Clone the repository with the following command:
$ git clone https://github.com/RoySimons96/Locksmith.git
Enter the repository by cd Locksmith.
Download the right version of bedtools
wget https://github.com/arq5x/bedtools2/releases/download/v2.30.0/bedtools.static.binary
Rename the file
mv bedtools.static.binary bedtools
Make sure the file has the executable permission
chmod a+x bedtools
Export the executable to PATH
export PATH=<Path to bedtools executable>:$PATH
Download the right version of mfeprimer
wget -c https://github.com/quwubin/MFEprimer-3.0/releases/download/v3.2.6/mfeprimer-3.2.6-linux-amd64.gz
Uncompress the file
gunzip mfeprimer-3.2.6-linux-amd64.gz
Rename the file
mv mfeprimer-3.2.6-linux-amd64 mfeprimer
Make sure the file has the executable permission
chmod a+x mfeprimer
Export the executable to PATH
export PATH=<Path to mfeprimer executable>:$PATH
Download tabix
wget https://github.com/samtools/htslib/releases/download/1.10/htslib-1.10.tar.bz2
Untar the tabix file
tar -xvf htslib-1.10.tar.bz2
Change working directory
cd htslib-1.10
Install tabix
make
make install
Export the executable to PATH
export PATH=<Path to tabix executable>:$PATH
Download libpng
wget http://downloads.sourceforge.net/project/libpng/libpng16/older-releases/1.6.2/libpng-1.6.2.tar.gz
Untar the libpng file
tar xvzf libpng-1.6.2.tar.gz
Change working directory
cd libpng-1.6.2/
Install libpng
./configure --prefix=`pwd`
make
make install
Define the location of the libpng directory
LIBPNGDIR=`pwd`
Download BLAT
cd ../
wget http://users.soe.ucsc.edu/~kent/src/blatSrc35.zip
unzip blatSrc35.zip
cd blatSrc/
Copy needed libpng files into BLAT directory
cp $LIBPNGDIR/png.h lib/
cp $LIBPNGDIR/pngconf.h lib/
cp $LIBPNGDIR/pnglibconf.h lib/
Install BLAT
make
Export the executable to PATH
export PATH=<Path to BLAT executable>:$PATH
- Download Conda with Python 3.9
wget https://repo.anaconda.com/archive/Anaconda3-2021.11-Linux-x86_64.sh
Install Conda
bash Anaconda3-2021.11-Linux-x86_64.sh
- Snakemake (at least v4.3.1) and biopython-1.79
Install Mamba into your Conda-based python distribution
conda install -n base -c conda-forge mamba
Activate the Conda base environment (which now includes Mamba).
conda activate base
Create a new conda environment called Locksmith with snakemake in it.
mamba create -c conda-forge -c bioconda -n snakemake Locksmith
Activate the Locksmith conda environment.
conda activate Locksmith
Check whether Snakemake is succesfully installed by running the following command:
snakemake --help
Install the following packages: biopython=1.79 .
conda install -c conda-forge biopython=1.79
Go into the Locksmith directory by:
cd <Path to Locksmith>
Make sure to activate the conda environment by:
conda activate Locksmith
snakemake --cores [amount of cores] --configfile [path to config file]
In order to perform a testrun of Locksmith go into the Locksmith directory and execute the following command:
snakemake --cores [amount of cores] --configfile test_data/config_test.json
Before running Locksmith several files are needed:
- Config file
This file is supplied in the Locksmith directory and needs to be completed with the correct local paths. For the other parameters a default value is in place.
- Chr2acc file:
A tab delimited file which shows the chromosome number and the corresponding accession number. The header consists of two columns. Header: #Chromosome Accession.version This file can be obtained from the NCBI FTP server e.g. https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.25_GRCh37.p13/GCF_000001405.25_GRCh37.p13_assembly_structure/Primary_Assembly/assembled_chromosomes/chr2acc
- Full genome.fa with right headers (create with generalize_genome_headers.py)
Obtain the Fasta sequence of the full reference genome.
Run the following command in order to change the headers into the chromosome numbers:
python scripts/preprocessing/generalize_genome_headers.py –g <Path to genome directory> -i <Path to chr2acc file>
- Split genome: chr_1.fa, chr_2.fa etc. (needed to create CpG bed files with Create_CpG_IDs_from_Reference_genome.py).
The files must be end with the following name format: ‘_[nr/X/Y].[fasta extension]’
Create CpG bed files for all chromosomes with the following command:
python scripts/preprocessing/create_cpg_ids_from_reference_genome.py –g <Path to chromosome fasta files with wildcard> -c <Path to chr2acc file>
- Target_list.bed
The target list is a bedfile with the following format:
Chromosome Start End CpG_ID Target strand
chr1 1232656 1232657 CpG23295 -
This file can be created with the following command:
python scripts/preprocessing/cg_illumina_to_target_bedfile.py -c <Path to Illumina cg identifier file> -i <Path to Infinium annotation file> -o <output_file_name> -p <Path to directory with CpG bedfiles>*.bed -e .bed
The Infinium annotation file can be obtained at e.g. https://zhouserver.research.chop.edu/InfiniumAnnotation/20180909/HM450/HM450.hg38.manifest.tsv.gz
The Illumina cg identifier file is a headerless file with only the Illumina cg identifier for each target per row.
-
snp_database.tbi
The tabix indexed file is a TAB-delimited file which shows the frequent SNPs in the chosen target regions. This file will be created automatically when it is not yet present in the working directory to save running time.
The configuration file which is needed to run Locksmith includes all initial parameters needed to create probes and select a probe panel.
"path_to_config_file": The path in which the configuration file can be found.
"path_to_scripts": The directory in which the Lockmith python scripts are situated.
"target_list_bedfile_path": The path to the bedfile which states the targets for probe design.
"acc_2_chrom_list_path": The path to the chr2acc file from the used reference genome. This file is needed in order to find frequent SNPs in the probe arms through the NCBI FTP server, without needing to download all of the SNP database.
"output_directory": The path of the directory in which the output files will be created.
"cpg_bed_path": The path (including wildcard) to the bedfiles of all CpG loci per chromosome.
"PATH_to_reference_genome_fasta": The Path to the reference genome Fasta file.
"ftp_path_snp_database": Path to the FTP server of the NCBI SNP database e.g. "ftp://ftp.ncbi.nih.gov/snp/latest_release/VCF/GCF_000001405.39.gz",
"target_range": Integer value of amount of basepairs to include on either side of the target CpG for probe design.
"snp_frequency_threshold": The threshold on the frequency of a SNP in order to count the SNP as frequent. e.g. 0.01
"dimer_exclusion_factor": The factor which determines how much dimers from the panel will be regarded as conflicting each iteration. This is a float between 0.0 and 1.0. To indicate that only one dimer must be chosen each iteration use the value 2.
"seed": The seed to use in random selection of the probes based on their cost. The seed makes sure that a Locksmith run with the exact same configuration yields the same results.
"backbone_sequence": Backbone sequence used for each probe in the panel. This consists of three parts, which are all 5’-3’ and will be appended to each other in the following order:
- "reverse_complement_universal_forward_primer": Sequence for annealing the forward primer during amplification of captured target.
- "common_region": Spacer sequence.
- "universal_reverse_primer": Sequence for annealing the reverse primer during amplification of captured target. "probe_specifics":
"probe_specifics": A list of parameters which are used for probe design.
- "min_arm_length": Minimum length of the upstream and downstream arms.
- "max_arm_length": Maximum length of the upstream and downstream arms.
- "min_target_length": Minimum length of the captured target sequence.
- "max_target_length": Maximum length of the captured target sequence
- "min_cg_percentage": Minimum CG percentage of the probe arms.
- "max_cg_percentage": Maximum CG percentage of the probe arms.
- "cpg_flanks": Amount of basepairs which must be flanking the target locus on both sides before annealing sites for the probe arms.
- "max_cpgs_in_arms": Maximum combined amount of CpGs in the probe arms which will be tolerated during probe design.
- "conversion": When "bisulfite", probes will be designed to target bisulfite converted gDNA, otherwise targeting gDNA.
"mfeprimer_hairpin_parameters": Parameters to use for hairpin check.
- "score_cutoff": Hairpin score threshold for reporting hairpins.
- "tm_cutoff": Hairpin Tm threshold for reporting hairpins.
"mfeprimer_dimer_parameters": Parameters to use for dimer check.
- "score_cutoff": Dimer score threshold for reporting dimers.
- "tm_cutoff": Dimer Tm threshold for reporting dimers.
"scoring_weights": Weights for calculating the probe cost.
- "hairpin": Hairpin weight
- "cpg": CpG weight
- "snp": SNP weight
- "tm": Tm weight
Formula: Hairpin_score * hairpin_weight + nr_of_cpgs_in_arms * cpg_weight + nr_of_snps_in_arms * snp_weight + Tm_difference_between_arms * tm_weight
"permutations": Maximum amount of iterations to be performed before choosing the panel with the least amount of dimers.
"max_threads": Maximum amount of threads that will be used by Locksmith
"existing_panel_csv_path": Path to chosen_panel.csv file when using an existing Locksmith panel to add probes to. (Default: False)