### Example Params File (YAML)

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

An example of a YAML file used to specify pipeline parameters. This format allows for clear organization of input, output, and configuration settings.

```yaml
input: <SAMPLESHEET>
outdir: <OUTDIR>
genome: 'GRCh37'
<...>
```

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

### Run nf-core/rnaseq Pipeline

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

Example command to run the nf-core/rnaseq pipeline with a test profile. This command is typically used for initial testing or demonstration purposes.

```bash
nextflow run nf-core/rnaseq -profile test_full,<docker/singularity/institute>
```

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

### Start RStudio Server

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/de_rstudio.md

Launches the RStudio server and keeps the Gitpod session active. Ensure sufficient sleep time for analysis.

```bash
sudo rstudio-server start && sleep 5000
```

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

### Install nf-metro and Dependencies

Source: https://github.com/nf-core/rnaseq/blob/master/docs/dev/metro_map.md

Installs the nf-metro package and cairosvg for rendering SVG to PNG. Ensure you have Python and pip installed.

```bash
pip install 'nf-metro>=0.5.4' cairosvg
```

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

### BBSplit FASTA List Configuration

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

This example shows the required format for the `--bbsplit_fasta_list` parameter, which specifies the short name and path to reference genomes for BBSplit indexing. This list is used to build the index for binning reads.

```bash
mm10,/path/to/mm10.fa
ecoli,/path/to/ecoli.fa
sarscov2,/path/to/sarscov2.fa
```

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

### Copy Static PNG to Documentation Directory

Source: https://github.com/nf-core/rnaseq/blob/master/docs/dev/metro_map.md

Copies the generated static PNG metro map to a specific subdirectory within the documentation for usage examples.

```bash
cp docs/images/nf-core-rnaseq_metro_map_grey.png \
  docs/usage/differential_expression_analysis/img/
```

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

### Run RiboDetector for rRNA Removal

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

Use this command to enable RiboDetector for rRNA removal. Ensure RiboDetector is installed and configured.

```bash
nextflow run nf-core/rnaseq --remove_ribo_rna --ribo_removal_tool ribodetector ...
```

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

### Example Generated Samplesheet with BAM Paths

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

This CSV format shows the structure of a samplesheet generated after the initial run, including paths to genome_bam and transcriptome_bam files.

```csv
sample,fastq_1,fastq_2,strandedness,seq_platform,seq_center,genome_bam,percent_mapped,transcriptome_bam
SAMPLE1,/path/sample1_R1.fastq.gz,/path/sample1_R2.fastq.gz,forward,ILLUMINA,,results/star_salmon/SAMPLE1.markdup.sorted.bam,85.2,results/star_salmon/SAMPLE1.Aligned.toTranscriptome.out.bam
SAMPLE2,/path/sample2_R1.fastq.gz,,reverse,ILLUMINA,,results/star_salmon/SAMPLE2.sorted.bam,92.1,results/star_salmon/SAMPLE2.Aligned.toTranscriptome.out.bam
```

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

### Load Required R Libraries

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/de_rstudio.md

Loads essential R packages for data manipulation (tidyverse), differential expression analysis (DESeq2), heatmap visualization (pheatmap), color palettes (RColorBrewer), and text label repulsion (ggrepel). Installs pheatmap if missing.

```r
#### Loading libraries ####

# tidyverse: collection of R packages for data manipulation, visualization and modeling

library("tidyverse")

# DESeq2: package for differential gene expression analysis

library("DESeq2")

# pheatmap: package for creating heatmaps, which will be used to visualise the results

install.packages("pheatmap") # To install the package missing in the current RStudio env

library("pheatmap")

# RColorBrewer: package for creating color palettes, which will be used to customise the heatmaps

library("RColorBrewer")

# ggrepel: package that provides geoms for ggplot2 to repel overlapping text labels in the plots

library("ggrepel")
```

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

### Custom Kallisto Quantification Arguments

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

Supply additional arguments to Kallisto quantification using '--extra_kallisto_quant_args'. This can be used to specify library strandedness, for example.

```bash
--extra_kallisto_quant_args "--fr-stranded"
```

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

### Step 1: Initial Run with BAM Generation

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

Run the pipeline to generate BAM files and a samplesheet for future reprocessing. Use the --save_align_intermeds flag to publish BAM files.

```bash
nextflow run nf-core/rnaseq \
  --input samplesheet.csv \
  --save_align_intermeds \
  --outdir results_initial \
  -profile docker
```

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

### Create R Project and Script

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/de_rstudio.md

Initializes a new R project and creates an R script file for analysis commands. The working directory should be set up correctly.

```r
#### Differential expression analysis with DESeq2 ####
```

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

### Launch nf-core/rnaseq Pipeline (Bash)

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

Execute the nf-core/rnaseq pipeline using a specific version, input samplesheet, output directory, genome, and aligner. Includes parameters to skip certain QC steps for faster execution.

```bash
nextflow run nf-core/rnaseq -r 3.12.0 \
--input /workspace/gitpod/training/data/reads/rnaseq_samplesheet.csv \
--outdir ./results_star_salmon \
--genome GRCh38chr21 \
--aligner star_salmon \
--pseudo_aligner salmon \
--skip_biotype_qc \
--skip_stringtie \
--skip_bigwig \
--skip_umi_extract \
--skip_trimming \
--skip_fastqc \
--skip_markduplicates \
--skip_dupradar \
--skip_rseqc \
--skip_qualimap

```

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

### Specify Samplesheet Input

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

Use the --input parameter to specify the path to your samplesheet file. This file must contain sample information, including FASTQ file locations and strandedness.

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

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

### Custom Salmon Quantification Arguments

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

Provide custom parameters to the Salmon quantification command via '--extra_salmon_quant_args'. For example, to enable sequence bias correction.

```bash
--extra_salmon_quant_args "--seqBias --gcBias"
```

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

### Disable fq lint validator

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

Use the extra_fqlint_args parameter to disable specific validators from fq. For example, disable the paired read name check by setting it to --disable-validator P001.

```bash
--extra_fqlint_args "--disable-validator P001"
```

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

### Per-sample Quantification with Salmon

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

This command demonstrates how to run the pipeline for per-sample quantification using Salmon, skipping alignment and merging steps. It's optimized for speed when only quantification is needed.

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    --pseudo_aligner salmon \
    --skip_alignment \
    --skip_quantification_merge \
    --outdir results
```

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

### Run nf-core/rnaseq Pipeline

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

Basic command to execute the nf-core/rnaseq pipeline. Requires specifying input samplesheet, output directory, genome annotation (GTF), and genome FASTA file. A profile for execution environment (e.g., Docker, Singularity) must also be provided.

```bash
nextflow run nf-core/rnaseq \
    --input <SAMPLESHEET> \
    --outdir <OUTDIR> \
    --gtf <GTF> \
    --fasta <GENOME FASTA> \
    -profile <docker/singularity/.../institute>
```

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

### R Design Formula Examples

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/theory.md

Specifies the sources of variation for the statistical model. The tilde (~) indicates a formula relationship. The order of variables does not affect results, but the main factor of interest is commonly placed last.

```r
design = ~ condition
```

```r
design = ~ sex + developmental_stage + condition
```

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

### Enable RustQC in nf-core/rnaseq

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

To enable RustQC, use the --use_rustqc flag. This flag automatically skips the individual QC tools that RustQC replaces.

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    --outdir results \
    --fasta genome.fa \
    --gtf annotation.gtf \
    --use_rustqc \
    -profile docker
```

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

### Run STAR Alignment with Parabricks GPU Acceleration

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

This command initiates the nf-core/rnaseq pipeline with Parabricks for GPU-accelerated STAR alignment. Ensure you have NVIDIA GPUs and the necessary container runtime (Docker/Singularity).

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    --outdir results \
    --fasta genome.fa \
    --gtf annotation.gtf \
    --use_parabricks_star \
    -profile docker
```

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

### Load Enrichment Analysis Libraries

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/de_rstudio.md

Loads necessary R libraries for functional enrichment analysis, including clusterProfiler for enrichment, org.Hs.eg.db for human gene annotation, and cowplot for plot manipulation. Includes installation for cowplot if missing.

```r
library(clusterProfiler)

library(org.Hs.eg.db)

install.packages("cowplot") # To install the package missing in the current RStudio env

library(cowplot)
```

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

### Basic Pipeline Execution

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

Standard command to run the pipeline with essential parameters for input, output, and genome data. Requires a samplesheet, output directory, GTF file, and genome FASTA file. Uses the 'docker' profile for execution.

```bash
nextflow run \
    nf-core/rnaseq \
    --input <SAMPLESHEET> \
    --outdir <OUTDIR> \
    --gtf <GTF> \
    --fasta <GENOME FASTA> \
    -profile docker
```

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

### Run Linting with nf-core/tools

Source: https://github.com/nf-core/rnaseq/blob/master/docs/CONTRIBUTING.md

Enforce nf-core guidelines by running lint tests on the pipeline directory. Failures or warnings will provide links to documentation for correction.

```bash
nf-core pipelines lint <pipeline_directory>
```

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

### Run RNA-Seq Pipeline with Prokaryotic Profile

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

This command initiates the nf-core/rnaseq pipeline with settings optimized for prokaryotic data, using Docker for containerization. Ensure your input files (samplesheet.csv, genome.fasta, annotation.gff3) are correctly specified.

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    --fasta genome.fasta \
    --gff annotation.gff3 \
    --outdir results \
    -profile prokaryotic,docker
```

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

### Set Sentieon License (File)

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

Use this command to set the Sentieon license secret when the license is provided as a file. The file content is base64-encoded.

```bash
nextflow secrets set SENTIEON_LICENSE_BASE64 $(cat <sentieon_license_file.lic> | base64 -w 0)
```

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

### Run nf-test Locally

Source: https://github.com/nf-core/rnaseq/blob/master/docs/CONTRIBUTING.md

Execute local tests using nf-test. Always include the 'test' profile explicitly, as this pipeline supports both CPU and GPU test profiles.

```bash
nf-test test --tag test --profile +docker --verbose
```

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

### Download Latest Ensembl Human Reference Files

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

Use these commands to download the latest Ensembl human reference FASTA and GTF files for GRCh38. Ensure consistency in reference resource usage to avoid compatibility issues.

```bash
latest_release=$(curl -s 'http://rest.ensembl.org/info/software?content-type=application/json' | grep -o '"release":[0-9]*' | cut -d: -f2)
wget -L ftp://ftp.ensembl.org/pub/release-${latest_release}/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa.gz
wget -L ftp://ftp.ensembl.org/pub/release-${latest_release}/gtf/homo_sapiens/Homo_sapiens.GRCh38.${latest_release}.gtf.gz
```

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

### Enable rRNA Removal with Bowtie2

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

Use this command to enable rRNA removal using the Bowtie2 tool. A rRNA database manifest file must be specified via --ribo_database_manifest.

```bash
nextflow run nf-core/rnaseq --remove_ribo_rna --ribo_removal_tool bowtie2 ...
```

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

### Configure Reference Genome and Annotations (Groovy)

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

Add this code to your `nextflow.config` file to define a new genome entry, specifying paths to FASTA, GFF, transcript FASTA, and pre-built STAR and Salmon indices.

```groovy
igenomes_base = '/workspace/gitpod/training/data/refs/'
genomes {
        'GRCh38chr21' {
            fasta                 = "${params.igenomes_base}/sequence/Homo_sapiens_assembly38_chr21.fasta"
            fasta_fai             = "${params.igenomes_base}/sequence/Homo_sapiens_assembly38_chr21.fasta.fai"
            gff                   = "${params.igenomes_base}/gencode_v29_chr21_parsed.gff"
            transcript_fasta      = "${params.igenomes_base}/gencode.v29.transcripts_chr21.fa"
            star_index            = "${params.igenomes_base}/star_index_chr21.tar.gz"
            salmon_index          = "${params.igenomes_base}/salmon_index_chr21.tar.gz"
	}
}

```

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

### Running Salmon in Isolation

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

Use the '--skip_alignment' parameter to run Salmon or Kallisto in isolation, without the standard alignment workflow. This is useful if you only need pseudoalignment and quantification.

```bash
--skip_alignment
```

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

### Rapid Quantification Profile

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

This command utilizes a pre-defined profile 'rapid_quant' to bundle common options for fast, per-sample quantification, including alignment-independent QC skips. It also specifies Docker for containerized execution.

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    -profile rapid_quant,docker \
    --outdir results
```

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

### Enable GPU Acceleration for RiboDetector

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

To leverage GPU acceleration with RiboDetector, use the `--use_gpu_ribodetector` flag. This requires a CUDA-enabled environment with PyTorch GPU support.

```bash
nextflow run nf-core/rnaseq --remove_ribo_rna --ribo_removal_tool ribodetector --use_gpu_ribodetector ...
```

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

### Check Sample Name Matching

Source: https://github.com/nf-core/rnaseq/blob/master/docs/usage/differential_expression_analysis/de_rstudio.md

Verify that sample names in the count data and metadata match and are in the correct order to prevent DESeq2 errors.

```r
all(colnames(dds$counts) %in% rownames(metadata)) # Must be TRUE

all(colnames(dds$counts) == rownames(metadata)) # Must be TRUE
```

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

### Set Computational Resources (Groovy)

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

Configure computational resource limits such as maximum CPUs, memory, and time by adding these parameters to the `params` directive in your `nextflow.config` file.

```groovy
params {
    max_cpus      = 2
    max_memory    = '6.5GB'
    max_time      = '2.h'
}

```

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

### Render Static SVG and PNG Metro Map

Source: https://github.com/nf-core/rnaseq/blob/master/docs/dev/metro_map.md

Generates a static SVG metro map and converts it to PNG using nf-metro and cairosvg. The `--theme light`, `--x-spacing`, `--y-spacing`, and `--no-straight-diamonds` options customize the map's appearance. The `--logo` option includes a pipeline logo.

```bash
nf-metro render assets/metro_map.mmd \
  -o docs/images/nf-core-rnaseq_metro_map_grey.svg \
  --theme light --x-spacing 60 --y-spacing 40 \
  --no-straight-diamonds \
  --logo docs/images/nf-core-rnaseq_logo_light.png
```

```python
python -c "import cairosvg; cairosvg.svg2png(
    url='docs/images/nf-core-rnaseq_metro_map_grey.svg',
    write_to='docs/images/nf-core-rnaseq_metro_map_grey.png', output_width=2265)"
```

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

### FASTP FASTQ Sampling

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

Use the `--extra_fastp_args` parameter to specify the `--reads_to_process` option for fastp. This allows you to limit the number of reads processed for testing or resource management.

```bash
--trimmer fastp --extra_fastp_args '--reads_to_process 10000'
```

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

### Pipeline Execution with Params File

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

Execute the pipeline using parameters defined in an external YAML or JSON file. This simplifies repeated runs with the same settings. Ensure the params file is correctly formatted and accessible.

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

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

### Render Animated SVG Metro Map

Source: https://github.com/nf-core/rnaseq/blob/master/docs/dev/metro_map.md

Creates an animated SVG metro map for use in the README file. The `--animate` flag enables animation. Other options are similar to static rendering.

```bash
nf-metro render assets/metro_map.mmd \
  -o docs/images/nf-core-rnaseq_metro_map_grey_animated.svg \
  --theme light --x-spacing 60 --y-spacing 40 --animate \
  --no-straight-diamonds \
  --logo docs/images/nf-core-rnaseq_logo_light.png
```

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

### Enable rRNA Removal with SortMeRNA

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

Use this command to enable rRNA removal using the default SortMeRNA tool. Ensure you have the necessary rRNA database configured.

```bash
nextflow run nf-core/rnaseq --remove_ribo_rna --ribo_removal_tool sortmerna ...
```

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

### Set Sentieon License (String)

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

Use this command to set the Sentieon license secret when the license is provided as a string (IP:Port). The license string is base64-encoded.

```bash
nextflow secrets set SENTIEON_LICENSE_BASE64 $(echo -n <sentieon_license_string> | base64 -w 0)
```

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

### Step 2: Reprocessing Run with BAM Input

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

Reprocess data using the generated samplesheet with BAM files, skipping the alignment step. The --skip_alignment flag ensures BAM files are used for post-processing and quantification.

```bash
nextflow run nf-core/rnaseq \
  --input samplesheets/samplesheet_with_bams.csv \
  --skip_alignment \
  --outdir results_reprocessed \
  -profile docker
```

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

### Enable Specific RustQC Modules

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

While RustQC includes Preseq and TIN by default, you can explicitly enable or disable specific RSeQC modules using these options. This is useful for fine-tuning the QC process.

```bash
--skip_preseq false \
--rseqc_modules bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication,tin
```

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

### Run Pipeline with GFFRead for Prokaryotic Genomes

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

Use this command to run the pipeline with the `--gffread_transcript_fasta` parameter, which is useful when RSEM fails to extract transcripts correctly, especially with non-standard annotation formats.

```bash
nextflow run nf-core/rnaseq \
    --input samplesheet.csv \
    --fasta genome.fasta \
    --gtf annotation.gtf \
    --gffread_transcript_fasta \
    --outdir results \
    -profile docker
```

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

### Pipeline Execution on ARM Architectures

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

Run the pipeline in an ARM-compatible mode by specifying the 'arm64' profile alongside 'docker'. This enables the use of ARM-compatible containers and adjusts Conda definitions for ARM operations. Note that this profile is experimental.

```bash
nextflow run \
    nf-core/rnaseq \
    --input <SAMPLESHEET> \
    --outdir <OUTDIR> \
    --gtf <GTF> \
    --fasta <GENOME FASTA> \
    -profile docker,arm64
```

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

### Custom STAR Alignment Parameters for 3′ Assays

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

Use these parameters to adjust STAR aligner settings for 3′ RNA-seq protocols like QuantSeq, by decreasing mismatch tolerance and clipping poly(A) tails. Pipeline defaults may be overridden.

```bash
--extra_star_align_args "--outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA"
```

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

### Set Nextflow Memory Options

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

Limit the memory allocated to the Nextflow Java virtual machine. Add this line to your environment configuration file (e.g., ~/.bashrc).

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