Functional Features Prediction and Visualization Tutorial

The purpose of this tutorial is to demonstrate how to predict and visualizing transcripts using IsoTV. The following inputs will be used to visualize the PDK2 gene: annotated transcriptome file (.gtf), transcript sequences (.fa/.fas), and isoform expression.

The data used for tutorial can be found in the GitHub under example/data.

The goal is to reconstruct the figure for the PDK2 gene from the IsoTV paper, which can also be found under example/PDK2_tutorial.pdf. It will assume that IsoTV has already been downloaded and installed.

Configuration

To follow this tutorial, the config_example_pdk2.yaml configuration file can be used. However, the file needs to be modified to include the full local paths for the required prediction tools.

Input file directory:

| -- IsoTV
    | -- example/
        | -- data/
            | -- genes.tab
            | -- PDK2.nanopore.gtf
            | -- PDK2.transcriptome.fas
            | -- PDK2.counts.txt
    ...

Paths for external tools to be changed (can be installed here ):

iupred2a_path: "/path/to/iupred2a/iupred2a.py"

brewery_path: "/path/to/Brewery/Brewery.py"

interproScan_path: "/path/to/my_interproscan/interproscan-5.38-76.0/interproscan.sh"

prositeScan_path: "/path/to/ps_scan/ps_scan.pl"
pfScan_path: "/path/to/ps_scan/pfscan"
prositeDat_path: "/path/to/prosite.dat"

For this tutorial, we are not basecalling or preprocessing ONT data, thus we set this parameters False. Annotation and quantification files are provided to complement the feature analysis, and thus set these to True. (This should already be set in the configuration file.)

basecalling: FALSE
preprocess: FALSE
annotation: TRUE
quantification: TRUE

Executing

The command below can be used to run IsoTV for this tutorial. This command needs to be executed from the IsoTV directory itself.

snakemake -j 32 Results/Output/test.pdf --configfile config_example_pdk2.yaml

This will use the config_example_pdk2.yaml configuration file, run prediction analysis on PDK2 using 32 cores, and force the pipeline to create Results/Output/test.pdf under example/.

However, the below commands is more apt for any future analysis. The difference here is that the filename for the output pdf file (i.e. test2.pdf) can be set directly from the command line:

snakemake -j 32 --configfile config_example_pdk2.yaml --config output_plots=test2.pdf

or from the configuration file (changing output_plots: "test2.pdf"):

snakemake -j 32 --configfile config_example_pdk2.yaml

Output

As the working directory has been set to example, all the intermediate, temporary, and output files will be created under example. The file that we are interested is test.pdf. Verify that this file is the same as example/PDK2_tutorial.pdf.

Output file directory:

| -- IsoTV
    | -- example/
        | -- Results/
            | -- Genes_temp/ (this folder is created and deleted)
            | -- Genes/
                | -- PDK2/
                    | -- PDK2_transcripts_filtered_analysis.txt
                    | -- PDK2_transcripts_stats.txt
                    | -- PDK2_functional_analysis.pdf
            | -- Output/
                | -- test.pdf
    ...