commands.txt

##### Calling bases

mkdir lambda

cd lambda

wget https://www.dropbox.com/s/eml7z2d82n3k8lq/lambda.tar.gz?dl=0 -O lambda.tar.gz

tar -xf lambda.tar.gz

rm lambda.tar.gz

conda install -c bioconda nanoplot 

NanoPlot --fastq pass.fastq

##### Filter reads

conda install -c bioconda filtlong

filtlong --min_mean_q 90 --min_length 3000 pass.fastq > ~/filtered/filtered.fq

##### Align reads to reference

source ~/git/pomoxis/venv/bin/activate

mini_align -i filtered.fq -r ~/refs/lambda_ref.fasta -P -p lambda -t 14

##### De novo assembly

mini_assemble -i ~/filtered/filtered.fq -o denovo -p lambda_denovo -t 14 -c

##### Error correction

conda create -n nanopolish -c bioconda -c conda-forge nanopolish 

mkdir ~/polished

cd ~/polished

mini_align -i ~/filtered/filtered.fq -r ~/denovo/lambda_denovo_final.fa -P -p polished -t 14

deactivate

##### Call variants

nanopolish index -d ~/lambda/ ~/filtered/filtered.fq

nanopolish variants --consensus -o ~/polished/polished.{1}.vcf -r ~/filtered/filtered.fq -b ~/polished/polished.bam -g ~/denovo/lambda_denovo_final.fa -q dcm,dam --min-candidate-frequency 0.1 -t 16

nanopolish vcf2fasta -g ~/denovo/lambda_denovo_final.fa polished.*.vcf > polished.fa

##### Get methylation data and map data

mkdir ~/mods

cd ~/mods

wget https://www.dropbox.com/s/1n31to1ac108apa/methylation_example.tar.gz?dl=0 -O methylation_example.tar.gz 

tar -xf methylation_example.tar.gz

cd methylation_example

source ~/git/pomoxis/venv/bin/activate

mini_align -i ~/mods/methylation_example/albacore_output.fastq -r ~/mods/methylation_example/reference.fasta -P -p methyl -t 16

deactivate

source activate nanopolish

nanopolish index -d fast5_files/ albacore_output.fastq

###### Call Methylation and calculate frequency

nanopolish call-methylation -t 15 -r albacore_output.fastq -b methyl.bam -g reference.fasta -w "chr20:5,000,000-10,000,000" > methylation_calls.tsv

git clone --recursive https://github.com/jts/nanopolish.git ~/git/nanopolish

~/git/nanopolish/scripts/calculate_methylation_frequency.py -i methylation_calls.tsv > methylation_frequency.tsv