nf-core/methylseq

You will need to create a design file with information about the samples in your experiment before running the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 3 columns, and a header row. See usage docs.

Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits. If set in your user config file (~/.nextflow/config) then you don't need to specify this on the command line for every run.

Use the --unmapped flag to set the --unmapped flag with Bismark align and save the unmapped reads to FastQ files.

By default, trimmed FastQ files will not be saved to the results directory. Specify this flag (or set to true in your config file) to copy these files to the results directory when complete.

If using a reference genome configured in the pipeline using iGenomes, use this parameter to give the ID for the reference. This is then used to build the full paths for all required reference genome files e.g. --genome GRCh38.

See the nf-core website docs for more details.

If you have no genome reference available, the pipeline can build one using a FASTA file. This requires additional time and resources, so it's better to use a pre-build index if possible. You can use the command line option --save_reference to keep the generated references so that they can be added to your config and used again in the future. If aligner is Bismark and bismark_index is specified, this parameter is ignored.

The FASTA index file (.fa.fai) is only needed when using the bwa_meth aligner. It is used by MethylDackel. If using Bismark this parameter is ignored.

Directory for a bwa-meth genome reference index. Only used when using the bwa-meth aligner.

Note that this is not a complete path, but the directory containing the reference. For example, if you have file paths such as /path/to/ref/genome.fa.bwameth.c2t.bwt, you should specify /path/to/ref/.

The nf-core/methylseq package is actually two pipelines in one. The default workflow uses Bismark with Bowtie2 as alignment tool: unless specified otherwise, nf-core/methylseq will run this pipeline.

Since bismark v0.21.0 it is also possible to use HISAT2 as alignment tool. To run this workflow, invoke the pipeline with the command line flag --aligner bismark_hisat. HISAT2 also supports splice-aware alignment if analysis of RNA is desired (e.g. SLAMseq experiments), a file containing a list of known splicesites can be provided with --known_splices.

The second workflow uses BWA-Meth and MethylDackel instead of Bismark. To run this workflow, run the pipeline with the command line flag --aligner bwameth.

By default, the pipeline only produces data for cytosine methylation states in CpG context. Specifying --comprehensive makes the pipeline give results for all cytosine contexts. Note that for large genomes (e.g. Human), these can be massive files. This is only recommended for small genomes (especially those that don't exhibit strong CpG context methylation specificity).

If specified, this flag instructs the Bismark methylation extractor to use the --comprehensive and --merge_non_CpG flags. This produces coverage files with information from about all strands and cytosine contexts merged into two files - one for CpG context and one for non-CpG context.

If using the bwa-meth workflow, the flag makes MethylDackel report CHG and CHH contexts as well.

Specify this parameter when working with PBAT (Post Bisulfite Adapter Tagging) libraries.

Using this parameter sets the --pbat flag when aligning with Bismark. This tells Bismark to align complementary strands (the opposite of --directional).

Additionally, this is a trimming preset equivalent to --clip_r1 6 --clip_r2 9 --three_prime_clip_r1 6 --three_prime_clip_r2 9

Use this parameter when working with RRBS (Reduced Representation Bisulfite Sequencing) data, that is digested using MspI.

Specifying --rrbs will pass on the --rrbs parameter to TrimGalore! See the TrimGalore! documentation to read more about the effects of this option.

This parameter also makes the pipeline skip the deduplication step.

Specify to run Bismark with the --slam flag to run bismark in SLAM-seq mode

NB: Only works with when using the bismark_hisat aligner (--aligner bismark_hisat)

Equivalent to --clip_r1 10 --clip_r2 10 --three_prime_clip_r1 10 --three_prime_clip_r2 10.

Also sets the --maxins flag to 1000 for Bismark.

Equivalent to --clip_r1 6 --clip_r2 6 --three_prime_clip_r1 6 --three_prime_clip_r2 6.

Also sets the --non_directional flag for Bismark.

Equivalent to --clip_r1 10 --clip_r2 15 --three_prime_clip_r1 10 --three_prime_clip_r2 10

Equivalent to --clip_r1 6 --clip_r2 6 --three_prime_clip_r1 2 --three_prime_clip_r2 2

Equivalent to --clip_r1 8 --clip_r2 8 --three_prime_clip_r1 8 --three_prime_clip_r2 8

Equivalent to --clip_r1 10 --clip_r2 10 --three_prime_clip_r1 10 --three_prime_clip_r2 10.

Also sets the --non_directional flag for Bismark.

By default, Bismark assumes that libraries are directional and does not align against complementary strands. If your library prep was not directional, use --non_directional to align against all four possible strands.

Note that the --single_cell and --zymo parameters both set the --non_directional workflow flag automatically.

By default, Bismark does not produce stranded calls. With this option the output considers all Cs on both forward and reverse strands and reports their position, strand, trinucleotide context and methylation state.

By default, Bismark is pretty strict about which alignments it accepts as valid. If you have good reason to believe that your reads will contain more mismatches than normal, this flags can be used to relax the stringency that Bismark uses when accepting alignments. This can greatly improve the number of aligned reads you get back, but may negatively impact the quality of your data.

Bismark uses the Bowtie alignment scoring mechanism to filter reads. Mismatches cost -6, gap opening -5 and gap extension -2. So, a threshold of-60 would allow 10 mismatches or ~ 8 x 1-2bp indels. The threshold is dependent on the length of reads, so a penalty value is used where penalty * bp read length = threshold.

The penalty value used by Bismark by default is 0.2, so for 100bp reads this would be a threshold of -20.

If you specifying the --relax_mismatches pipeline flag, Bismark instead uses 0.6, or a threshold of -60. This adds the Bismark flag --score_min L,0,-0.6 to the alignment command.

The penalty value can be modified using the --num_mismatches pipeline option.

Customise the penalty in the function used to filter reads based on mismatches. The parameter --relax_mismatches must also be specified.

See the parameter documentation for --relax_mismatches for an explanation.

Use to discard any methylation calls with less than a given read coverage depth (in fold coverage) during Bismark's bismark_methylation_extractor step.

For paired-end reads it is theoretically possible that read_1 and read_2 overlap. To avoid scoring overlapping methylation calls twice, this is set to true by default. (Only methylation calls of read 1 are used since read 1 has historically higher quality basecalls than read 2). Whilst this option removes a bias towards more methylation calls in the center of sequenced fragments it may de facto remove a sizable proportion of the data. To count methylation data from both reads in overlapping regions, set this to false.

Ignore the first <int> bp from the 5' end of Read 1 (or single-end alignment files) when processing the methylation call string. This can remove e.g. a restriction enzyme site at the start of each read or any other source of bias (such as PBAT-Seq data).

Ignore the first <int> bp from the 5' end of Read 2 of paired-end sequencing results only. Since the first couple of bases in Read 2 of BS-Seq experiments show a severe bias towards non-methylation as a result of end-repairing sonicated fragments with unmethylated cytosines (see M-bias plot), it is recommended that the first couple of bp of Read 2 are removed before starting downstream analysis. Please see the section on M-bias plots in the Bismark User Guide for more details.

Ignore the first <int> bp from the 5' end of Read 2 of paired-end sequencing results only. Since the first couple of bases in Read 2 of BS-Seq experiments show a severe bias towards non-methylation as a result of end-repairing sonicated fragments with unmethylated cytosines (see M-bias plot), it is recommended that the first couple of bp of Read 2 are removed before starting downstream analysis. Please see the section on M-bias plots in the Bismark User Guide for more details.

Ignore the last <int> bp from the 3' end of Read 1 (or single-end alignment files) when processing the methylation call string. This can remove unwanted biases from the end of reads.

Specify to run Bismark with the --known-splicesite-infile flag to run splice-aware alignment using HISAT2. A .gtf file has to be provided from which a list of known splicesites is created by the pipeline

NB: This only works when using the bismark_hisat aligner with --align

Specify to run Bismark with the --local flag to allow soft-clipping of reads. This should only be used with care in certain single-cell applications or PBAT libraries, which may produce chimeric read pairs. (See Wu et al.).

For example, if --minins 60 is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as --maxins is also satisfied). A 19-bp gap would not be valid in that case.

Default: no flag (Bismark default: 0).

For example, if --maxins 100 is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as --minins is also satisfied). A 61-bp gap would not be valid in that case.

Default: not specified. Bismark default: 500.

Sets --CX during methylation extraction and --nome during the coverage2cytosine step.

Will also force the coverage2cytosine step to run.

Run MethylDackel with the --ignore_flags option, to ignore SAM flags.

Run MethylDackel with the --methyl_kit option, to produce files suitable for use with the methylKit R package.

Setting this option could be useful if you want calculate coverage stats over a list of regions, i.e. for targeted methylation sequencing data.

Deduplication removes PCR duplicate reads after alignment. Specifying this option will skip this step, leaving duplicate reads in your data.

Note that this is turned on automatically if --rrbs is specified.