SRAtoVCF

A pipeline for converting short-read DNA data from SRA into VCF files

Prerequisite

• Pyhthon 3.6+
• edirect 12.5
• sra-tools 3.0.1
• Trimmomatic 0.39
• FastQC 0.12.1
• bwa-mem2 2.2.1
• samtools 1.17
• gatk 4.4.0.0
• cwltool 1.0+

Using Docker container

• Docker

Using Singularity container

• Singularity or SingularityCE or Apptainer

On HPC

• conda

The input parameter in the workflow (main.cwl)

The table shows the parameter settings for workflow.

Input	Description
ref_genome	File path. Reference genome
SRA_acession	String. SRA accession for the data you want to process.
input_adapters_file	File path. The file contains the adapter sequence for Trimmomatic to trim.
removeDuplicates	Boolean. Whether to remove duplicates.
ReadGroupID	String. Read group identifier. ID must be unique.
ReadGroupLibrary	String. DNA preparation library identifier.
ReadGroupPlatformUnit	String. Platform Unit.
ReadGroupPlatform	String. Platform/technology used to produce the read.
ReadGroupSampleName	String. The name of the sample sequenced in this read group.
CREATE_INDEX	Boolean. Whether or not to create an index file for the BAM file after adding readgroup information.
creat_variant_index	Boolean. Whether to create index file for HaplotypeCaller output.
select_type_INDEL	String. To select the INDEL form the HaplotypeCaller output vcf file.
filter_expression_SNP	String. The criteria for filtering SNPs.
filter_expression_INDEL	String. The criteria for filtering INDELs.
exclude_filtered	Boolean. Whether to remove duplicates.
creat_index	Boolean. Whether to create index file for the SortVcf vcf output.

*The parameters to create an index file should not be changed, and it should keep with TRUE.

Running on HPC (Cere)

Step 1. Set up conda environment

First, we need to install the runner for the pipeline called cwltool to compile the pipeline. Therefore we have to set up a conda environment.

Load the latest version minconda module and creat an environment called cwltool.

module load miniconda
conda create --name cwltool

Next time just has to activate the environment. To activate the environment:

conda activate cwltool

Now we are inside the cwltool environment and can install the cwltool.

conda install -c conda-forge cwltool
conda install -c conda-forge cwl-utils #dependency

Step 2. Download the singularity

Due to running on HPC, we need to use the singularity rather than the docker.

First, create a folder in your working directory to store the singularities that needed for the workflow and run the following code:

module load apptainer (singularityCE #or singularity (depend on the HPC))
cwl-docker-extract --singularity DIRECTORY ../workflow/main.cwl
export CWL_SINGULARITY_CACHE=your/sifDirectory/Path

If your computing node has internet, you can skip the cwl-docker-extract step and just load the apptainer (singularityCE) module.

There are a few things to set up when using singularity.

1. Set CWL_SINGULARITY_CACHE. to the folder containing all .sif files. This will make the cwl runtime automatically fetch the singularity container with this path.
2. Set APPTAINER_CACHEDIR. The default path is under the home directory, but the process will generate many files, resulting in insufficient storage space in the home directory. Therefore, this path needs to be in the project directory. (Using singularityCE or singularity APPTAINER_CACHEDIR -> SINGULARITY_CACHEDIR)

Step3. Run the workflow

Download the organisum related SRA metadata on NCBI

cwltool --singularity bioproject_meta_download.cwl --species_name Apis mellifera --output_file bee.tsv

Prepared the reference genome index file

cwltool --singularity [path to]/workflow/prep_ref.cwl --ref_genome PATH-to-ReferenceFile

Processing the specific SRA file to vcf

cwltool --singularity [path to]/SRAtoVCF/workflow/main.cwl ../SRAtoVCF/workflow/main.yml

Running on local PC

Using Docker change the code to:

#original cwltool --singularity ....
cwltool --no-match-user ...

Using local software (already have all the tools on local PC) change the code to:

#original cwltool --singularity ....
cwltool --no-container ...

Build a shared conda environment and singularity image files

For multiple users to run the pipeline on HPC, it can create a shared conda environment for all the users. It can save some storage space.

For conda environment Ref.

Step 1. Create a conda environment in a shared directory.

conda create --prefix=SharedDirectoryPath/envname

Step 2. Set up the environment variable and module in conda environment

Create the file for the environment setting.

cd SharedDirectoryPath/envname
mkdir -p ./etc/conda/activate.d
mkdir -p ./etc/conda/deactivate.d
touch ./etc/conda/activate.d/env_vars.sh
touch ./etc/conda/deactivate.d/env_vars.sh

Edit the environment setting when using the environment (./etc/conda/activate.d/env_vars.sh) as follow:

#!/bin/sh
module load apptainer
export CWL_SINGULARITY_CACHE=/project/nal_genomics/shared_files/SRAtoVCF/sif

If there are other settings you want to add, you can modify the content yourself. Edit the environment setting (./etc/conda/deactivate.d/env_vars.sh) as follow:

#!/bin/sh
module unload apptainer
unset CWL_SINGULARITY_CACHE

It will reset the setting when we leave the conda environment.

Step 3. install cwltool

conda activate SharedDirectoryPath/envname

Now we are inside the cwltool environment and can install the cwltool.

conda install -c conda-forge cwltool
conda install -c conda-forge cwl-utils #dependency

You can check if the environment settings are right:

module list
#output
#Currently Loaded Modules:
  #1) miniconda/4.12.0   2) apptainer/1.2.2
echo $CWL_SINGULARITY_CACHE
#output
#/PathtoSharedSiFFolder

Step 4. Leave conda environment

conda deactivate

For shared singularity image files

Step 1. Create a folder to store the singularity image files (sifs) in a shared directory and pull the sifs.

cd PathtoStoretheSingularityImageFiles
mkdir sif
cd sif
cwl-docker-extract --singularity . Pathto/workflow/main.cwl

Step 2. Set the environment variable in conda env_var.sh file.

Just like the conda environment Step 2. However, the APPTAINER_CACHEDIR still have to be setted by each user, because it has to be unique to user. Ref.

Example

In the test example, we utilize the yeast data as an example. The example folder contains the reference sequence of R64, and the processed SRA accession is SRR23631020.

First, create the index file for the reference genome. (Remember to complete the setting first and run the command in the example directory.)

 cwltool --singularity ../workflow/prep_ref.cwl --ref_genome GCF_000146045.2_R64_genomic.fna

Second, generate the example YAML file for the main.cwl (main variant calling pipeline) by generate_example_yml.sh.

sh generate_example_yml.sh.
#it will print out Enter git clone path:
#if your git clone path is /path/to/git/clone you should just enter it.

Finally, run the main.cwl by following the command.

cwltool --singularity ../workflow/main.cwl example.yaml