This repository contains all the software and scripts used for the following study.

Garcia-Nieto PE, Morrison AJ, Fraser HB. The somatic mutation landscape of the human body. BioRxiv. 2019. link

Garcia-Nieto PE, Morrison AJ, Fraser HB. The somatic mutation landscape of the human body. Genome Biology. 2019. link

The contents of this repository encompass:

• A method to call DNA somatic mutations from the GTEx v7 RNA-seq data:
  • Mapping raw reads to the human genome Hg19.
  • Calling mutations.
  • Identifying and removing false-positive mutations
• All analyses included in the study:
  • Method validation using exome sequencing data
  • General characterization of the landscape of mutations.
  • Phenotypic associations.
  • Molecular associations -- chromatin and gene expression.
  • Strand asymmetry analysis.
  • Selection dynamics.
  • Assessment of cancer-like characteristics of somatic mutations

This document is not intended to be a thorough description of the methods or results. It is a guide that serves as a reproducibility reference for the code used in the aforementioned study.

For a complete description of the methods and results please refer to the publication.

Requirements

• Unix -- all software was run on bash in Ubuntu 14.04
• snakemake -- for all pipelines
• STAR 2.5.2a -- sequence aligner
• kent from ucscGenomeBrowser
• BCFtools 1.6
• bedtools 2.28
• samtools 1.6
• R 3.4:
  • MASS
  • MatrixEQTL
  • Only output alignments in library
  • Rsamtools
  • Rtsne
  • biomaRt
  • cluster
  • dndscv
  • ggplot2
  • ggrepel
  • gplots
  • gtools
  • metap
  • proto
  • rSubmitter
  • reshape
  • rjson
  • seqinr
  • topGO
  • Tidyverse
  • Bioconductor:
    • GenomicRanges
    • Biostrings
    • GenomicFeatures
    • GenomicRanges
    • Homo.sapiens
    • SNPlocs.Hsapiens.dbSNP144.GRCh37
    • org.Hs.eg.db
• python 3.6
  • pysam
  • pandas
  • numpy
  • scipy

Overview

All raw RNA reads were downloaded from dbGaP using GTEx v7 data.

The execution outline is as follows:

1. Setting up configuration files and downloading all required auxiliary files.
2. Creating a genome index for the STAR sequence aligner.
3. Mapping to the human genome.
4. Creation of read pileup files for positions having two different sequence calls.
5. Per-position addition of potential artifacts for the alternate allele.
6. Mutation calling and removal of likely false-positives.
7. Elimination of potential systematic artifacts (Panel of Normals).
8. Elimination of hyper-mutated samples.
9. High-level analyses.

An in-depth description on how to execute each of these steps is described below.

Setting up

Downloading auxiliary files

Follow this link

Download file, Uncompress it, and move it to a static location. These files are a variety of genome files, gene annotations, data from external databases (e.g. COSMIC, Roadmap epigenomics, TCGA), RNA edit info, etc.

Configuring

Modify the file config.json, all buckets should be self-explanatory. The file is setup as it was used for the original study, which was run on the Sherlock cluster at Stanford University.

It's worth noting the following:

• genomeDir is the path for the genome STAR index (see mapping for details in how to make it)
• Any bucket containing */auxiliaryFiles/[...] should be replaced with the location of the downloaded and uncompressed file above: new_path/auxiliaryFiles/[...]
• vcfFile and vcfFileCommon are paths to the genotype vcf files from the GTEx project, please access those through dbGaP, feel free to contact me for any guidance on this.
• projectDir and scratchDir should be identical, this the root path where all results will be stored.
• Most other buckets can be left unmodified

Cluster and parallelization set up

Most pipelines are designed to run jobs in parallel. There are some extra files and scripts to run snakemake on a SLURM cluster:

• The file cluster.json contains rule-specific specifications and can be used on snakemake.
• submit.py is a custom submission script to be used for job submission on snakemake.
• jobState is a bash script to be used by snakemake to check for sate of jobs, this script should be added to the bash $PATH

Creating genome index for STAR

This is the only step not included as a download or a step in any pipeline due to large memory and disk usage.

Inputs

• Human genome Hg19 with common SNPs masked as "N". Included in the auxiliaryFiles, the path to the file should be the genomeFasta bucket in config.json
• Genome annotation in GTF format. Included in the auxiliaryFiles, the path to the file should be the auxiliaryFiles:GTF_gtex bucket in config.json

Outputs

• Genome index for STAR

Execution

Replace variables ($) with the associated value in the config.json file

STAR --runThreadN 16 --runMode genomeGenerate --genomeDir ${genomeDir} --genomeFastaFile ${genomeFasta} --sjdbGTFfile ${auxiliaryFiles:GTF_gtex} --sjdbOverhang 99

Mapping raw RNA-seq reads and creating pileup files

The following pipeline includes:

• Mapping to the human genome, sorting and indexing bam files, removing duplicate reads.
• Creating coverage maps.
• Creating read pileup files for positions covered by two different sequence calls.
• Adding per-site bias information to the pileup files: read position bias, mapping quality bias, sequence quality bias, strand bias, variant distance bias.

A full description of these biases can be found in the Methods section of the publication.

Inputs

• Raw rna-seq paired fastq files. These should be downloaded from dbGaP GTEx v7, please contact me for support on this. The path to these files should be the fastqDir bucket in config.json. File names should have the format {sample}_1.fastq.gz {sample}_2.fastq.gz ``nformation. Included in the auxiliaryFiles download, the path to the file should be the `sraTable` bucket in `config.json`
• Genome size table. Included in the auxiliaryFiles download, the path to the file should be the genomeSizeFile bucket in config.json
• A variety supporting files included in auxiliaryFiles

Outputs

• Sorted, bam files without duplicate reads. Files will be located in $mappingDir/{tissue}/{sample}_RmdupSortedAligned.out.bam, where $mappingDir is the mappingDir bucket in config.json
• Coverage maps $root/depth_bam/{sample}.bed.gzip. Where $root is the projectDir bucket in config.json
• Pileup files for positions with two sequence calls $pileupDir/{tissue}/{sample}.txt. Where $pileupDir is relative and is the pileupDir bucket in config.json

Execution

Linear execution (this is not feasible as it would take a very large time to finish):

cd mappingMutationCalling_pipeline
snakemake --snakefile SnakefilePileup.smk --printshellcmds --keep-going --restart-times 2 

Parallel execution in a SLURM cluster:

cd mappingMutationCalling_pipeline
snakemake --snakefile SnakefilePileup.smk --restart-times 2 --keep-going --max-jobs-per-second 3 --max-status-checks-per-second 0.016 --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --keep-going --cluster "../submit.py"

Mutation calling

The following pipeline includes:

• Calling mutations based on coverage and number of reads supporting alt allele, while incorporating sequencing error probabilities.
• Labelling and removing false positives:
  • Black-listed regions.
  • RNA edits.
  • Splicing junction errors.
  • Sequencing errors.
  • All biases described in the previous section.
• Remove mutations from systematic artifacts based on a pseudo Panel of Normals (PoN).
• Remove hypermutated samples.

A full description of the false-positive removal process can be found in the Methods section of the publication.

Inputs

``o sequence calls $pileupDir/{tissue}/{sample}.txt. From the previous section.

• A variety of supporting files included in auxiliaryFiles

Outputs

• Mutation maps per sample ${mutationCountDir:map}/{tissue}/n6_0.0_0.7/{sample}.txt.
• Counts of mutations following a 6-type profile (C>T, C>G, C>T, C>A, T>G, T>A, T>C) $mutationCountDir:$context/{tissue}/n6_0.0_0.7/{sample}.txt
• Counts of mutations with pentanucleotide context per sample $mutationCountDir:$context/{tissue}/n6_0.0_0.7/{sample}.txt

Execution

Linear execution:

cd mutationCalling
snakemake --snakefile --printshellcmds --keep-going --restart-times 2 

Parallel execution in a SLURM cluster:

cd mutationCalling
snakemake --restart-times 2 --keep-going --max-jobs-per-second 3 --max-status-checks-per-second 0.016 --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --keep-going --cluster "../submit.py"

General analyses

``lysis and plotting scripts for:

• Correction of mutations based on sequencing depth.
• Artifact analysis.
• Mutation statistics -- variant allele frequency, mutation density across subjects and samples, etc.
• Cross-tissue comparison of mutation maps.
• Phenotypic associations (age, ethnicity, sex).
• Mutation profile analysis (tSNE).
• Gene expression association with mutation load.
• Association between gene expression and mutation load.

These analyses span Figures 1,2,4 in the publication.

Inputs

• Mutation maps and counts from above
• A variety of supporting files included in auxiliaryFiles

Outputs

• A variety of plots and text files. These files will be located within the paths described in the generalMutationAnalyses bucket from config.json

Execution

Linear execution:

cd generalMutationAnalyses
snakemake --keep-going --restart-times 2 --nolock 

Parallel execution in a SLURM cluster:

cd generalMutationAnalyses
snakemake --restart-times 2 --keep-going --max-jobs-per-second 3 --max-status-checks-per-second 0.016 --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --keep-going --cluster "../submit.py"

Chromatin analyses

The following pipeline includes data analysis and plotting scripts for:

• Downloading signal chromatin maps for selected tissues and chromatin marks from the Roadmap epigenomics project.
• Processing of chromatin signal.
• Association analysis between chromatin and mutation load.

These analyses are included in Figure 2 of the publication.

Inputs

• Mutation maps and counts from above
• A variety of supporting files included in auxiliaryFiles

Outputs

• A variety of plots and text files. These files will be located within $projectDir/chromatin/, where $projectDir is the projectDir bucket in `config.json

Execution

Linear execution:

cd chromatin
snakemake --keep-going --restart-times 2 --nolock 

Parallel execution in a SLURM cluster:

cd chromatin
snakemake --restart-times 2 --keep-going --max-jobs-per-second 3 --max-status-checks-per-second 0.016 --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --keep-going --cluster "../submit.py"

Strand bias and selection analyses

The following pipeline includes data analysis and plotting scripts for:

• Processing and analysis of mutations in the transcribed and non-transcribed strands.
• Selection analyses using dN/dS.
• Selection analyses using VAF comparison.

These analyses are included in Figures 1,3 of the publication.

Inputs

• Mutation maps and counts from above
• A variety of supporting files included in auxiliaryFiles

Outputs

• A variety of plots and text files. These files will be located within the paths in the selectionDir bucket from config.json

Execution

Linear execution:

cd selectionAnalyses
snakemake --keep-going --restart-times 2 --nolock 

Parallel execution in a SLURM cluster:

cd selectionAnalyses
snakemake --restart-times 2 --nolock --printshellcmds --keep-going --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --cluster "../submit.py"

Cancer analyses

The following pipeline includes data analysis and plotting scripts for:

• Enrichment of COSMIC mutations in mutation maps
• Mutations on cancer driver genes
• Selection analyses on cancer mutations and driver genes using dN/dS and VAF comparisons

These analyses are included in Figures 5 of the publication.

Inputs

• Mutation maps and counts from above
• A variety of supporting files included in auxiliaryFiles

Outputs

• A variety of plots and text files. These files will be located within the paths in the cancer bucket from config.json

Execution

Linear execution:

cd cancer
snakemake --keep-going --restart-times 2 --nolock 

Parallel execution in a SLURM cluster:

cd cancer
snakemake --restart-times 2 --nolock --printshellcmds --keep-going --cluster-config ../cluster.json --cluster-status jobState --jobs 500 --cluster "../submit.py"