annotation-nf¶

The annotation-nf pipeline performs variant annotation for the VCF at the isotype level using both SnpEff and BCFtools/csq (BCSQ).

Pipeline overview¶

-------------    
ANNOTATION-NF
-------------

nextflow andersenlab/annotation-nf --debug

nextflow andersenlab/annotation-nf --vcf=hard-filtered.vcf --species=c_elegans --divergent_regions=divergent_regions_strain.bed

    parameters           description                                              Set/Default
    ==========           ===========                                              ========================
    --debug              Set to 'true' to test                                    ${params.debug}
    --species            Species: 'c_elegans', 'c_tropicalis' or 'c_briggsae'     ${params.species}
    --vcf                hard filtered vcf to calculate variant density           ${params.vcf}
    --divergent_regions  (Optional) Divergent region bed file                     ${params.divergent_regions}
    --reference          Reference used based on species and project              ${params.reference}
    --output             (Optional) output folder name                            ${params.output}

    username                                                                      ${"whoami".execute().in.text}

    HELP: http://andersenlab.org/dry-guide/pipeline-annotation-nf

Software Requirements¶

• The latest update requires Nextflow version 20.0+. On QUEST, you can access this version by loading the nf20_env 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

Usage¶

For more info about running Nextflow pipelines in the Andersen Lab, check out this page

Testing on Quest¶

This command uses a test dataset

nextflow run andersenlab/annotation-nf --debug

Running on Quest¶

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

You should run this in a screen session.

nextflow run andersenlab/annotation-nf --vcf <path_to_vcf> --species <species> --divergent_regions <path_to_file>

Parameters¶

--debug¶

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

For example:

nextflow run andersenlab/annotation-nf --debug

--vcf¶

Path to the hard-filter, isotype VCF (output from post-gatk-nf)

--species¶

Choose from c_elegans, c_briggsae, or c_tropicalis. Species will specifiy a default reference genome. You can select a different one if you prefer (see below)

--divergent_regions¶

This is the divergent_regions_strain.bed file output from the post-gatk-nf pipeline. This file is used to add a column to the flat file if the variant is within a divergent region. Currently, C. elegans is the only species with divergent regions, if running for another species, do not provide a divergent_regions file and the pipeline will ignore it.

--reference, --project, --ws_build (optional)¶

By default, the reference genome is set by the species parameter. If you don't want to use the default, you could change the project and/or ws_build. As long as the genome is in the proper location on quest (for more, see the genomes-nf pipeline), this will work. Alternatively, you could provide the path to a reference of your choice.

Defaults: - C. elegans - /projects/b1059/data/c_elegans/genomes/PRJNA13758/WS276/c_elegans.PRJNA13758.WS276.genome.fa.gz - C. briggsae - /projects/b1059/data/c_briggsae/genomes/QX1410_nanopore/Feb2020/c_briggsae.QX1410_nanopore.Feb2020.genome.fa.gz - C. tropicalis - /projects/b1059/data/c_tropicalis/genomes/NIC58_nanopore/June2021/c_tropicalis.NIC58_nanopore.June2021.genome.fa.gz

--ncsq_param (optional)¶

This parameter is necessary for correct annotation using BCSQ for variants with many different annotations (like found in divergent regions). In 20210121 we found that the default value of 224 was sufficient, but as more strains are added this number might need to increase. If there is an issue, you should see a warning error from BCFtools and they should suggest what to change this parameter to.

Output¶

├── strain_vcf
│   ├── {strain}.{date}.vcf.gz
│   └── {strain}.{date}.vcf.gz.tbi
└── variation
    ├── WI.{date}.hard-filter.isotype.snpeff.vcf.gz
    ├── WI.{date}.hard-filter.isotype.snpeff.vcf.gz.tbi
    ├── snpeff.stats.csv
    ├── WI.{date}.hard-filter.isotype.bcsq.vcf.gz
    ├── WI.{date}.hard-filter.isotype.bcsq.vcf.gz.tbi
    ├── WI.{date}.hard-filter.isotype.bcsq.vcf.gz.stats.txt 
    └── WI.{date}.strain-annotation.bcsq.tsv

Data storage¶

Cleanup¶

Once the pipeline has complete successfully and you are satisfied with the results, the final data can be moved to their final storage place on Quest accordingly:

• Both the strain_vcf and the variation folders can be moved to /projects/b1059/data/{species}/WI/variation/{date}/vcf
• If applicable, all snpeff .bed files (HIGH, LOW, MODERATE, etc.) can be moved to /projects/b1059/data/{species}/WI/variation/{date}/tracks/ (As of 20210901 this is no longer being produced for CeNDR)

Updating CeNDR¶

Check out the CeNDR page for more information about updating a new data release for CeNDR.

Updating NemaScan¶

Once a new CeNDR release is ready, it is important to also update the genome-wide association mapping packages to ensure users can appropriately analyze data from new strains as well as old strains. Here is a list of things that need to be updated:

• The default vcf should be changed to the newest release date (i.e. from 20200815 to 20210121). Users will still have the option to use an earlier vcf.
• Ensure strain annotation flat file is stored in the proper file location to be accessed, both on QUEST and on GCP for CeNDR/local users.