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RRBS

This workflow is designed for the analysis of Reduced Representation Bisulfite Sequencing (RRBS) data. It performs adapter trimming, alignment, methylation quantification, and quality control.

Workflow Inputs​

  • Workflow Name (Optional): A name for your workflow to help identify it in the list of processed workflows.
  • FASTQ Folder: The folder containing your FASTQ files, including forward (R1), reverse (R2), and UMI (I1) reads. Files should follow the naming convention <Sample Name>_R1.fastq.gz, <Sample Name>_R2.fastq.gz, and <Sample Name>_I1.fastq.gz.
  • Sample Genome Index (TAR): Path to the reference genome index for the sample. The default is a pre-built rat genome index, but you can override it with your own.
  • Spike-in Genome Index (TAR): Path to the reference genome index for the spike-in control (e.g., lambda phage). The default is a pre-built lambda genome index, but you can override it with your own.
  • PhiX Genome Index (TAR): Path to the Bowtie2 reference index for PhiX. The default is a pre-built PhiX index, but you can override it with your own.
  • Output Report Name: A name or prefix to use for the generated reports and pipeline outputs.

Workflow Steps​

The RRBS pipeline consists of several steps, each with additional configurable options:

1. Pre-Trim FastQC​

This step assesses the initial quality of raw reads before adapter trimming.

  • Additional Configurable Options:
  • pretrim_fastqc_ncpu: Number of CPU cores.
  • pretrim_fastqc_ramGB: Memory allocation in GB.
  • pretrim_fastqc_disk: Disk space allocation in GB.

2. Attach UMI​

Appends UMI information from the I1 file to the read names in R1 and R2 files for downstream analysis.

  • Additional Configurable Options:
  • attach_umi_ncpu: Number of CPU cores.
  • attach_umi_ramGB: Memory allocation in GB.
  • attach_umi_disk: Disk space allocation in GB.

3. Trim Galore (Regular Adapters)​

Removes standard adapter sequences from the reads.

  • Additional Configurable Options:
  • trim_reg_adapt_ncpu: Number of CPU cores.
  • trim_reg_adapt_ramGB: Memory allocation in GB.
  • trim_reg_adapt_disk: Disk space allocation in GB.

4. Trim Diversity Adapters​

Removes NuGen-specific diversity adapters.

  • Additional Configurable Options:
  • trim_diversity_adapt_ncpu: Number of CPU cores.
  • trim_diversity_adapt_ramGB: Memory allocation in GB.
  • trim_diversity_adapt_disk: Disk space allocation in GB.

5. Post-Trim FastQC​

Evaluates the quality of reads after adapter trimming.

  • Additional Configurable Options:
  • posttrim_fastqc_ncpu: Number of CPU cores.
  • posttrim_fastqc_ramGB: Memory allocation in GB.
  • posttrim_fastqc_disk: Disk space allocation in GB.

6. MultiQC​

Aggregates and summarizes results from FastQC and Trim Galore steps.

  • Additional Configurable Options:
  • multiqc_ncpu: Number of CPU cores.
  • multiqc_ramGB: Memory allocation in GB.
  • multiqc_disk: Disk space allocation in GB.

7. Align Trimmed Reads (Sample & Spike-in)​

Aligns trimmed reads to both the sample and spike-in reference genomes using Bismark.

  • Additional Configurable Options (for both Sample and Spike-in):
  • align_trim_sample/spike_in_ncpu: Number of CPU cores.
  • align_trim_sample/spike_in_ramGB: Memory allocation in GB.
  • align_trim_sample/spike_in_disk: Disk space allocation in GB.

8. Mark UMI Duplicates (Sample & Spike-in)​

Identifies and tags UMI duplicates in the aligned BAM files.

  • Additional Configurable Options (for both Sample and Spike-in):
  • tag_udup_sample/spike_in_ncpu: Number of CPU cores.
  • tag_udup_sample/spike_in_ramGB: Memory allocation in GB.
  • tag_udup_sample/spike_in_disk: Disk space allocation in GB.

9. Mark PCR Duplicates (Sample & Spike-in)​

Identifies and marks PCR duplicates using Picard's MarkDuplicates.

  • Additional Configurable Options (for both Sample and Spike-in):
  • mark_dup_sample/spike_in_ncpu: Number of CPU cores.
  • mark_dup_sample/spike_in_ramGB: Memory allocation in GB.
  • mark_dup_sample/spike_in_disk: Disk space allocation in GB.

10. Quantify Methylation (Sample & Spike-in)​

Quantifies methylation levels using Bismark's methylation extractor.

  • Additional Configurable Options (for both Sample and Spike-in):
  • quant_methyl_sample/spike_in_ncpu: Number of CPU cores.
  • quant_methyl_sample/spike_in_ramGB: Memory allocation in GB.
  • quant_methyl_sample/spike_in_disk: Disk space allocation in GB.

11. Bowtie2 PhiX Alignment​

Aligns reads to the PhiX genome using Bowtie2 to assess the level of PhiX contamination.

  • Additional Configurable Options:
  • bowtie2_phix_ncpu: Number of CPU cores.
  • bowtie2_phix_ramGB: Memory allocation in GB.
  • bowtie2_phix_disk: Disk space allocation in GB.

12. SAMTools Mapped​

Calculates the percentage of reads mapped to different chromosomes and contigs.

  • Additional Configurable Options:
  • chrinfo_ncpu: Number of CPU cores.
  • chrinfo_ramGB: Memory allocation in GB.
  • chrinfo_disk: Disk space allocation in GB.

13. Collect QC Metrics​

Gathers and summarizes quality control metrics from various steps in the pipeline.

  • Additional Configurable Options:
  • collect_qc_ncpu: Number of CPU cores.
  • collect_qc_ramGB: Memory allocation in GB.
  • collect_qc_disk: Disk space allocation in GB.

14. Merge Results​

Combines reports and QC metrics files from all samples.

  • Additional Configurable Options:
  • merge_results_ncpu: Number of CPU cores.
  • merge_results_ramGB: Memory allocation in GB.
  • merge_results_disk: Disk space allocation in GB.

Workflow Outputs​

The RRBS pipeline produces several outputs, including:

  • QC Report: A comprehensive report containing quality control metrics from all steps of the pipeline.
  • Alignment BAM files: Aligned reads in BAM format for both sample and spike-in.
  • Methylation quantification files: Files containing methylation levels for each cytosine in the genome.
  • Bismark reports: Reports generated by Bismark, including alignment and methylation extraction summaries.
  • MultiQC report: Aggregated report summarizing results from FastQC and Trim Galore.

Additional Notes​

  • The default settings for the workflow are suitable for most RRBS datasets, but you may need to adjust them based on the specific characteristics of your data and computational resources.
  • You can find more detailed information about each step of the pipeline and the tools used in the corresponding tool documentation.