SIMONE (Structural varIants and Methylation using lONg rEads)

Snakemake pipeline for investigating structural variation and methylation using long-reads and tumor-control samples

Description

SIMONE uses minimap2 to align long reads to the human reference genome. SV identification is performed using sniffles and cuteSV. Methylation frequencies are calculated using nanopolish. Regions with differential methylation (tumor vs control) are identified by means of pycoMeth.

Get Data

To get the Oxford Nanopore PromethION ultra long reads:

pip install gdown 

then run:

cd SIMONE
mkdir -p data
cd data
# get GRCh38 reference (hg38 with decoys)
wget http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/GRCh38_reference_genome/GRCh38_full_analysis_set_plus_decoy_hla.fa
#index the reference genome
samtools faidx GRCh38_full_analysis_set_plus_decoy_hla.fa
#get chromosomes that we need to exclude later
cut -f1 GRCh38_full_analysis_set_plus_decoy_hla.fa.fai | tail -n +25 > GRCh38_full_analysis_set_plus_decoy_hla.exclude.txt
#get chromosomes we need
cut -f1 GRCh38_full_analysis_set_plus_decoy_hla.fa.fai | head -24 > classic.chrs.txt
# get GRCh38 gff
wget http://ftp.ensembl.org/pub/release-105/gff3/homo_sapiens/Homo_sapiens.GRCh38.105.gff3.gz
bgzip -d Homo_sapiens.GRCh38.105.gff3.gz
sed -i -e '/#/! s/^/chr/' Homo_sapiens.GRCh38.105.gff3
#get control fastq
gdown https://drive.google.com/uc?id=1fbdD4TZMSrx8LbeF0bsfwYr4WqDc8yX0
#get tumor fastq
gdown https://drive.google.com/uc?id=1tQfbXyTyxyc-XEuaKHaFKGry1RVQMcZa
#get control,tumor fast5
gdown https://drive.google.com/drive/folders/1kHrnQrGYfPFWFMpsOAOANdBUpVxeULDE --folder
cd -

The control and the tumor files are 1G.pass.fastq.gz (control) and 1S.pass.fastq.gz (tumor) with correponding fast5 files.

RUN

To use nanopolish with conda env:

export HDF5_PLUGIN_PATH=/usr/local/hdf5/lib/plugin

then run:

Align FASTQ to GRCh38

snakemake align --cores 20 --use-conda 

Calculate/plot coverage statistics

snakemake depth --cores 20 --use-conda

Call SVs with sniffles, cuteSV and make a consensus SVs callset selecting only tumor SVs

snakemake svcall --cores 20 --use-conda

Methylation analysis with nanopolish and pycoMeth analysis for comparing methylation status between normal and tumor

snakemake meth --cores 20 --use-conda