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gatk4 RNA variant calling pipeline

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## Introduction

**nf-core/rnavar** is a bioinformatics pipeline for RNA variant calling analysis following GATK4 best practices.

## Pipeline summary

1. Merge re-sequenced FastQ files ([`cat`](http://www.linfo.org/cat.html))
2. Read QC ([`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/))
3. (Optionally) Extract UMIs from FASTQ reads ([`UMI-tools`](https://github.com/CGATOxford/UMI-tools))
4. (Optionally) HLATyping from FASTQ reads ([`Seq2HLA`](https://github.com/TRON-Bioinformatics/seq2HLA))
5. Align reads to reference genome ([`STAR`](https://github.com/alexdobin/STAR))
6. Sort and index alignments ([`SAMtools`](https://sourceforge.net/projects/samtools/files/samtools/))
7. Duplicate read marking ([`Picard MarkDuplicates`](https://gatk.broadinstitute.org/hc/en-us/articles/360037052812-MarkDuplicates-Picard))
8. Scatter one interval-list into many interval-files ([`GATK4 IntervalListTools`](https://gatk.broadinstitute.org/hc/en-us/articles/4409917392155-IntervalListTools-Picard-))
9. Splits reads that contain Ns in their cigar string ([`GATK4 SplitNCigarReads`](https://gatk.broadinstitute.org/hc/en-us/articles/4409917482651-SplitNCigarReads))
10. Estimate and correct systematic bias using base quality score recalibration ([`GATK4 BaseRecalibrator`](https://gatk.broadinstitute.org/hc/en-us/articles/4409897206043-BaseRecalibrator), [`GATK4 ApplyBQSR`](https://gatk.broadinstitute.org/hc/en-us/articles/4409897168667-ApplyBQSR))
11. Convert a BED file to a Picard Interval List ([`GATK4 BedToIntervalList`](https://gatk.broadinstitute.org/hc/en-us/articles/4409924780827-BedToIntervalList-Picard-))
12. Call SNPs and indels ([`GATK4 HaplotypeCaller`](https://gatk.broadinstitute.org/hc/en-us/articles/4409897180827-HaplotypeCaller))
13. Merge multiple VCF files into one VCF ([`GATK4 MergeVCFs`](https://gatk.broadinstitute.org/hc/en-us/articles/4409924817691-MergeVcfs-Picard-))
14. Index the VCF ([`Tabix`](http://www.htslib.org/doc/tabix.html))
15. Filter variant calls based on certain criteria ([`GATK4 VariantFiltration`](https://gatk.broadinstitute.org/hc/en-us/articles/4409897204763-VariantFiltration))
16. Annotate variants ([`BCFtools Annotate`](https://samtools.github.io/bcftools/bcftools.html), [`snpEff`](https://pcingola.github.io/SnpEff/se_introduction/), [Ensembl VEP](https://www.ensembl.org/info/docs/tools/vep/index.html))
17. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks ([`MultiQC`](http://multiqc.info/), [`R`](https://www.r-project.org/))

### Summary of tools and version used in the pipeline

| Tool       | Version |
| ---------- | ------- |
| BCFTools   | 1.22    |
| BEDTools   | 2.31.1  |
| cat        | 9.5     |
| EnsemblVEP | 115.2   |
| FastQC     | 0.12.1  |
| GATK       | 4.6.2.0 |
| GffRead    | 0.12.7  |
| HTSlib     | 1.21    |
| Mosdepth   | 0.3.10  |
| MultiQC    | 1.33    |
| Picard     | 3.4.0   |
| SAMtools   | 1.22.1  |
| Seq2HLA    | 2.3     |
| SnpEff     | 5.3.0a  |
| STAR       | 2.7.11b |
| Tabix      | 1.21    |
| UMI-tools  | 1.1.6   |

## Usage

> [!NOTE]
> If you are new to Nextflow and nf-core, please refer to [this page](https://nf-co.re/docs/get_started/environment_setup/overview) on how to set-up Nextflow. Make sure to [test your setup](https://nf-co.re/docs/get_started/run-your-first-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_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz
```

Each row represents a fastq file (single-end) or a pair of fastq files (paired end).

Now, you can run the pipeline using:

```console
nextflow run nf-core/rnavar -profile <docker/singularity/podman/shifter/charliecloud/conda/institute> --input samplesheet.csv  --outdir <OUTDIR> --genome GRCh38
```

> [!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/running/run-pipelines#using-parameter-files).

For more details and further functionality, please refer to the [usage documentation](https://nf-co.re/rnavar/usage) and the [parameter documentation](https://nf-co.re/rnavar/parameters).

## Pipeline output

To see the results of an example test run with a full size dataset refer to the [results](https://nf-co.re/rnavar/results) tab on the nf-core website pipeline page.
For more details about the output files and reports, please refer to the
[output documentation](https://nf-co.re/rnavar/output).

## Credits

rnavar was originally written by Praveen Raj and Maxime U Garcia at [The Swedish Childhood Tumor Biobank (Barntumörbanken), Karolinska Institutet](https://ki.se/forskning/barntumorbanken).
Nicolas Vannieuwkerke at [CMGG](https://www.cmgg.be/en/) later joined and helped with further development (1.1.0 and forward).

Maintenance is now lead by Maxime U Garcia (before at [Seqera](https://seqera.io), now at [National Genomics Infrastructure](https://ngisweden.scilifelab.se/))

Main developers:

- [Maxime U Garcia](https://github.com/maxulysse)
- [Nicolas Vannieuwkerke](https://github.com/nvnieuwk)

We thank the following people for their extensive assistance in the development of this pipeline:

- [Harshil Patel](https://github.com/drpatelh)
- [Nicolás Schcolnicov](https://github.com/nschcolnicov)
- [Ömer An](https://github.com/bounlu)
- [Phil Ewels](https://github.com/ewels)
- [Praveen Raj](https://github.com/praveenraj2018)
- [Sarah Maman](https://github.com/SarahMaman)

## Contributions and Support

If you would like to contribute to this pipeline, please see the [contributing guidelines](docs/CONTRIBUTING.md).

For further information or help, don't hesitate to get in touch on the [Slack `#rnavar` channel](https://nfcore.slack.com/channels/rnavar) (you can join with [this invite](https://nf-co.re/join/slack)).

## Citations

If you use nf-core/rnavar for your analysis, please cite it using the following doi: [10.5281/zenodo.6669636](https://doi.org/10.5281/zenodo.6669636)

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).

rnavar

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