K-mer based variant detection. v1.0.0-gamma.
Developed by Stephanie Georges, MS
Based on the thesis project of Andrew Farrell, PhD
Marth Lab, University of Utah
Publication in preparation
For questions and feature requests, please contact [email protected]
RUFUS is a reference-free, K-mer based variant detection algorithm, for short-read DNA sequence data. RUFUS is intended to run on a high performance computing (HPC) cluster with singularity installed. At a high level, you'll need to download the pre-built singularity container (detailed below) and set up a batch script corresponding to your resource manager. If your HPC system uses SLURM, you can utilize the provided helper functions to create your SBATCH scripts (detailed below).
RUFUS currently supports a single subject sample, multiple control samples, and only accepts GRCh38 as a reference genome. The samples must be in BAM format (though may be unaligned). The reference genome must be in FASTA format, and must be indexed by BWA. If the BWA indexes are not detected in the same directory as the reference genome, RUFUS will create them.
RUFUS has two stages: a variant calling stage, and a post-processing stage. Separation of the stages is necessary because the calling stage may be run in a windowed fashion, requiring multiple parallel RUFUS jobs over all of the windows. The combination stage must wait to proceed until all calling jobs are complete. Algorithmic runtime increases roughly linearly with sample coverage. Generally with whole-genome mode, a 100x sample run will take 1 day. Windowed mode completes significantly faster.
The pre-built RUFUS singularity container may be obtained from Zenodo. To download:
curl "https://zenodo.org/records/13871423/files/rufus_v1.0.0-gamma" -o rufus.sif
RUFUS requires the following data to run:
- A subject sample in BAM format (this may be unaligned)
- One or more control samples in BAM format (these may be unaligned)
- A reference fasta file (this must be indexed by BWA) - for use in reporting the called variants. It's recommended to provide the BWA indexes in the same data directory if you have them to save time creating them during the RUFUS run.
To create the BWA indexes, run the following commands:bwa index -a bwtsw {REFERENCE.fa} samtools faidx {REFERENCE.fa}
All of the above required files, as well as any optional ones, must be located in a single directory, which will be mounted to the singularity container.
RUFUS will, by default, output the following files in the same bound directory containing the input data:
- A VCF file containing the called variants
- A supplemental directory with:
- A pre-filtered VCF file
- A BAM file containing the raw reads containing the mutant kmers
- A BAM file containing the assembled contigs from the raw reads containing the mutant kmers
- A hash table containing the unique subject kmers and their counts
RUFUS has two execution stages:
- The calling stage, invoked by the following
singularity exec --bind {PATH_TO_LOCAL_DATA_DIR}:/mnt {PATH_TO_RUFUS_CONTAINER}/rufus.sif bash /opt/RUFUS/runRufus.sh [-s|--subject <arg>] [-r|--ref <arg>] [-t|--threads <arg>] [-k|--kmersize <arg>] [-m|--min <arg>] [-h|--help] [-c|<controls-1>] ... [-c|<controls-n>] ...OPTIONS
With the following usage:
Required Arguments:
-s,--subject: single bam file (may be unaligned) containing the subject of interest
-c,--controls: bam file (may be unaligned) for the sequence data of the control sample (can be used multiple times, e.g. -c control1 -c control2)
-r,--ref: file path to the desired reference file
-t,--threads: number of threads to use (min 3)
Optional Arguments:
-k,--kmersize: length of k-mer to use (defaults to 25)
-m,--min: overwrites the minimum k-mer depth count to call variant (defaults to 5)
-e,--exclude: Jhash file of kmers to exclude from mutation list (can be used multiple times, e.g. -e Jhash1 -e Jhash2)
-f,--refhash: Jhash file containing reference hashList
-h,--help: Print help
- The post-processing stage, invoked by the following
singularity exec --bind {PATH_TO_LOCAL_DATA_DIR}:/mnt {PATH_TO_RUFUS_CONTAINER}/rufus.sif bash /opt/RUFUS/post_process/post_process.sh [-w window_size] [-r reference] [-subject] [-c control1,control2,control3...] [-d source_dir]
With the following usage:
Required Arguments:
-w window_size The size of the window used in the RUFUS run
-r reference The reference used in the RUFUS run
-c controls The control bam files used in the RUFUS run
-s subject_file The name of the subject file: must be the same as that supplied to the RUFUS run
-d source_dir The source directory where the vcf(s) made by the calling stage are located
Optional Arguments:
-h help Print help message
The SLURM helper script automatically creates the two SLURM batch scripts necessary to run RUFUS on a SLURM-managed HPC cluster, as well as a bash script to execute them. To use:
- Execute the helper script (see full usage options below):
singularity exec {PATH_TO_RUFUS_CONTAINER}/rufus.sif bash /opt/RUFUS/singularity/setup_slurm.sh [-s subject] [-c control1,control2,control3...] [-b genome_build] [-a slurm_account] [-p slurm_partition] ...OPTIONS
- Then execute the generated bash script:
bash launch_rufus.sh
The full usage options for the helper script are as follows:
Required Arguments:
-d data_directory The directory containing the subject, control, and reference files to be used in the run
-s subject The subject sample of interest; must be located in data_directory
-c control(s) A single control or comma-delimited array of multiple controls; must be located in data_directory
-b genome_build The desired genome build; currently only supports GRCh38
-r reference The reference file matching the genome build; must be located in data_directory
-a slurm_account The account for the slurm job
-p slurm_partition The partition for the slurm job
-l slurm_job_array_limit The maximum amount of jobs slurm allows in an array
Optional Arguments:
-m kmer_depth_cutoff The amount of kMers that must overlap the variant to be included in the final call set
-w window_size The size of the windows to run RUFUS on, in units of kilabases (KB); allowed range between 500-5000; defaults to single run of entire genome if not provided
-f reference_hash: Jhash file containing reference kMer hash list
-x exclude_hash: Single or comma-delimited list of Jhash file(s) containing kMers to exclude from unique hash list
-y path_to_rufus_container If not provided, will look in current directory for rufus.sif
-z rufus_threads Number of threads provided to RUFUS; defaults to 36
-e email The email address to notify with slurm updates
-q slurm_job_queue_limit The maximum amount of jobs able to be ran at once; defaults to 20
-t slurm_time_limit The maximum amount of time to let the slurm job run; defaults to 7 days for full run, or one hour per window (DD-HH:MM:SS)
-h help Print usage
Notes on SLURM arguments:
This script utilizes SLURM arrays to batch RUFUS call runs and thus requires the SLURM job array limit to comply with user settings. To find your SLURM job array limit:
scontrol show config | grep "MaxArraySize"
To maximize parallelism, filling in the slurm job queue limit (-q) is recommended. You can find your limit by typing:
scontrol show config | grep "default_queue_depth"
singularity exec /home/my_container_path/rufus.sif bash /opt/RUFUS/singularity/setup_slurm.sh -d /home/my_data_dir/ -s subject.bam -c control_a.bam, control_b.bam -r GRCh38_reference.fa -a my-slurm-account -p my-slurm-partition -w 1000 -t "00:30:00" -m 5 -l 20 -z 36 -e "[email protected]" -f /home/my_container_path/
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