- Introduction of DNAMarkMaker
- Installation
- Usage
- command : target_SNP_selection
- command : ARMS_preparation
- command : tri_ARMS
- command : tetra_ARMS
- command : CAPS
- The input file format
- The example of execution
- The output file format
DNAMarkMaker is a tool to develop ARMS and/or CAPS markers that target SNPs between cultivars/lines, utilizing data from next-generation sequencing.
- python >=3.9.13
- samtools
- primer3-py
You can install DNAMarkMaker using bioconda.
conda install -c bioconda dnamarkmaker
Alternatively, if you want to create DNAMarkMaker specific environment.
conda create -n DNAMarkMaker -c bioconda dnamarkmaker
conda activate DNAMarkMaker
An error is detected if conda's python is not 3.9.13 or later.
conda create -n DNAMarkMaker python=3.9.13 # Also You can select later 3.9.13
conda activate DNAMarkMaker
conda install -c bioconda dnamarkmaker
'DNAMarkMaker' offers five commands. Users can specify which command to run using the '-w' option. Here is a summary of each command.
Purpose: Identify SNPs from the BAM file, which contains alignment data of two breeds.
Description: This command should be the first one executed when using DNAMarkMaker, as it lays the foundation for subsequent marker design by selecting target SNPs.
Required options
-Abam ABAM Full path of A bam
-Bbam BBAM Full path of B bam
-reference REFERENCE Full path of reference fasta
-position POSITION Target chromosome position [chr:start:end]
-o OUTPUT_DIR Output directory
Additional options
-Aname ANAME A name (A)
-Bname BNAME B name (B)
-min_depth MIN_DEPTH Minimum depth of target SNP (10)
-max_depth MAX_DEPTH Maximum depth of target SNP (99)
-minMQ MINMQ Minimum mapping quality detected from bam (0)
-minBQ MINBQ Minimum base quality detected from bam (13)
-Bhetero BHETERO Whether to target heterozygous SNP in B (no)
-Bsim BSIM B simulation file
-Cbam CBAM Full path of C bam
-Csim CSIM C simulation file
Purpose: Design breed-specific primers for the creation of ARMS markers.
Description: This command utilizes the inter-cultivar SNP information previously identified to design primers specific to each breed or line, preparing for the next stage of ARMS marker development.
Required options
-o OUTPUT_DIR Output directory
Additional options
-recipe RECIPE Full path of primer recipe file
Purpose: Develop tri-ARMS markers.
Description: Leveraging the primers designed in the ARMS_preparation phase, this command facilitates the development of tri-ARMS markers, which involve three primers for amplification.
Required options
-o OUTPUT_DIR Output directory
Additional options
-recipe RECIPE Full path of primer recipe file
-PCR_max_size PCR_MAX_SIZE Maximum size of PCR product (700)
-PCR_min_size PCR_MIN_SIZE Minimum size of PCR product (100)
-SNP_dist SNP_DIST Target SNP distance (100:300)
-make_html MAKE_HTML Whether to html file (yes)
Purpose: Develop tetra-ARMS markers.
Description: Similarly to the tri_ARMS command, this command uses the primers from ARMS_preparation but develops tetra-ARMS markers, which utilize four primers in the amplification process.
Required options
-o OUTPUT_DIR Output directory
Additional options
-first_size FIRST_SIZE Size of first band (100:500)
-second_size SECOND_SIZE Size of second band (600:1000)
-make_html MAKE_HTML Whether to html file (yes)
Purpose: Create CAPS markers.
Description: This command supports the development of CAPS markers by introducing specific restriction enzymes to the identified inter-cultivar SNP information, resulting in markers that are identified based on the cleavage patterns of these enzymes on the DNA fragments.
Required options
-o OUTPUT_DIR Output directory
-restriction_enzyme RESTRICTION_ENZYME Full path of restriction enzyme file
Additional options
-recipe RECIPE Full path of primer recipe file
-PCR_max_size PCR_MAX_SIZE Maximum size of PCR product (1000)
-PCR_min_size PCR_MIN_SIZE Minimum size of PCR product (500)
-fragment_min_size FRAGMENT_MIN_SIZE Minimum fragment size of restricted PCR product (200)
-make_html MAKE_HTML Whether to html file (yes)
Band size of PCR product amplified by developped marker
A sorted binary format file that stores NGS reads alignment data bai file.
A fasta format file for alignmnet.
A txt file contains space-separated arbitrary simulation confidence intervals for each depth. Users can obtain this from the provided URL (https://github.com/SegawaTenta/DNAMarkMaker_manual/tree/main/simulation) and modify the values if required for specific analyses.
A txt file contains the options used by the primer design tool, primer3. The default version of this file can be downloaded from the provided URL (https://github.com/SegawaTenta/DNAMarkMaker_manual/blob/main/primer_recipe/primer_recipe.txt), but users can also tailor the values inside to suit specific needs or experimental conditions.
A txt file contains space-separated arbitrary the name of restriction enzyme and recognizing sequense (https://github.com/SegawaTenta/DNAMarkMaker_manual/blob/main/restriction_enzyme/restriction_enzyme.txt) and modify the contents if required for specific analyses.
ex1) Homozygous plant
DNAMarkMaker -w target_SNP_selection \
-reference Full/path/to/referance.fasta \
-Abam Full/path/to/A.bam \
-Bbam Full/path/to/B.bam \
-position chr1:10000:50000 \
-o example1
ex2) Autotetraploid plants (terget of simplex in B)
DNAMarkMaker -w target_SNP_selection \
-reference Full/path/to/referance.fasta \
-Abam Full/path/to/A.bam \
-Bbam Full/path/to/B.bam \
-position chr1:10000:50000 \
-o example3 \
-min_depth 59 \
-max_depth 300 \
-Bhetero yes\
-Bsim downloaded/sim_simplex_AAAa_95.txt
ex3) Heterozygous plants
DNAMarkMaker -w target_SNP_selection \
-reference Full/path/to/referance.fasta \
-Abam Full/path/to/A.bam \
-Bbam Full/path/to/B.bam \
-Cbam Full/path/to/F1.bam \
-position chr1:10000:50000 \
-o example3 \
DNAMarkMaker -w ARMS_preparation \
-o example1
DNAMarkMaker -w tri_ARMS \
-o example1
DNAMarkMaker -w tetra_ARMS \
-o example1
DNAMarkMaker -w CAPS \
-restriction_enzyme downloaded/restriction_enzyme.txt\
-o example1
The formats and contents of the output files align with those delineated in the manual for the GUI version, available at URL(https://github.com/SegawaTenta/DNAMarkMaker_manual/blob/main/Manual.pdf).