TX2P - Transcript to protein

TX2P allows for automated integration of mass spectometry data in long-read RNA-sequencing workflows. It does so by predicting open reading frames for each transcripts within an input GTF/GFF file, translates it into peptide sequences and then searches for those predicted proteins in mass spec datasets using MetaMorpheus.

NOTE! Currently TX2P is only set up to work with GRCh38. Chromsome IDs need to be matching those in UCSC hg38

Prerequisites

Before installing TX2P, you need to have Docker installed on your system. Docker is a platform that allows you to create, deploy, and run applications in containers. More information about Docker can be found at https://www.docker.com/. Follow the instructions below to install Docker if you have not already:

Installing Docker

For Linux:

1. Open a Terminal: Use your Linux distribution's package manager to install Docker. The command varies depending on the distribution:

   Ubuntu/Debian:

   sudo apt-get install docker-ce docker-ce-cli containerd.io
   

   Fedora:

   sudo dnf -y install docker-ce
   

   CentOS:

   sudo yum install docker-ce docker-ce-cli containerd.io
   

2. Start the Docker Service: Use sudo systemctl start docker to start the Docker service.

3. Verify Installation: Check if Docker is installed correctly by typing docker --version in the terminal.

For Windows

See detailed steps here: WINDOWS

Getting Started:

Download the two docker images:

docker pull murphydaviducl/getorf:latest

docker pull murphydaviducl/metamorpheusdocker:latest

Clone the directory. From command line simply run the following command from the directory where you wish to install the repo:

git clone https://github.com/MurphyDavid/TX2P

cd TX2P

metamorpheusdocker is a copy of MetaMorpheus as availible from https://smith-chem-wisc.github.io/MetaMorpheus/ or https://github.com/smith-chem-wisc/MetaMorpheus If desired you should be able to use an updated version from those sources. Please cite https://doi.org/10.1021/acs.jproteome.7b00873

Input

Transcript file

TX2P requires a TSV/CSV file with the transcript IDs in a single column, one ID per line.

Example format:

PB.26.5
ENST00000000001
ENST00000000002
STRNG003
STRNG004
ENCL00000000005
ENCL00000000006

Transcript structures

TX2P also requires an input GTF/GFF file with the transcript structures to test. This file needs transcript_id specified in the follwong format, with IDs matching those in the above TSV/CSV file.

Example format:

chr1	PacBio	transcript	1785288	1890877	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1785288	1787053	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1787322	1787437	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1789053	1789269	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1817837	1817875	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1825397	1825499	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1839190	1839238	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	exon	1890820	1890877	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	CDS	1787331	1787437	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	CDS	1789053	1789269	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	CDS	1817837	1817875	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";
chr1	PacBio	CDS	1825397	1825453	.	-	.	gene_id "ENSG00000078369.18"; transcript_id "PB.26.5";

Testing:

Getting ORFs

TODO: allow only alphanumeric charaters as transcript ids.

current_folder=$(pwd)

docker run -v $current_folder:/current_folder  --entrypoint Rscript murphydaviducl/getorf /getcds/getCDSandAAseq.R --gtf /current_folder/test.gtf --transcript /current_folder/test_transcripts.tsv --output /current_folder/output_ORFs.csv

Parameter	Description
--gtf	Path to the GTF/GFF file containing transcript structures to test.
--transcript	Path to a TSV/CSV file with a single column (no headings) listing transcript IDs of interest.
--output	Path to the output CSV file containing predicted Open Reading Frames (ORFs).

Searching mass spec data.

Download a mass spec file for testing.

wget ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/09/PXD020044/H_Luh_ND_3.raw

Create an output folder

current_folder=$(pwd)

mkdir testoutput

cd ./testoutput

Run on the example mass spec data.

docker run -v $current_folder:/current_folder -it --entrypoint dotnet murphydaviducl/metamorpheusdocker /metamorpheus/CMD.dll -o /current_folder/testoutput -t /current_folder/mmconfig/Task2-CalibrateTaskconfig.toml /current_folder/mmconfig/Task4-GPTMDTaskconfig.toml current_folder/mmconfig/Task5-SearchTaskconfig.toml -d /current_folder/test.fasta -s /current_folder/H_Luh_ND_3.raw

Parameter	Description
-o	Output directory where the results will be stored.
-t	Paths to MetaMorpheus configuration files (Task2, Task4, Task5).
-d	Path to the FASTA file containing sequences of transcripts of interest.
-s	Path to the mass spectrometry file(s) for analysis.

You can add almost any number of additional mass spec files, it ss recommended to include a full dataset to allow good calibration, though we suggest keeping it to less than 200GB. Provide the Docker access to additional folders by adding more in the format -v folder:/mountname. The paths to additional raw files need to be given in terms of the mounted path inside the Docker container.

Supported formats are

• Thermo .raw

• mzML file in centroid mode.

• .mgf

Using the provided example files, once the tool has run, you should now have a folder that looks like this:

The Task3 folder should look like this:

The AllQuantifiedProteinGroups should have one entry like this:

Usage

If you are trying to confirm that a transcript in an organism is producing a protein, it's suggested to include the full known proteome for that organism just as you would a contaminants file. For humans, the relevant fasta file can be downloaded from UniProt: https://www.uniprot.org/help/downloads

docker run -v $current_folder:/current_folder -it --entrypoint dotnet mymodifiedmetamorpheus:latest /metamorpheus/CMD.dll -o /current_folder/testoutput -t /current_folder/mmconfig/Task2-CalibrateTaskconfig.toml /current_folder/mmconfig/Task4-GPTMDTaskconfig.toml current_folder/mmconfig/Task5-SearchTaskconfig.toml -d [Fasta file for transcripts of interest] /current_folder/contaminants.fasta [Fasta file of known proteins for organism of interest] -s [complete list of mass spec files]

Parameter	Description
-d	Path to the FASTA file containing sequences of transcripts of interest.
/current_folder/contaminants.fasta	Path to the FASTA file containing sequences of contaminants.
[Fasta file of known proteins for organism of interest]	Path to the FASTA file of known proteins for the organism of interest.
-s	Path to the complete list of mass spectrometry files for analysis.

Citation

Please cite our manuscript: