HotSpot3D

This 3D proximity tool can be used to identify mutation hotspots from linear protein sequence and correlate the hotspots with known or potentially interacting domains, mutations, or drugs. Mutation-mutation and mutation-drug clusters can also be identified and viewed.

Usage

    Program:     HotSpot3D - 3D mutation proximity analysis program.
    Version:     V1.0.0
     Author:     Beifang Niu, John Wallis, Adam D Scott, & Sohini Sengupta

Usage: hotspot3d [options]

       Preprocessing
         prep      --  Run preprocessing steps 2-7 (beta)

         drugport  --  0) Parse drugport database (OPTIONAL)
         uppro     --  1) Update proximity files
         calroi    --  2) Generate region of interest (ROI) information
         statis    --  3) Calculate p_values for pairs of mutations
         anno      --  4) Add region of interest (ROI) annotation
         trans     --  5) Add transcript annotation
         cosmic    --  6) Add COSMIC annotation to proximity file
         prior     --  7) Prioritization

       Analysis

         search    --  0) 3D mutation proximity searching
         post      --  1) Post-processing of 3D proximity searching output
         cluster   --  2) Determine mutation-mutation and mutation-drug clusters
         sigclus   --  3) Determine significance of clusters (BETA/OPTIONAL)
         summary   --  4) Summarize clusters (OPTIONAL)
         visual    --  5) Visulization of 3D proximity (OPTIONAL)

Support

For user support please email [email protected]

Update

To reinstall code (in some cases, may need --sudo):

cpanm --reinstall HotSpot3D-#.tar.gz

Install (Ubuntu 14.04.01)

Prerequisites:

In order to install HotSpot3D package, first install CPANM

(cpanm - get, unpack build and install modules from CPANM)

NOTE: Some steps may require adding --force to install successfully.

sudo apt-get install cpanminus

Another way to install cpanminus is to just download it, as per the installer
    
	curl -LO http://xrl.us/cpanm

	chmod +x cpanm

Or by using cpan

	cpan App::cpanminus

Intall Perl5 local lib

cpanm --local-lib=~/perl5 local::lib && eval $(perl -I ~/perl5/lib/perl5/ -Mlocal::lib)

Intall LWP::Simple module

sudo apt-get install libwww-perl

Intall Test::Most module

wget http://search.cpan.org/CPAN/authors/id/O/OV/OVID/Test-Most-0.34.tar.gz

cpanm Test-Most-0.34.tar.gz

Install HotSpot3D package:

git clone https://github.com/ding-lab/hotspot3d

cd hotspot3d

cpanm HotSpot3D-#.#.tar.gz


Installations under some organizations may use an internal perl version.

To make use of the /usr/ perl, edit the first line of ~/perl5/bin/hotspot3d.

from: #!/org/bin/perl

to: #!/usr/bin/perl)

Example - Preprocessing

1. (Optional) Run drugport module to parse Drugport data and generate a drugport parsing results flat file :

    hotspot3d drugport --pdb-file-dir=pdb_files_dir
   

2. Run 3D proximity calculation that also updates any existing preprocessed data (default launches LSF jobs) :

    hotspot3d uppro --output-dir=preprocessing_dir --pdb-file-dir=pdb_files_dir --drugport-file=drugport_parsing_results_file 1>hotspot3d.uppro.err 2>hotspot3d.uppro.out
   

3. Calculate protein domain information for each UniProt ID (make sure all uppro jobs have finished!) :

    hotspot3d calroi --output-dir=preprocessing_dir
   

4. Significance determination calculation :

    hotspot3d statis --output-dir=preprocessing_dir
   

5. Add protein domain annotation information to 3D proximity information :

    hotspot3d anno --output-dir=preprocessing_dir
   

6. Choose transcripts based on the alignment between Uniprot sequence and human peptides sequences :

    hotspot3d trans --output-dir=preprocessing_dir
   

7. Add cosmic v67 information to 3D proximity results :

    mkdir preprocessing_dir/cosmic
   
    cp COSMIc/cosmic_67_for_HotSpot3D_missense_only.tsv.bz2 ./preprocessing_dir/cosmic/
   
    cd ./preprocessing_dir/cosmic/ 
   
    bzip2 -d cosmic_67_for_HotSpot3D_missense_only.tsv.bz2
   
    hotspot3d cosmic --output-dir=preprocessing_dir
   

8. Prioritization :

    hotspot3d prior --output-dir=preprocessing_dir --p-value-cutoff=0.1 --3d-distance-cutoff=20 --linear-distance-cutoff=0.5
   

Example - Analysis

3D proximity searching based on prioritization results and visualization

1. Proximity searching (acquire proximity information for input mutations):

    hotspot3d search --maf-file=your.maf --prep-dir=preprocessing_dir
   

2. Post-processing of pairwise data (required for cluster step):

    hotspot3d post --maf-file=your.maf
   

3. Cluster pairwise data:

    hotspot3d cluster --collapsed-file=3D_Proximity.pairwise.singleprotein.collapsed --pairwise-file=3D_Proximity.pairwise --maf-file=your.maf
   

4. Cluster significance calculation:

    hotspot3d sigclus --prep-dir=preprocessing_dir --pairwise-file=3D_Proximity.pairwise --clusters-file=3D_Proximity.pairwise.singleprotein.collapsed.clusters
   

5. Clustering Summary:

    hotspot3d summary --clusters-file=3D_Proximity.pairwise.singleprotein.collapsed.clusters
   

6. Visualization (works with PyMol):

    hotspot3d visual --pairwise-file=3D_Proximity.pairwise --clusters-file=3D_Proximity.pairwise.singleprotein.collapsed.clusters --pdb=3XSR
   

Annotations

Check out scripts/ for various annotation scripts to add more details to the .clusters file.

HGNC download can be found here: http://www.genenames.org/cgi-bin/genefamilies/.

Information on the Ensembl .gtf can be found here: http://useast.ensembl.org/info/website/upload/gff.html, and downloads can be found at the Ensembl ftp site, ftp://ftp.ensembl.org/pub/.

See the scripts/README.annotations for more details.

Tips

Mutation file - Standard .maf with custom coding transcript and protein annotations (ENST00000275493 and p.L858R)

There are only a handful of columns necessary from .maf files. They are:

	Hugo_Symbol
	
	Chromosome
	
	Start_Position
	
	End_Position
	
	Variant_Classification
	
	Reference_Allele
	
	Tumor_Seq_Allele1
	
	Tumor_Seq_Allele2
	
	Tumor_Sample_Barcode

And two non-standard columns:

	a transcript ID column
	
	a protein peptide change column (HGVS p. single letter abbreviations, ie p.T790M)

Current Annotation Support:

	Transcript ID - Ensembl coding transcript ID's (ENST)

	Gene name - HUGO symbol

Clustering with different pairs data:

	For intra you need to include the singleprotein pairs without DrugPort results/pairs.

	For inter you need complex pairs without DrugPort pairs.

	For DrugPort only, do not include singleprotein or complex pairs; include only DrugPort pairs.

	For intra+inter you can concatenate the singleprotein and complex pairs without DrugPort pairs.

	For intra+DrugPort include singleprotein pairs and DrugPort pairs.

	For inter+DrugPort include complex pairs and DrugPort pairs.

	For intra+inter+DrugPort include a concatenated singleprotein and complex pairs file with the DrugPort pairs.

NOTE: that if concatenating pairs files, you should take care with removing the second header that will appear in the middle of the file. The .pairwise file contains both intra and inter pairs, so it can be used when involving intra or inter clustering.

Clustering based on different distance measures:

    There are some pairs found on multiple structures. In HotSpot3D versions v0.6.2 and earlier, clustering only used the shortest distance among different structures (shortest structure distance, SSD). In HotSpot3D versions v0.6.3 and later, clustering can be done using the average distance among different structures (average structure distance, ASD), and this is now default.