post-gatk-nf

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 CaeNDR.

Pipeline overview

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    parameters              description                                            Set/Default
    ==========              ===========                                            ========================
    --debug                 Use --debug to indicate debug mode                     (optional)
    --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)
    --postgatk              Run post-GATK steps                                    true
    --pca                   Run PCA analysis                                       false

Software Requirements

• The latest update requires Nextflow version 24+. On Rockfish, you can access this version by loading the nf24_env conda environment prior to running the pipeline command:

module load python/anaconda
source activate /data/eande106/software/conda_envs/nf24_env

Usage

Note: if you are having issues running Nextflow or need reminders, check out the Nextflow page.

Testing on Rockfish

This command uses a test dataset

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

Running on Rockfish

You should run this in a screen or tmux session.

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

Parameters

-profile

There are three configuration profiles for this pipeline.

• rockfish - Used for running on Rockfish (default).
• quest - Used for running on Quest.
• local - Used for local development.

Note

If you forget to add a -profile, the rockfish profile will be chosen as default

--debug

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

For example:

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

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

Debugging for PCA:

You can debug the PCA pipeline with the following data/command:

nextflow run -latest andersenlab/post-gatk-nf --vcf ./test_data/WI.20220404.hard-filter.vcf.gz --species c_elegans --sample_sheet ./test_data/sample_sheet_2.tsv --eigen_ld 0.8,0.6 --anc XZ2019 --pca true -resume

--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 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. 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/ or --vcf_folder /projects/b1059/data/c_elegans/WI/variation/20210121/vcf/

--species (optional)

default = c_elegans

Options: c_elegans, c_briggsae, or c_tropicalis

PCA

The PCA steps can be run either with the full pipeline or independently. To run only PCA use -- pca true --postgatk false

The input VCF is filtered to bi-alleleic snps with no missing genotypes. A LD filtering threshold is required and LD filtering is performed using plink. You can also filter for singletons by specifying the --singletons

PCA is performed using smartPCA. Parameters to control outlier threshold or removal iterations are desribed below.

--postgatk (default: true)

Set to false to skip post-gatk steps

--pca (default: false)

Set to true to run PCA analysis

--snv_vcf (pca)

File path to SNV-filtered VCF

--pops (pca)

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, 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

Relevant Docker Images

• andersenlab/postgatk (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/postgatk.Dockerfile or .github/workflows/build_postgatk_docker.yml GitHub actions will create a new docker image and push if successful
• andersenlab/tree (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/tree.Dockerfile or .github/workflows/build_tree_docker.yml GitHub actions will create a new docker image and push if successful
• andersenlab/pca (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/pca.Dockerfile or .github/workflows/build_pca_docker.yml GitHub actions will create a new docker image and push if successful
• andersenlab/r_packages (link): Docker image is created manually, code can be found in the dockerfile repo.

Make sure that you add the following code to your ~/.bash_profile. This line makes sure that any singularity images you download will go to a shared location on /vast/eande106 for other users to take advantage of (without them also having to download the same image).

# add singularity cache
export SINGULARITY_CACHEDIR='/vast/eande106/singularity/'