MutPredMerge

Workflow that integrates and parallelizes use of the MutPred suite with VCF files.

Content

Motivation
Installation
Usage
Other Resources
Citation

Motivation

The MutPred suite is a collection of machine learning tools that predict the pathogenicity of protein-coding variants to infer molecular mechanisms of disease. MutPred takes in a fasta formatted amino acid sequences as the primary input. The challenges with this input format are that isolated amino acid sequences cannot be easily assigned to genomic locations and the genomics community (including clinical genomics) works in the chromosomal space and uses VCF (Varant Call Format) as their primary file format.

The MutPred suite offers several advantages over other functional prediction methods:

Tools in the MutPred suite have been shown to be among the top-performing methods in independent assessments and community-wide experiments
Apart from general pathogenicity scores, the MutPred tools return a ranked list of putative molecular mechanisms, thus generating hypotheses for further experimental follow-up.

Hence, if MutPred were to ingest a VCF containing DNA sequence variants and perform its calculations on those variants that map to conincal forms of conceptually translated amino acid sequences, then MutPred's acceptance and use would increase in the genomics community to ultimately aid in clinical DNA sequence analaysis. In particular MutPred's prediction capabiliies could improve the annotation of variants of uncertain significance.

The above goal can be acheived through a scalable integrated workflow that combines genomic tools designed to annotate data stored in VCF files with tools in the MutPred Suite. In terms of performance, this workflow should be able to analyze VCF files containing an order of 100,000 variants in a few hours and be extensible with respect to VCF annotation. Because MutPred tools operate on individual amino acid sequences, and in some cases subsequences, scalablity can be achieved through standard parallelization using multiple computer nodes within a cluster. Making such a system portable for wide use is enabled by employing a standardized worflow system and containerizing tools that have complex installation requirements.

Workflow alt text

Link to Workflow

Future Directions

Implement support for other annotation tools - SNPeff, VARANT, OpenCRAVAT
Think about containerization and accessibility to users
Scale up to cloud and HPC environments

Installation and Dependencies

Snakemake

Snakemake: Current conda installation of Snakemake has datrie dependency failure: See bug: (1) https://bitbucket.org/snakemake/snakemake/issues/934/installation-failed-in-python-37 and (2) https://github.com/pytries/datrie/issues/52

Update datrie dependency first.

wget https://github.com/pytries/datrie/archive/0.7.1.tar.gz
tar xf 0.7.1.tar.gz
cd datrie-0.7.1
./update_c.sh 
python3.7 setup.py build
python3.7 setup.py install

Normal conda installation of Snakemake should work after this.

conda install -c bioconda -c conda-forge snakemake

MutPred Suite

To install the MutPred Suite, run the install bash script in the main directory. Beware, the full MutPred suite, when unzipped, takes 90 Gb to store.

bash install.sh

If you would like to install the different tools yourself, you can download them here:

MutPred2
MutPredLOF
MutPred-Indel

Put them into the tools directory and use tar -xvzf to unzip the tarballs. You will need to find and download the MATLAB MCR in order to run MutPredLOF and MutPred-Indel. MutPred2 comes with a MATLAB MCR so copying that folder into the other two is an option. Just pointing to the MutPred2 MCR directory creates path issues.

Annovar

Due to licensing issues, we can't include Annovar in the source code. Go to Annovar and fill out the form to receive a link to download the tool. Add the package to the tools folder.

Go to the main annovar directory (/tools/annovar/) and run the command:

perl annotate_variation.pl -downdb -buildver hg19 -webfrom annovar refGene humandb/

This will download the necessary dependency files for this pipeline.

Usage

Running MutPredMerge

To use the pipeline, edit the top three variables in the Snakefile:

MAIN_DIR = "/path/to/MutPredMerge/"
VCFFILE  = "/path/to/example.vcf" # your target VCF file
BASE     = "example"

Make sure the BASE variable is the same as the vcf filename without the ".vcf"

To run the pipleline type

snakemake

Depending on your computational resources, each variant can take 2 minutes or more to score. If you have many variants, we recommend you run the pipeline in the background.

nohup snakemake &

Parallelization

MutPredMerge automatically handles the parallelization process. Just run snakemake and assign a number of cores to the program.

snakemake --cores 10  #or your choice of available cores

Beware, MutPredLOF and MuPredIndel require substantial memory space. Future iterations of this project will add known memory limits needed for each of these programs, but currently, make sure you have at least 15 Gb of RAM available for every 6 threads (cores) you are running. This is the limit that we have found in our stress tests for this module.

Output

The pipeline outputs two files into the data folder: example.annotated.vcf and example.scored.vcf. Example.annotated.vcf is just a copy of the original vcf file but with the scored variants annotated with the outputs of the different MutPred tools. The unscored variants are ignored and no changes are made, but the scored variants will have six new variable values in the INFO section. Example.scored.vcf is a vcf file with just the scored variants with the six new variable values in the INFO section.

The six new variable values in INFO.

INFO=<ID=MPMANN,Number=1,Type=String,Description="Annotation from ANNOVAR in the transcript and protein space">
INFO=<ID=MPMTOOL,Number=1,Type=String,Description="Name of software run from MutPred suite: MP2 for MutPred2 (missense), MPL for MutPred-LOF (loss-of-function) and MPI for MutPred-Indel (non-frameshifting indels)">
INFO=<ID=MPMSCORE,Number=1,Type=Float,Description="General (pathogenicity) prediction score">
INFO=<ID=MPMMECH,Number=.,Type=String,Description="Predicted molecular mechanisms that meet software threshold">
INFO=<ID=MPMPROB,Number=.,Type=Float,Description="Posterior probability for each molecular mechanism">
INFO=<ID=MPMPVAL,Number=.,Type=Float,Description="P-value for each molecular mechanism">

Example Annotated Variant

The pipeline outputs the above variables in the following form.

MPMANN=line1855|NM_152486|c.T1027C;MPMTOOL=MP2;MPMSCORE=0.078;MPMMECH=Gain_of_Intrinsic_disorder,Altered_MoRF,Gain_of_Helix;MPMPROB=0.51,0.30,0.27;MPMPVAL=1.5e-03,0.02,0.05

Interpretation of the output for the annotated variant

MPMANN=line1855|NM_152486|c.T1027C
- Annovar annotated the variant with the RefSeq accession NM_152486, the HGVS notation c.T1027C, and the line number of the variant in the .exonic_variant_function file.
MPMTOOL=MP2
- The variant is a missense variant that was scored by MutPred2 (MP2).
MPMSCORE=0.078
- MutPred2 predicted the variant has a pathogenicity score of 0.078 (very low)
MPMMECH=Gain_of_Intrinsic_disorder, Altered_MoRF, Gain_of_Helix
- MutPred2 predicted that the protein has three molecular mechanisms that are impacted by the variant.
MPMPROB=0.51, 0.30, 0.27
- Each of the predicted mechanisms have cooresponding probabilities for how likely these mechanisms are actually affected.
MPMPVAL=1.5e-03, 0.02, 0.05
- Each of the predicted mechanisms have cooresponding p-values for how likely these mechanisms are actually affected.

These variables are added to the end of the existing INFO strings

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT
chr1    877831  .   T   C   1485.12 PASS    AB=0;ABP=0;AC=4;ADP=45;AF=1;AN=4;AO=48;CIGAR=1X;DP=49;DPB=49;DPRA=0;EPP=11.8771;EPPR=0;GTI=0;HET=0;HOM=1;LEN=1;MEANALT=2;MQM=60;MQMR=0;NC=0;NS=1;NUMALT=1;ODDS=70.0487;PAIRED=1;PAIREDR=0;PAO=0;PQA=0;PQR=0;PRO=0;QA=1694;QR=0;RO=0;RPL=25;RPP=3.19126;RPPR=0;RPR=23;RUN=1;SAF=20;SAP=5.9056;SAR=28;SF=0,1;SRF=0;SRP=0;SRR=0;TYPE=snp;WT=0;MPMANN=line1855|NM_152486|c.T1027C;MPMTOOL=MP2;MPMSCORE=0.078;MPMMECH=Gain_of_Intrinsic_disorder,Altered_MoRF,Gain_of_Helix;MPMPROB=0.51,0.30,0.27;MPMPVAL=1.5e-03,0.02,0.05 GT:ADF:RO:PVAL:GL:GQ:RD:RBQ:SDP:QA:RDR:RDF:ABQ:AO:AD:DP:QR:ADR:FREQ

Resources

Slides

Cite

In Process: Manuscript

Pejaver V, Urresti J, Lugo-Martinez J, Pagel KA, Lin GN, Nam H, Mort M, Cooper DN, Sebat J, Iakoucheva LM, Mooney SD, Radivojac P. MutPred2: inferring the molecular and phenotypic impact of amino acid variants. bioRxiv 134981; doi: https://doi.org/10.1101/134981.

NCBI-Hackathons / MutPredMerge

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