In this tutorial, we will perform methylation calling using dorado on the ONT methylation benchmark sample. We will use the Escherichia coli str. K-12 substr. MG1655 sample for this analysis. E.coli exhibits 5mC methylation in CCWGG context. We will detect these 5mC methylation marks using E.coli sample from the benchmarking dataset.

• Installation
• Data Access and preparation
• Running methylation calling

First, we have to install dorado basecaller which we will use for methylation basecalling using dorado models. We can install dorado using following command on linux terminal :

wget https://cdn.oxfordnanoportal.com/software/analysis/dorado-1.1.1-linux-x64.tar.gz
tar -xvzf dorado-1.1.1-linux-x64.tar.gz

# download dorado models

# basecalling model : 
dorado-1.1.1-linux-x64/bin/dorado download --model dna_r10.4.1_e8.2_400bps_sup@v5.0.0

# modification model
dorado-1.1.1-linux-x64/bin/dorado download --model dna_r10.4.1_e8.2_400bps_sup@v5.0.0_5mC_5hmC@v3

If you want a more recent version of dorado/dorado models, you may refer to the Dorado Github repository.

Samtools can be downloaded and installed from here: https://www.htslib.org/download/

Modkit can be downloaded and installed from here: https://github.com/nanoporetech/modkit

We will need to download following required data:

• Raw signal data (POD5 files)
• Reference genome file (FASTA file)

Below are the steps to download the required data : The following command requires you to have AWS CLI installed.

1. Create a directory to store the data

   mkdir ONT_meth_tutorial
   

2. Download sample POD5 file

   aws s3 sync --no-sign-request s3://ont-basemod-benchmark-data/Raw/pod5/bacteria/Ecoli_WT_5kHz/ ./ONT_meth_tutorial/pod5/Ecoli_WT_5kHz/
   

3. Download genome fasta file

   aws s3 cp --no-sign-request s3://ont-basemod-benchmark-data/Analysis/Reference/ecoli.fa.gz ./ONT_meth_tutorial
   

dorado-1.1.1-linux-x64/bin/dorado \
    basecaller dna_r10.4.1_e8.2_400bps_sup@v5.0.0 \
    ./ONT_meth_tutorial/pod5/Ecoli_WT_5kHz \
    --modified-bases-models dna_r10.4.1_e8.2_400bps_sup@v5.0.0_5mC_5hmC@v3 --min-qscore 10 \
    --reference ONT_meth_tutorial/ecoli.fa.gz -r > Ecoli_WT_hac_5mC_5hmC_v5r3.bam

samtools sort \
    Ecoli_WT_hac_5mC_5hmC_v5r3.bam \
    -o Ecoli_WT_hac_5mC_5hmC_v5r3_sorted.bam  \
    --write-index

modkit pileup \
    --ref ONT_meth_tutorial/ecoli.fa.gz \
    --ignore h \
    Ecoli_WT_hac_5mC_5hmC_v5r3_sorted.bam \
    Ecoli_WT_hac_5mC_5hmC_v5r3_modkit.bed

Modkit pileup command returns a bedMethyl file containing genome coordinates for the cytosine locations (columns 1-3) and corresponding methylation frequencies (column 11). The first few lines of the file are shown below.

Please refer to the modkit documentation for more information regarding the output file.