Pipeline generating coverage graphs by aligning sequencing reads to a selected viral reference genome, visualizing the depth of coverage across the genome.
Pipeline generating coverage graphs by aligning sequencing reads to a selected viral reference genome, visualizing the depth of coverage across the genome.
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## Introduction
**nf-core/seqcoverage** is a bioinformatics pipeline designed to visualize sequencing coverage of viral reads. Users can select from a predefined set of genomes or specify any genome available in the NCBI RefSeq dataset using its accession number. The pipeline generates a coverage plot for the selected virus, providing clear insights into sequencing depth and distribution.
1. Sample QC ([`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/))
2. Read classification to viral database ([`Kraken2`](https://ccb.jhu.edu/software/kraken2/))
3. Filter reads for virus of interest
4. Align reads to chosen virus genome ([`Minimap2`](https://github.com/lh3/minimap2/))
5. Generate coverage graphs ([`Samtools coverage`](http://www.htslib.org/doc/samtools))
## Usage
> [!NOTE]
> If you are new to Nextflow and nf-core, please refer to [this page](https://nf-co.re/docs/usage/installation) on how to set-up Nextflow.Make sure to [test your setup](https://nf-co.re/docs/usage/introduction#how-to-run-a-pipeline) with `-profile test` before running the workflow on actual data.
First, prepare a samplesheet with your input data that looks as follows:
`samplesheet.csv`:
```csv
sample,fastq_1,fastq_2
CONTROL_PAIRED_ENDS,S1_L002_R1_001.fastq,S1_L002_R2_001.fastq
CONTROL_SINGLE_END,S1_L002_001.fastq,
```
Each row represents a pair of fastq files (paired-end sequencing data). The input files should be in fastq or fastq.gz format.
Now, you can run the pipeline using:
```bash
nextflow run nf-core/seqcoverage \
-profile \
-c custom.conf \
--input samplesheet.csv \
--outdir
```
Example `custom.conf` might look like this:
```bash
params {
viruses = ['COVID_OC43', 'SARS_CoV_2', 'RSV_A']
kraken2_db = 'path/to/kraken2db'
viruses_csv = 'pipeline/dir/assets/viruses.csv'
minimap2_db = 'path/to/minimap2db.fasta'
}
```
Full list of in-build viruses available for use are within the `viruses.csv` file in assets and are as following:
- SARS_CoV_2
- InfluenzaA
- InfluenzaB
- RSV_A
- Rhinovirus_A
- Rhinovirus_B
- Rhinovirus_C
- Rhinovirus_NAT001
- Parainfluenza1
- Parainfluenza3
- Parainfluenza4
- Human_Metapneumovirus
- Adenovirus2
- Adenovirus5
- Adenovirus1
- Adenovirus7
- Adenovirus35
- AdenovirusD
- AdenovirusB2
- AdenovirusB1
- AdenovirusA
- AdenovirusE
- AdenovirusF
- AdenovirusD
- Adenovirus54
- COVID_OC43
- COVID_229E
- COVID_NL63
- COVID_HKU1
- Bocavirus3
- Mastadenovirus_A
- Mastadenovirus_C
- Monkeypox
It is also possible to use accession numbers for the virus of choice and will depend on availability within the minimap2 database of use (suggested NCBI Viral RefSeq).
> [!WARNING]
> Please provide pipeline parameters via the CLI or Nextflow `-params-file` option. Custom config files including those provided by the `-c` Nextflow option can be used to provide any configuration _**except for parameters**_; see [docs](https://nf-co.re/docs/usage/getting_started/configuration#custom-configuration-files).
For more details and further functionality, please refer to the [usage documentation](https://nf-co.re/seqcoverage/usage) and the [parameter documentation](https://nf-co.re/seqcoverage/parameters).
## Pipeline Parameters
### **Input and Output Options**
- **`input`**: Path to input samplesheet. (csv)
- **`outdir`**: Directory for pipeline output.
### **Pipeline-Specific Options**
- **`viruses`**: List of target viruses for analysis. (eg. viruses = ['COVID_OC43', 'SARS_CoV_2', 'RSV_A'])
- **`clusterOpts`**: Additional options for cluster execution.
- **`kraken2_db`**: Path to the Kraken2 database.
- **`viruses_csv`**: CSV file containing virus metadata. (in-build list available in assets folder or the pipeline)
- **`minimap2_db`**: Path to the Minimap2 reference genome.
### **Configuration & Validation**
- **`config_profile_name`**: Custom profile name.
- **`config_profile_description`**: Profile description.
- **`config_profile_contact`**: Contact information for profile.
- **`config_profile_url`**: URL for profile documentation.
- **`validate_params`**: Enable schema validation (`true` by default).
### **Pipeline Execution & Output**
- **`publish_dir_mode`**: Mode for output file handling (`copy` by default).
- **`email`**: Email address for notifications.
- **`email_on_fail`**: Send email only on failure.
- **`plaintext_email`**: Send email in plaintext format.
- **`monochrome_logs`**: Disable colored logs if `true`.
- **`hook_url`**: Webhook URL for notifications.
- **`help` / `help_full`**: Show help messages.
- **`version`**: Show pipeline version.
### **MultiQC Options**
- **`multiqc_config`**: Custom MultiQC config file.
- **`multiqc_title`**: Title for the MultiQC report.
- **`multiqc_logo`**: Custom logo for MultiQC.
- **`max_multiqc_email_size`**: Maximum email size for MultiQC reports (`25MB`).
- **`multiqc_methods_description`**: Description of methods used in MultiQC.
### **Execution Profiles**
#### **Debugging**
- **Profile**: `debug`
- **Description**: Enables debugging logs and keeps temporary files.
#### **Containerized Execution**
- **Profile**: `docker`
- Runs the pipeline using Docker.
- **Profile**: `singularity`
- Uses Singularity for containerization.
#### **Cluster Execution**
- **Profile**: `slurm`
- Runs jobs on a Slurm cluster.
## Pipeline output
The pipeline generates several output files, with the most important being the HTML report. This report provides a detailed summary of the analysis results, including the presence of majority and minority variants of mutations as defined by the HIVdb Stanford database.
### Key Outputs
- **Coverage Graphs**: Samtools coverage graphs for each chosen virus per sample
- **Location**: `generate_coverage/`
- **FastQC Reports**: Quality control reports generated by FastQC for the input sequencing data.
- **Location**: `fastqc/`
- **Kraken2 Classification**: Files for kraken2 classification including kraken2 report, classified and unclassified reads
- **Location**: `kraken2_kraken2/`
- **Minimap2 Alignment**: Minimap2 alignment files for each sample including index files
- **Location**: `minimap2_align/`
- **Split Reads**: Split reads for each sample by the viral genome of choice
- **Location**: `split_by_genome/`
- **MultiQC Report**: MultiQC report summarizing the results of the pipeline
- **Location**: `multiqc/`
- **Pipeline Info**: Information about the pipeline run including the command and parameters used
- **Location**: `pipeline_info/`
## Credits
nf-core/seqcoverage was originally written by Magdalena Dabrowska.
We thank the following people for their extensive assistance in the development of this pipeline:
## Contributions and Support
If you would like to contribute to this pipeline, please see the [contributing guidelines](.github/CONTRIBUTING.md).
For further information or help, don't hesitate to get in touch on the [Slack `#seqcoverage` channel](https://nfcore.slack.com/channels/seqcoverage) (you can join with [this invite](https://nf-co.re/join/slack)).
## Citations
An extensive list of references for the tools used by the pipeline can be found in the [`CITATIONS.md`](CITATIONS.md) file.
You can cite the `nf-core` publication as follows:
> **The nf-core framework for community-curated bioinformatics pipelines.**
>
> Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.
>
> _Nat Biotechnol._ 2020 Feb 13. doi: [10.1038/s41587-020-0439-x](https://dx.doi.org/10.1038/s41587-020-0439-x).
