createivf

The createivf uses snakemake to automate the generation of breakpoint read support from Nanopore sequenced reads.

Installation

Download the package:

git clone https://github.com/simpsonlab/createivf.git

The pipeline uses conda to manage dependencies:

conda env create -f createivf/workflows/envs/environment.yaml

Once the conda dependencies have completed installing, activate the conda environment:

conda activate createivf

Basecalling is performed with guppy. A Nvidia GPU is required on the server to speed up the process of generating FASTQ files using the CUDA set of libraries.

Usage

To run the workflow, individual run steps should be executed followed by running breakpoints which occur on the merged run data for a single sample.

Configuration

To configure the pipeline, a config.yaml file needs to be created with various parameters defined:

run_name: "run_name"
run_root: "/path/to/ont/runs"
analysis_root: "/path/to/analysis/root/directory"
sample: "samplename"
reference: "/path/to/reference/genome/fasta"
basecaller: "guppy_basecaller"
guppy_config: "dna_r9.4.1_450bps_hac.cfg"
num_callers: "8"
gpu_runner_per_device: "4"
chunks_per_runner: "512"
device: "'cuda:0 cuda:1'"
fast5_dir: "/path/to/fast5/files"
metadata: "/path/to/sample_cytogenetics.tsv"
cytobands: "/path/to/hg38.cytoBand.composite.txt"

The guppy_basecaller is not available as part of the conda package and must be installed separately. The following parameters are set for the GPU version of guppy:

• basecaller
• guppy_config
• num_callers
• gpu_runner_per_device
• chunks_per_runner
• device

There are two required files for running breakpoint analysis:

• metadata which is a tab separated file containing sample, band for region1 and band for region2
• cytobands which contains the genomic regions and their corresponding cytogenetic bands

The cytobands file that is downloaded from the UCSC site requires an additional column to work with the analysis pipeline. The script workflow/scripts/fix_cytoband_file.py can be used to append the additional column.

Run the workflow

The basecaller uses guppy and a GPU to convert FAST5 files to FASTQ files.

snakemake -s /path/to/createivf/workflow/Snakefile --cores 1 all_basecall

Once basecalling has completed, the remainder of the pipeline can be executed on a standard server. Individual runs can be executed using the following to generate BAM files for each run for a given sample:

snakemake -s /path/to/createivf/workflow/Snakefile --cores 8 all_map

Once alignment has completed, the all_breakpoint rule will execute the merging of individual runs per sample into a single sample BAM file and run breakpoint detection on the merged file:

snakemake -s /path/to/createivf/workflow/Snakefile --cores 8 all_breakpoint

This will merge each run BAM file for a given sample into a single sample BAM file and breakpoint read information:

sample/merged.sorted.bam
sample/bp1.reads
sample/bp2.reads

Credits and Acknowledgements

The script (abyss-fac.pl) to generate read stats was obtained from:

https://github.com/bcgsc/abyss/blob/master/bin/abyss-fac.pl

The cytogenetic band file was obtained from UCSC. A column was appended to the file that prefixed the band with chr.

License

MIT