### Install Nextflow Edge Release from Scratch

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Download and install a Nextflow edge release from scratch. This involves exporting the desired version, downloading the installer, and moving the binary to a system-wide location.

```bash
export NXF_VER="20.11.0-edge"
wget -qO- get.nextflow.io | bash
sudo mv nextflow /usr/local/bin/
```

--------------------------------

### Example Params File

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

A sample YAML file defining pipeline input, output directory, and reference genome.

```yaml
input: './samplesheet.csv'
outdir: './results/'
genome: 'GRCh37'
<...>
```

--------------------------------

### Run Pipeline with Test Profile

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Execute the pipeline with the 'test' profile and a specified environment profile to verify setup.

```bash
nextflow run nf-core/methylseq \
  --outdir <OUTDIR> \
  --genome GRCh38 \
  -profile test,<docker/singularity/podman/shifter/charliecloud/conda/institute>
```

--------------------------------

### Run nf-core/methylseq Pipeline

Source: https://github.com/nf-core/methylseq/blob/master/README.md

Execute the pipeline using default parameters, specifying the input samplesheet, output directory, and genome reference. Use a profile for environment setup (e.g., Docker, Singularity).

```bash
nextflow run nf-core/methylseq --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>
```

--------------------------------

### Example Command Error

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This snippet shows a typical command error encountered during pipeline execution, including the command that failed and the working directory. It serves as an example for debugging.

```console
Command error:
    .command.sh: line 9:  30 Killed    STAR --genomeDir star --readFilesIn WT_REP1_trimmed.fq.gz --runThreadN 2 --outFileNamePrefix WT_REP1. <TRUNCATED> 
Work dir:
    /home/pipelinetest/work/9d/172ca5881234073e8d76f2a19c88fb

Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`
```

--------------------------------

### Samplesheet Example with Multiple Runs

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Illustrates how to format a samplesheet when a single sample has multiple FASTQ files across different runs or lanes. The 'sample' identifier must be consistent across all entries for the same sample.

```csv
sample,fastq_1,fastq_2,genome
SRR389222,SRR389222_sub1.fastq.gz,,
SRR389222,SRR389222_sub2.fastq.gz,,
SRR389222,SRR389222_sub3.fastq.gz,,
Ecoli_10K_methylated,Ecoli_10K_methylated_R1.fastq.gz,Ecoli_10K_methylated_R2.fastq.gz,
```

--------------------------------

### Running the Methylseq Pipeline with Docker

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This command executes the nf-core/methylseq pipeline using the Docker containerization. It specifies the input samplesheet, output directory, and reference genome. Ensure you have Nextflow and Docker installed.

```bash
nextflow run nf-core/methylseq --input ./samplesheet.csv --outdir ./results --genome GRCh38 -profile docker
```

--------------------------------

### BWA-Meth Internal Command Translation

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage/bs-seq-primer.md

This example illustrates the internal translation performed by BWA-Meth, showing how the user command is converted to a piped command for BWA-MEM. This process avoids creating temporary files.

```bash
bwa mem -p ref.fa.bwameth.c2t '<python bwameth.py c2t A.fq B.fq'
```

--------------------------------

### Samplesheet Header Example

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Defines the required header row for the samplesheet CSV file, specifying the columns for sample identifier, FASTQ files, and genome reference.

```csv
sample,fastq_1,fastq_2,genome
```

--------------------------------

### Example Samplesheet for Methylseq Pipeline

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This CSV formatted samplesheet includes both single-end and paired-end data. Ensure the first three columns (sample, fastq_1, fastq_2) adhere to the specified format. The 'genome' column is optional.

```csv
sample,fastq_1,fastq_2,genome
CONTROL_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz,
CONTROL_REP2,AEG588A2_S2_L002_R1_001.fastq.gz,AEG588A2_S2_L002_R2_001.fastq.gz,
CONTROL_REP3,AEG588A3_S3_L002_R1_001.fastq.gz,AEG588A3_S3_L002_R2_001.fastq.gz,
TREATMENT_REP1,AEG588A4_S4_L003_R1_001.fastq.gz,,
TREATMENT_REP2,AEG588A5_S5_L003_R1_001.fastq.gz,,
TREATMENT_REP3,AEG588A6_S6_L003_R1_001.fastq.gz,,
TREATMENT_REP3,AEG588A6_S6_L004_R1_001.fastq.gz,,
```

--------------------------------

### Run BWA-Meth with BWA-MEM2 (GPU)

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This command initiates the pipeline using BWA-Meth with the BWA-MEM2 algorithm, leveraging GPU acceleration. Ensure the 'gpu' profile is active and specify the aligner and input samplesheet.

```bash
nextflow run nf-core/methylseq --aligner bwameth --use_mem2 --profile gpu --input samplesheet.csv --genome GRCh38
```

--------------------------------

### Run Pipeline with Params File

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Execute the pipeline using a YAML or JSON parameters file for repeated configurations. Avoid using '-c' for parameters.

```bash
nextflow run nf-core/methylseq -profile docker -params-file params.yaml
```

--------------------------------

### On-the-fly C-to-T conversion with BWA-MEM2

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage/bs-seq-primer.md

This command demonstrates how to perform on-the-fly C-to-T and G-to-A conversions for FASTQ files using a Python script with BWA-MEM2. Both converted files are streamed directly into the aligner, producing standard SAM alignment output.

```bash
bwa-mem2 mem -p ref.fa.bwameth.c2t '<python bwameth.py c2t A.fq B.fq'
```

--------------------------------

### Run BWA-Meth with BWA-MEM2 algorithm (CPU)

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This command initiates the nf-core/methylseq pipeline using the BWA-Meth aligner with the BWA-MEM2 algorithm for CPU processing. It requires specifying the input samplesheet and the reference genome.

```bash
nextflow run nf-core/methylseq --aligner bwameth --use_mem2 --input samplesheet.csv --genome GRCh38
```

--------------------------------

### Pipeline Configuration Summary

Source: https://github.com/nf-core/methylseq/blob/master/assets/email_template.txt

Outputs the pipeline configuration parameters in a formatted list. Each parameter is listed with its key and value.

```Groovy
<% out << summary.collect{ k,v -> " - $k: $v" }.join("\n") %>
```

--------------------------------

### Samplesheet CSV Format

Source: https://github.com/nf-core/methylseq/blob/master/README.md

Prepare a CSV file with sample information including FASTQ file paths and genome reference. Supports single-end and paired-end reads.

```csv
sample,fastq_1,fastq_2,genome
SRR389222_sub1,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub1.fastq.gz,,
SRR389222_sub2,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub2.fastq.gz,,
SRR389222_sub3,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub3.fastq.gz,,
Ecoli_10K_methylated,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R1.fastq.gz,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R2.fastq.gz,
```

--------------------------------

### Run Pipeline with Edge Release (Direct)

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Execute a pipeline using a specific Nextflow edge version directly on the command line. The version is specified inline before the 'nextflow run' command.

```bash
NXF_VER="20.11.0-edge" nextflow run nf-core/rnaseq -profile test,docker -r 3.0
```

--------------------------------

### Specify Samplesheet Input

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This parameter points to the location of the samplesheet file, which is crucial for defining the samples and their associated FASTQ files for the analysis.

```bash
--input '[path to samplesheet file]'
```

--------------------------------

### Global Resource Limits Configuration

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Configure maximum resources (CPUs, memory, time) for all pipeline processes by default. This is useful for setting system-wide caps.

```nextflow
process {
    resourceLimits = [
        cpus: 4,
        memory: '15.GB',
        time: '1.h'
    ]
}
```

--------------------------------

### Run Targeted Sequencing Analysis

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This command enables targeted sequencing analysis, filtering methylation data to specified regions and collecting hybrid-capture metrics. It requires specifying the aligner, target regions file, and enabling the relevant flags.

```bash
nextflow run nf-core/methylseq \
  --input samplesheet.csv \
  --genome GRCh38 \
  --aligner bismark \
  --run_targeted_sequencing \
  --target_regions_file genome_target_regions.bed \
  --collecthsmetrics
```

--------------------------------

### bwa-mem Workflow Output Structure

Source: https://github.com/nf-core/methylseq/blob/master/docs/output.md

This structure outlines the output for the bwa-mem alignment workflow, including FastQC, alignment, deduplication, Rastair processing for conversion rates, and pipeline information.

```text
bwamem/
├── bwamem
│   ├── alignments
│   │   └── samtools_stats
│   └── deduplicated
│       └── picard_metrics
├── fastqc
│   ├── Ecoli_10K_methylated_1_fastqc.html
│   ├── Ecoli_10K_methylated_2_fastqc.html
│   └── zips
├── multiqc
│   └── bwamem
├── pipeline_info
│   ├── execution_report_2024-12-13_05-36-34.html
│   ├── execution_timeline_2024-12-13_05-36-34.html
│   ├── execution_trace_2024-12-13_05-36-34.txt
│   ├── nf_core_methylseq_software_mqc_versions.yml
│   ├── params_2024-12-13_05-36-43.json
│   └── pipeline_dag_2024-12-13_05-36-34.html
├── rastair
│   ├── call
│   │   └── Ecoli_10K_methylated.rastair_call.txt
│   ├── mbias
│   │   └── Ecoli_10K_methylated.rastair_mbias.txt
│   ├── mbiasparser
│   │   └── Ecoli_10K_methylated.rastair_mbias_processed.csv
│   └── methylkit
│       └── Ecoli_10K_methylated.rastair_methylkit.txt.gz
└── trimgalore
    ├── fastqc
    └── logs
```

--------------------------------

### Specify Pipeline Version

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Run a specific version of the pipeline using the '-r' flag to ensure reproducibility.

```bash
nextflow run nf-core/methylseq -r 1.3.1
```

--------------------------------

### Run Pipeline with Edge Release (Export)

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Set the Nextflow edge version as an environment variable before running the pipeline. This method is an alternative to specifying the version inline.

```bash
export NXF_VER="20.11.0-edge"
nextflow run nf-core/rnaseq -profile test,docker -r 3.0
```

--------------------------------

### Add Custom Arguments to Modules

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Append additional arguments to module commands, such as '--scaffolds' for Bismark, to bypass OS file handle limits.

```bash
nextflow run nf-core/methylseq --scaffolds
```

--------------------------------

### Bismark Workflow Output Structure

Source: https://github.com/nf-core/methylseq/blob/master/docs/output.md

This structure shows the directories and files generated when the Bismark alignment workflow is used. It includes results from FastQC, alignment, deduplication, methylation calls, and reports.

```text
bismark/
├── bismark
│   ├── alignments
│   ├── deduplicated
│   ├── methylation_calls
│   ├── reports
│   └── summary
├── fastqc
│   ├── Ecoli_10K_methylated_1_fastqc.html
│   ├── Ecoli_10K_methylated_2_fastqc.html
│   └── zips
├── multiqc
│   └── bismark
├── pipeline_info
│   ├── execution_report_2024-12-13_05-38-05.html
│   ├── execution_timeline_2024-12-13_05-38-05.html
│   ├── execution_trace_2024-12-13_05-38-05.txt
│   ├── nf_core_methylseq_software_mqc_versions.yml
│   ├── params_2024-12-13_05-38-14.json
│   └── pipeline_dag_2024-12-13_05-38-05.html
└── trimgalore
    ├── fastqc
    └── logs
```

--------------------------------

### bwa-meth Workflow Output Structure

Source: https://github.com/nf-core/methylseq/blob/master/docs/output.md

This structure details the output directories and files for the bwa-meth alignment workflow. It includes FastQC, alignment, deduplication, methylation calling with Methyldackel, and pipeline information.

```text
bwameth/
├── bwameth
│   ├── alignments
│   └── deduplicated
├── fastqc
│   ├── Ecoli_10K_methylated_1_fastqc.html
│   ├── Ecoli_10K_methylated_2_fastqc.html
│   └── zips
├── methyldackel
│   ├── Ecoli_10K_methylated.markdup.sorted_CpG.bedGraph
│   └── mbias
├── multiqc
│   └── bwameth
├── pipeline_info
│   ├── execution_report_2024-12-13_05-36-34.html
│   ├── execution_timeline_2024-12-13_05-36-34.html
│   ├── execution_trace_2024-12-13_05-36-34.txt
│   ├── nf_core_methylseq_software_mqc_versions.yml
│   ├── params_2024-12-13_05-36-43.json
│   └── pipeline_dag_2024-12-13_05-36-34.html
└── trimgalore
    ├── fastqc
    └── logs
```

--------------------------------

### BWA-Meth Alignment Command

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage/bs-seq-primer.md

This command demonstrates how BWA-Meth maps bisulfite-converted reads to an in silico converted reference. It shows the typical command structure for aligning paired-end reads.

```bash
python bwameth.py --reference ref.fa A.fq B.fq
```

--------------------------------

### Embed Pipeline Logo in Email

Source: https://github.com/nf-core/methylseq/blob/master/assets/sendmail_template.txt

Reads a PNG logo file from the project directory, encodes it in Base64, and prepares it for embedding in an email.

```Groovy
<% out << new File("$projectDir/assets/nf-core-methylseq_logo_light.png").
    bytes.
    encodeBase64().
    toString().
    tokenize( '\n' )*.
    toList()*. 
    collate( 76 )*.
    collect { it.join() }.
    flatten().
    join( '\n' ) %>
```

--------------------------------

### Update Pipeline Cache

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Fetch the latest version of the pipeline from GitHub to ensure you are running the most recent code.

```bash
nextflow pull nf-core/methylseq
```

--------------------------------

### Attach MultiQC Report to Email

Source: https://github.com/nf-core/methylseq/blob/master/assets/sendmail_template.txt

Conditionally attaches a MultiQC report to an email if the file exists and its size is within a specified limit. The report is Base64 encoded.

```Groovy
<% if (mqcFile){
def mqcFileObj = new File("$mqcFile")
if (mqcFileObj.length() < mqcMaxSize){
out << """
--nfcoremimeboundary
Content-Type: text/html; name=\"multiqc_report\"
Content-Transfer-Encoding: base64
Content-ID: <mqcreport>
Content-Disposition: attachment; filename=\"${mqcFileObj.getName()}\" 

${mqcFileObj.
    bytes.
    encodeBase64().
    toString().
    tokenize( '\n' )*.
    toList()*. 
    collate( 76 )*.
    collect { it.join() }.
    flatten().
    join( '\n' )}
"""
}}
%>
```

--------------------------------

### Process-Specific Resource Limits

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Set resource limits for a specific pipeline process using its name. This allows fine-grained control over resource allocation for individual steps.

```nextflow
process {
  withName: 'BISMARK_ALIGN' {
    resourceLimits = [
        cpus: 4,
        memory: '15.GB',
        time: '1.h'
    ]
  }
}
```

--------------------------------

### Set Nextflow JVM Memory Options

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Limit the memory allocated to the Nextflow Java Virtual Machine. This is typically added to your shell's environment configuration file like ~/.bashrc.

```bash
NXF_OPTS='-Xms1g -Xmx4g'
```

--------------------------------

### Override Process Memory Limit

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Increase the memory allocated to a specific process, such as 'BISMARK_ALIGN', by overriding its default or configured limit. This is useful when a process requires more memory than initially allocated.

```nextflow
process {
  withName: 'BISMARK_ALIGN' {
    memory = 100.GB
  }
}
```

--------------------------------

### Override Docker Container for a Process

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Use this configuration to specify a different Docker container for a particular Nextflow process. Identify the process name and set the desired container image.

```nextflow
process {
    withName: PANGOLIN {
        container = 'quay.io/biocontainers/pangolin:3.0.5--pyhdfd78af_0'
    }
}
```

--------------------------------

### Override Singularity Container for a Process

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

This configuration allows you to specify an alternative Singularity container for a Nextflow process. Ensure you use the correct URL for the Singularity image.

```nextflow
process {
    withName: PANGOLIN {
        container = 'https://depot.galaxyproject.org/singularity/pangolin:3.0.5--pyhdfd78af_0'
    }
}
```

--------------------------------

### Conditional Email Body

Source: https://github.com/nf-core/methylseq/blob/master/assets/email_template.txt

Generates the email body based on the pipeline's success or failure status. Includes exit status and error messages on failure.

```Groovy
<% if (success) {
    out << "## nf-core/methylseq execution completed successfully! ##"
} else {
    out << """####################################################
## nf-core/methylseq execution completed unsuccessfully! ##
####################################################
The exit status of the task that caused the workflow execution to fail was: $exitStatus.
The full error message was:

${errorReport}
"""
}
%>
```

--------------------------------

### Override Conda Environment for a Process

Source: https://github.com/nf-core/methylseq/blob/master/docs/usage.md

Configure a specific Conda environment for a Nextflow process. This is useful for managing software dependencies that are available via Conda.

```nextflow
process {
    withName: PANGOLIN {
        conda = 'bioconda::pangolin=3.0.5'
    }
}
```

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