tutorial.md

Tutorial on an example dataset

In this tutorial, we provide step-by-step instructions to run the benchmarking pipeline. The necessary pod5 and references are provided in the example folder:

• Pod5 files - example/pod5
  
• Reference - example/references/H.pylori_J99_ATCC700824.fa

You can also run the pipeline using your own dataset by replacing the example dataset folder with your dataset folder and downloading the appropriate genome file from UCSC.

Creating conda environment

Setting up the environment

1. sudo apt update && sudo apt install samtools openjdk-17-jdk git wget -y (if on a debian machine)

2. Install docker

   • setup docker to run without sudo

     sudo groupadd docker
     sudo usermod -aG docker $USER
     newgrp docker

   • test if everything is working:

     docker run hello-world
     

3. Install apptainer: https://apptainer.org/

   on a debian machine:

    sudo add-apt-repository -y ppa:apptainer/ppa
    sudo apt update
    sudo apt install -y apptainer-suid
   

4. Install conda:

   wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O miniconda.sh
   bash miniconda.sh -b -u
   ~/miniconda3/bin/conda init bash 
   source ~/.bashrc 
   conda tos accept
   

5. Install nextflow

   curl -s https://get.nextflow.io | bash
   echo 'alias nextflow='$(realpath nextflow) >> ~/.bashrc
   source ~/.bashrc
   

6. Pull github repo:

   git clone https://github.com/SowpatiLab/ONT_Methylation_Benchmarking.git 
   cd ONT_Methylation_Benchmarking
   

7. Index reference files All the reference files are expected to be indexed first.

       samtools faidx example/references/H.pylori_J99_ATCC700824.fa
       cut -f 1,2 \
           example/references/H.pylori_J99_ATCC700824.fa.fai \
           >  example/references/H.pylori_J99_ATCC700824.genome
   

   Note: This is for the example reference file in the example folder. All other references need to be indexed in the same fashion.

8. Running the nextflow workflow:

   # change directory to example
   cd example
   
   # run nextflow with conda 
   nextflow run ../benchmark_nextflow \
       -params-file config.yaml \
       -profile conda --conda_prefix $CONDA_PREFIX \
       --threads 10 \
       --memory  32.GB
   
   # running with docker profile
   # only run once 
   
   docker pull sowpati/ont-methylation-benchmarking:latest
   
   [ ! -d tooling/rockfish_models ] && mkdir -p tooling/rockfish_models
   docker run -v $(pwd):$(pwd) -w $(pwd) \
       sowpati/ont-methylation-benchmarking:latest rockfish download \
           -m 5kHz -s tooling/rockfish_models
   
   # run nextflow with docker
   nextflow run ../benchmark_nextflow \
       -params-file config.yaml \
       -profile docker \
       --threads 10 \
       --memory  32.GB
   
   # run nextflow with apptainer
   nextflow run ../benchmark_nextflow \
       -params-file config.yaml \
       -profile apptainer \
       --threads 10 \
       --memory  32.GB
   

9. Installing Snakemake

   # option 1:
   conda create \
       -c conda-forge \
       -c bioconda -c nodefaults \
       -n benchmark_env snakemake
   
   # option 2:
   conda env create \
       -n benchmark_env \
       -f benchmark_nextflow/envs/benchmarking_env.yml
   
   # activate conda env
   conda activate benchmark_env
   

10. Running the snakemake workflow:

    ## ensure snakemake is installed 
    snakemake --use-apptainer \
        --apptainer-args "--nv " \
        -s ../benchmark_snakemake/snakefile \
        --cores 10
    

Final folder structure

.
├── benchmark_nextflow         # nextflow  workflow lives here
├── benchmark_snakemake        # snakemake workflow lives here
├── output
|   │   ├── bam                # bam files go here
|   │   ├── sorted_mod               # mod bams
|   │   ├── sorted_mod_cleansed      # filtered mod bams
|   │   ├── sorted_move              # move table bams
|   │   ├── sorted_move_cleansed     # filtered move table bams
|   │   └── sorted_move_fnord        # move table bams sorted by pod5 id
│   ├── intermediary
|   │   ├── blow5
|   │   └── fastq
│   ├── meta                  # final output beds for each tool
|   │   ├── deepbam
|   │   ├── deepmod2
|   │   ├── deepplant
|   │   ├── dorado
|   │   ├── f5c
|   │   ├── f5c_stranded
|   │   └── rockfish
│   ├── qc
|   │   ├── mosdepth
|   │   ├── nanoplot
|   │   ├── nanoq
|   │   └── nanostat
│   └── tool_out
|       ├── deepbam
|       ├── deepmod2
|       ├── deepplant
|       ├── dorado
|       ├── f5c
|       └── rockfish
├── pod5                       # input raw signal files
├── references                 # references files go here
├── tooling                    # tool install dir
│   ├── DeepBAM_models
│   ├── deepmod2
│   ├── DeepPlant_models
│   ├── dorado
│   ├── dorado_models
│   ├── f5c
│   ├── modkit
│   ├── rockfish
│   └── rockfishmodels
├── config.yaml                # controls for the workflow
├── references.yaml            # reference file map
└── work                       # working folder generated by nextflow

Note: For more detailed usage of the workflow check the nextflow.md and snakemake.md files