/nf_methylseq

A Nextflow pipeline to align and quantify Methylation (Bisulfite) sequencing data.

Primary LanguageNextflow

Bisulfite Sequencing Pipeline

A Nextflow pipeline to align and quantify Methylation (Bisulfite) sequencing data.

The pipeline was created to run on the ETH Euler cluster and it relies on the server's genome files. Thus, the pipeline needs to be adapted before running it in a different HPC cluster.

Pipeline steps

  1. FastQC
  2. FastQ Screen
  3. Trim Galore
  4. FastQC
  5. Bismark
  6. Bismark filter non-conversion [Optional]
  7. Bismark deduplication
  8. Bismark methylation extractor
  9. coverage2cytosine [Optional]
  10. Bismark2report
  11. Bismark2summary
  12. MultiQC

Required parameters

Path to the folder where the FASTQ files are located.

--input /cluster/work/nme/data/josousa/project/fastq/*fastq.gz

Output directory where the files will be saved.

--outdir /cluster/work/nme/data/josousa/project

Genomes

  • Reference genome used for alignment.

    --genome

    Available genomes:

        GRCm39 # Default
        GRCm38
        GRCh38
        GRCh37 
        panTro6
        CHIMP2.1.4
        BDGP6
        susScr11
        Rnor_6.0
        R64-1-1
        TAIR10
        WBcel235
        E_coli_K_12_DH10B
        E_coli_K_12_MG1655
        Vectors
        Lambda
        PhiX
        Mitochondria
  • Option to use a custom genome for alignment by providing an absolute path to a custom genome file.

    --custom_genome_file '/cluster/work/nme/data/josousa/project/genome/CHM13.genome'

    Example of a genome file:

    name           GRCm39
    species        Mouse
    bismark        /cluster/work/nme/genomes/Mus_musculus/Ensembl/GRCm39/Sequence/BismarkIndex/          

FastQ Screen optional parameters

  • Option to provide a custom FastQ Screen config file.
    --fastq_screen_conf '/cluster/work/nme/software/config/fastq_screen.conf' # Default

Bismark optional parameters

  • Option to set the alignment mode to local.

    --local

    In this mode, it is not required that the entire read aligns from one end to the other. Rather, some characters may be omitted (“soft-clipped”) from the ends in order to achieve the greatest possible alignment score.

  • Option to write all reads that could not be aligned to a file in the output directory.

    --unmapped

  • Option to write all reads which produce more than one valid alignment with the same number of lowest mismatches or other reads that fail to align uniquely to a file in the output directory.

    --ambiguous

Skipping and adding options

  • Option to skip FastQC, TrimGalore, and FastQ Screen. The first step of the pipeline will be the Bismark alignment. --skip_qc

  • Option to skip FastQ Screen. --skip_fastq_screen

  • Option to skip Bismark deduplication. --skip_deduplication

  • Option to add Bismark filter non-conversion before deduplication (if selected) and before Bismark methylation extractor. --add_filter_non_conversion

Extra arguments

Acknowledgements

This pipeline was adapted from the Nextflow pipelines created by the Babraham Institute Bioinformatics Group and from the nf-core pipelines. We thank all the contributors for both projects. We also thank the Nextflow community and the nf-core community for all the help and support.