K-mer based variant detection.
Andrew Farrell PhD
Research Associate
Department of Human Genetics
USTAR Center for Genetic Discovery
Eccles Institute of Human Genetics
University of Utah School of Medicine
15 North 2030 East, Room 7140
Salt Lake City, UT 84112-5330
Email: [email protected]
http://marthlab.org/
If you get errors during the installation contact me at at [email protected] or submit an issue.
This project is still under development and not fully stable but free to use and any feedback is welcome.
Note: RUFUS is only supported on linux environments.
1) Download
git clone https://github.com/jandrewrfarrell/RUFUS.git
cd RUFUS
2) Build
mkdir bin
cd bin
cmake ../
make
** DEPENDENCE ON THE 4.9.2 COMPILER HAS BEEN REMOVED.**
RUFUS requires Samtools and bamtools. Please make sure that they are globally installed in your environment.
To make sure that RUFUS was successfully built, we provide users with a test run script to run RUFUS on a small test set of data with a small test reference, and default parameters. To test RUFUS, simply run
cd testRun
bash runTest.sh
NOTE: Make sure that runTest.sh is called directly from the testRun directory, or the testRun script will not be able to find the appropriate resources.
All data for this run is contained in the resources dir, and nothing needs to be provided by the user to test RUFUS.
At the end of a successfull test run, you should see a file named
testRun/Child.bam.generator.V2.overlap.hashcount.fastq.bam.vcf
This file should contain a single varient call. The call should look exactly as follows:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT ./Child.bam Mother.bam Father.bam
5:177630000 12896 X-DeNovo T G 25 PASS RN=NODE_Child.bam.generator.V2_0_L273_D22:10:12::MH0;MQ=60;cigar=273M;SB=0.454545;CVT=X;HD=-1_-1_-1_-1_-1_19_-1_19_19_-1_-1_-1_-1_-1_20_20_19_-1_-1_18_-1_18_-1_-1_18_-1_-1_;AO=19;VT=X GT:DP:RO:AO 0/1:39:20:19 0/0:23:23:0 0/0:23:23:0
If you were unable to reproduce this call, something went wront with the RUFUS install, and you should get the test run to work before proceeding further. If you are unable to reproduce a successfull test run, please contact me at [email protected]
RUFUS is primarily used to find mutations unique to a proband sample, that are not found in the control samples
Usage:
./runRufus.sh [-s|--subject <arg>] [-c|--controls][<controls-1>] ... [<controls-n>] [-t|--threads <arg>] [-k|--kmersize <arg>] [-r|--ref <arg>] [-m|--min <arg>]
[-h|--help]
-s,--subject: bam file containing the subject of interest (REQUIRED)
-c, --controls: bam files containing the control subjects (REQUIRED)
-t,--threads: number of threads to use (REQUIRED) (min 3)
-k,--kmersize: size of Kmer to use (REQUIRED)
-r,--ref: file path to the desired reference file to create VCF (REQUIRED)
-m,--min: overwrites the minimum k-mer count to call variant (OPTIONAL, Do not provide a min unless you are sure what you want)
-h,--help: HELP!!!!!!!!!!!!!!!
The command line should look something like this:
bash runRufus.sh --subject Child.bam --controls Mother.bam --controls Father.bam --kmersize 25 --threads 40 --ref human_reference_v37_decoys.fa
or
bash runRufus.sh -s Child.bam -c Mother.bam -c Father.bam -k 25 -t 40 -r human_reference_v37_decoys.fa
The flags can be provided any any order.
RUFUS can take any number control files (Must provide atleast one).
Each control Bam file requires a [-c|--controls] flag infront of the control file
For Example:
bash runRufus.sh -s tumorT1.bam -c tumorT0.bam -k 25 -t 40 -r human_reference_v37_decoys.fa
or
bash runRufus.sh -s Proband.bam -c Mother.bam -c Father.bam -c Sibling1.bam -c Sibling2.bam -k 25 -t 40 -r human_reference_v37_decoys.fa
We recommend a kmer size of 25, 40 threads, and to NOT provide RUFUS with the optional --min parameter
After RUFUS has identified reads containing mutant kmers, the reads must be aligned to a reference fasta file. Any fasta file can be used as a reference, as long as the fasta file has been indexed for BWA. If a fasta has been indexed by bwa, there will be reference files with the following extensions: pac, .ann, .abm, .bwt, sa. In order to prepare a reference fasta for bwa, simply type:
bwa index -a bwtsw reference.fa
samtools faidx reference.fa
This will produce the BWA index files, and the fasta file index respectively. Make sure that the bwa index files and the fasta index file are in the same directory as reference.fa
In order for RUFUS to run on a fresh Ubuntu build, all of the following packages must be installed:
General
sudo apt-get update
sudo apt-get install python
sudo apt-get install cmake
sudo apt-get install wget
GCC-4.9 (c/c++ compiler)
sudo apt-get install build-essential
sudo add-apt-repository ppa:ubuntu-toolchain-r/test
sudo apt-get install g++-4.9
zlib (file compression library)
sudo apt-get install zlib1g-dev
sudo apt-get install libbz2-dev
bzlib (bz2 file compression library)
sudo apt-get install libbz2-dev
sudo apt-get install liblzma-dev
bc (floating point precision library)
sudo apt-get install bc
Curse (terminal control library)
sudo apt-get install libncurses5-dev