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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.
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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
- The latest update requires Nextflow version 24+. On Rockfish, you can access this version by loading the
nf24_envconda environment prior to running the pipeline command:
module load python/anaconda
source activate /data/eande106/software/conda_envs/nf24_env
Note: if you are having issues running Nextflow or need reminders, check out the Nextflow page.
This command uses a test dataset
nextflow run -latest andersenlab/post-gatk-nf --debug
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>
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
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
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
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.
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/
default = c_elegans
Options: c_elegans, c_briggsae, or c_tropicalis
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.
Set to false to skip post-gatk steps
Set to true to run PCA analysis
File path to SNV-filtered VCF
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.
LD thresholds to test for PCA. Can provide multiple with --eigen_ld 0.8,0.6,0.4
Ancestor strain to use for PCA.
Note
Make sure this strain is in your VCF
default - popgen-YYYYMMDD
A directory in which to output results. If you have set --debug, the default output directory will be popgen-YYYYMMDD-debug.
├── 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
andersenlab/postgatk(link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made toenv/postgatk.Dockerfileor.github/workflows/build_postgatk_docker.ymlGitHub actions will create a new docker image and push if successfulandersenlab/tree(link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made toenv/tree.Dockerfileor.github/workflows/build_tree_docker.ymlGitHub actions will create a new docker image and push if successfulandersenlab/pca(link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made toenv/pca.Dockerfileor.github/workflows/build_pca_docker.ymlGitHub actions will create a new docker image and push if successfulandersenlab/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/'