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# Post-gatk-nf
# post-gatk-nf

Pipeline for simple popgen analysis.
This pipeline performs population genetics analyses (such as identifying shared haplotypes and divergent regions) at the isotype level. The VCFs output from this pipeline are used within the lab and also released to the world via CeNDR.

## Typical use for debugging:

```
nextflow main.nf --debug
# Pipeline overview
```
### Typical use for new vcf:

```
nextflow main.nf --vcf path_to_vcf.vcf.gz --sample_sheet path_to_sample_sheet.tsv --species <species>
```
### Parameters
* * * * ** * * * * * * * * * * * * * *
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parameters description Set/Default
========== =========== ========================
--debug Use --debug to indicate debug mode (optional)
--vcf Hard filtered vcf to calculate variant density (required)
--vcf_folder Folder to hard and soft filtered vcf (required)
--sample_sheet TSV with column iso-ref strain, bam, bai (no header) (required)
--species Species: 'c_elegans', 'c_tropicalis' or 'c_briggsae' c_elegans
--output Output folder name. popgen-date (in current folder)
### Overview
```

![Pipeline-overview](img/post-gatk.drawio.svg)

## Software Requirements

* The latest update requires Nextflow version 20.0+. On QUEST, you can access this version by loading the `nf20` conda environment prior to running the pipeline command:

```
module load python/anaconda3.6
source activate /projects/b1059/software/conda_envs/nf20_env
```

Alternatively you can update Nextflow by running:

```
nextflow self-update
```

**This pipeline currently only supports analysis on Quest, cannot be run locally**


# Usage

**For more info about running Nextflow pipelines in the Andersen Lab, check out [this page](http://andersenlab.org/dry-guide/2021-12-01/quest-nextflow/)**

## Testing on Quest

*This command uses a test dataset*

```
nextflow run andersenlab/post-gatk-nf --debug
```

## Running on Quest

You should run this in a screen session.

### Profiles

There are now three ways to run this pipeline:

1. `-profile standard` (default): runs original processes including subseting VCF and divergent and haplotype calls.
- sample_sheet, vcf_folder, (species)
2. `-profile pca`: does not run the original post-gatk processes, only the PCA analysis. *Note: requires different parameters*
- snv_vcf, species, anc, eigen_ld, pops
3. `-profile standard --pca`: runs all processes including subseting VCF, divergent and haplotype calls, PCA analysis of isotypes. *Requires additional parameters relating to PCA*
- sample_sheet, vcf_folder, species, anc, eigen_ld
- Note: the `-profile standard` is optional, just adding the `--pca` param is enough.

```
nextflow run andersenlab/post-gatk-nf --vcf <path_to_vcf> --sample_sheet <path_to_sample_sheet>
```

# Parameters

## --debug

You should use `--debug true` for testing/debugging purposes. This will run the debug test set (located in the `test_data` folder).

![Overview of post-gatk-nf](https://github.com/AndersenLab/post-gatk-nf/blob/main/img/post-gatk-nf-flow.png?raw=true)
For example:

```
nextflow run andersenlab/post-gatk-nf --debug -resume
```

Using `--debug` will automatically set the sample sheet to `test_data/sample_sheet.tsv`

## --sample_sheet

A custom sample sheet can be specified using `--sample_sheet`. The `sample sheet` is generated from the sample sheet used as input for [`wi-gatk-nf`](https://github.com/AndersenLab/wi-gatk) with only columns for strain, bam, and bai subsetted. **Make sure to remove any strains that you do not want to include in this analysis.** (*i.e. subset to keep only ISOTYPE strains*)

Remember that in `--debug` mode the pipeline will use the sample sheet located in `test_data/sample_sheet.tsv`.

!!! Important
There is no header for the sample sheet!

The `sample sheet` has the following columns:

* __strain__ - the name of the strain
* __bam__ - name of the bam alignment file
* __bai__ - name of the bam alignment index file

!!! Note
As of 20210501, bam and bam.bai files for all strains of a particular species can be found in one singular location: `/projects/b1059/data/{species}/WI/alignments/` so there is no longer need to provide the location of the bam files.


## --vcf_folder

Path to the **folder** containing both the hard-filtered and soft-filtered vcf outputs from [`wi-gatk`](https://github.com/AndersenLab/wi-gatk). VCF should contain **ALL** strains, the first step will be to subset isotype reference strains for further analysis.

!!! Note
This should be the **path to the folder**, we want to isotype-subset both hard and soft filtered VCFs. For example: `--vcf_folder /projects/b1059/projects/Katie/wi-gatk/WI-20210121/variation/`

### --species (optional)

__default__ = c_elegans

Options: c_elegans, c_briggsae, or c_tropicalis

### --snv_vcf (pca profile)

File path to SNV-filtered VCF

### --pops (pca profile)

Strain list to filter VCF for PCA analysis. No header:

| AB1 |
| --- |
| CB4856 |
| ECA788 |

!!!Note
If you run the standard profile with pca this file will be automatically generated to include all isotypes.

### --eigen_ld (pca)

LD thresholds to test for PCA. Can provide multiple with `--eigen_ld 0.8,0.6,0.4`

### --anc (pca)

Ancestor strain to use for PCA.

*Note: Make sure this strain is in your VCF*

### --output (optional)

__default__ - `popgen-YYYYMMDD`

A directory in which to output results. If you have set `--debug true`, the default output directory will be `popgen-YYYYMMDD-debug`.

# Output

```
├── ANNOTATE_VCF
│   ├── ANC.bed.gz
│   ├── ANC.bed.gz.tbi
│   ├── Ce330_annotated.vcf.gz
| └── Ce330_annotated.vcf.tbi
├── EIGESTRAT
│   └── LD_{eigen_ld}
│      ├── INPUT_FILES
│ │ └── *
│      ├── OUTLIER_REMOVAL
│ │ ├── eigenstrat_outliers_removed_relatedness
│ │ ├── eigenstrat_outliers_removed_relatedness.id
│ │ ├── eigenstrat_outliers_removed.evac
│ │ ├── eigenstrat_outliers_removed.eval
│ │ ├── logfile_outlier.txt
│ │ └── TracyWidom_statistics_outlier_removal.tsv
│      └── NO_REMOVAL
│ └── same as outlier_removal
├── pca_report.html
├── divergent_regions
│   ├── Mask_DF
│   │   └── [strain]_Mask_DF.tsv
| └── divergent_regions_strain.bed
├── haplotype
│   ├── haplotype_length.pdf
│   ├── sweep_summary.tsv
│   ├── max_haplotype_genome_wide.pdf
│   ├── haplotype.pdf
│   ├── haplotype.tsv
│   ├── [chr].ibd
│   └── haplotype_plot_df.Rda
├── tree
│   ├── WI.{date}.hard-filter.isotype.min4.tree
│   ├── WI.{date}.hard-filter.isotype.min4.tree.pdf
│   ├── WI.{date}.hard-filter.min4.tree
│   └── WI.{date}.hard-filter.min4.tree.pdf
├── NemaScan
│   ├── strain_isotype_lookup.tsv
│   ├── div_isotype_list.txt
│   ├── haplotype_df_isotype.bed
│   ├── divergent_bins.bed
│   └── divergent_df_isotype.bed
└── variation
   ├── WI.{date}.small.hard-filter.isotype.vcf.gz
  ├── WI.{date}.small.hard-filter.isotype.vcf.gz.tbi
   ├── WI.{date}.hard-filter.isotype.SNV.vcf.gz
   ├── WI.{date}.hard-filter.isotype.SNV.vcf.gz.tbi
   ├── WI.{date}.soft-filter.isotype.vcf.gz
   ├── WI.{date}.soft-filter.isotype.vcf.gz.tbi
   ├── WI.{date}.hard-filter.isotype.vcf.gz
   └── WI.{date}.hard-filter.isotype.vcf.gz.tbi
```

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