This repository contains xr-seq and damage-seq workflows.

• This workflow is prepared using Snakemake workflow management system and conda

• To run the workflow, you should have conda installed for environment management. All the other packages including Snakemake and their dependencies can be obtained automatically through environments prepared for each step of the workflow. You can follow the installation steps from the link.

• Initially, you should clone the repository and navigate into the directory:

  git clone https://github.com/CompGenomeLab/xr-ds-seq-snakemake.git
      
  cd xr-ds-seq-snakemake
  

• Next, you should create a conda environment with the defined packages. Install mamba and create the environment using mamba:

  conda install -c conda-forge mamba
  
  mamba create -c bioconda -c conda-forge -c r -n repair snakemake=6.3.0 python=3.8 rust=1.50 sra-tools=2.11.0
  
  conda activate repair
  

• You should install boquila tool to simulate repair and damage reads.

This workflow is prepared according to the structure recommended by Snakemake:

• config/: contains the configuration files.

• logs/: contains the log files of each step. This folder will automatically appear when you run the workflow.

• report/: contains the description files of figures, which will be used in reports.

• resources/: contains samples/ where the raw XR-seq and Damage-seq data are stored, input/ where the input files are stored, and ref_genomes/ where the reference genome files are stored. Reference genome files can be automatically produced by the workflows, if they are properly defined in the config files.

• results/: contains the generated files and figures. This folder will automatically appear when you run the workflow.

• workflow/: contains envs/ where the environments are stored, rules/ where the Snakemake rules are stored, and scripts/ where the scripts used inside the rules are stored.

Before running the workflow, you should edit the configuration files. For both XR-seq and Damage-seq workflows, there are 2 configuration files: config_(XR/DS)_initial.yaml and config_(XR/DS).yaml. The configuration file with "initial" prefix shouldn't be modified by the user since they are containing configuration settings that are common for all XR-seq and Damage-seq experiments. For more detail about these configuration files, check out the readme file in config/ directory. The parameters for "config_(XR/DS).yaml" as below:

• sample: The name of the sample file w/o the extension. Multiple sample names can be given in the below format:

  sample: 
      - "SAMPLE_1"
      - "SAMPLE_2"
      - "SAMPLE_3"
  

  • Using the given sample name, the workflow will look for {SAMPLE}.fastq.gz as raw data. Therefore, the fastq file must be gzipped before running the workflow.

  • If the layout of the given sample is paired-end, the workflow will look for {SAMPLE}_R1.fastq.gz, {SAMPLE}_R2.fastq.gz and {SAMPLE}_1.fastq.gz, {SAMPLE}_2.fastq.gz as raw data. Therefore, paired-end sample files must contain _R1/2 or _1/2 suffixes and the suffixes should not be given in the configuration file to the sample.

• damage_type: Damage type of each sample should be provided here in the same order of the samples:

  damage_type: 
      - "64"
      - "CPD"
      - "oxaliplatin"
  

  • Currently damages below are available can be provided as (case-insensitive):

    • (6-4)PP: 64, 64pp, (6-4)pp, 6-4pp;
    • CPD: CPD;
    • Cisplatin: cisplatin;
    • Oxaliplatin: oxaliplatin.

• srr: Contains 2 parameters: enabled, codes. enabled is a boolean variable (can only be True or False) and if it is True, then the pipeline will retrieve the raw data by sra-toolkit the defining the SRR code in the codes parameter.

  srr: 
  enabled: True
  codes:
      - "SRRXXXXXXX"
  

  • Each downloaded raw data with the SRR codes will be named as the corresponding sample name in the sample parameter.

  • If a sample have multiple SRR codes, then the code should be provided in the given format: SRRXXXXXXX:SRRXXXXXXX:SRRXXXXXXX

• input: Contains 4 parameters: exist, files, sample, srr. This parameter is used for boquila, which can take input files for more accurate read simulations. If you don't have any input file, you can set exist as False. files parameter contains the names of input files w/o the extension. sample parameter contains the indeces of samples that will use the input of the same order. Lastly, srr can be used for downloading inputs from sra-toolkit. It works the same was as the srr parameter above. As a whole inputs can be provided as:

  input:
      exist: True
      files:
          - "input0"
          - "input1"
          - "input2"  
      sample: 
          - "0-3" # input0 will be used for sample 0,1,2,3
          - "4, 7" # input1 will be used for sample 4,7
          - "5, 6" # input2 will be used for sample 5,6
      srr: 
          enabled: True
          codes:
          - "SRRXXXXXXX" # srr of input0
          - "SRRXXXXXXX" # srr of input1
          - "SRRXXXXXXX" # srr of input2
  

• Genome parameters: The parameters build, species, datatype, and release are defined to retrieve correct reference genome from ensembl. For more information, you can check out the link.

  • If genome_download is set to False, then a fasta file expected to be provided as resources/ref_genomes/${build}/genome_${build}.fa, where ${build} should be the string given in build parameter.

  • In the same manner, If bowtie2_build is set to False, then a build should be provided in resources/ref_genomes/${build}/Bowtie2/, where ${build} should be the string given in build parameter. File name before the extensions must be genome_${build}. Lastly to properly set bowtie2_build to False, genome_download must be set to False as well.

• filter: This parameter is used to filter chromosomes by the given regex.

After adjusting the configuration file, you can run the workflow from xr-ds-seq-snakemake directory.

• For XR-seq:

  snakemake -pr --use-conda --cores 64 --snakefile Snakefile_XR.smk --keep-going --rerun-incomplete 
  

• For Damage-seq:

  snakemake -pr --use-conda --cores 64 --snakefile Snakefile_DS.smk --keep-going --rerun-incomplete 
  

snakemake -pr --use-conda --profile config/slurm --snakefile Snakefile_DS.smk --keep-going --rerun-incomplete 

To generate detailed HTML report files, the code below should be run after workflow:

snakemake --report report.zip