported as SVGs.
IsoVis screenshot 1

IsoVis screenshot 2

Visualisation output:

The IsoVis visualisation displays separate tracks for peptides, ORFs and transcripts. Users can limit their analysis to specific peptides, ORFs or transcripts of interest by hiding irrelevant parts of the visualization and using the peptide and ORF stacks to highlight specific features.

Overlapping ORFs are shown using a hatching pattern. Coding regions are represented as thick dark grey boxes. Peptides uniquely mapped to ORFs, transcripts, and genes are indicated in orange, cyan, and blue, respectively. Multi-mapping peptides are dark grey.

For additional IsoVis details, refer to the documentation here.