#bio-polyploid-tools
##Introduction This tools are designed to deal with polyploid wheat. The first tool is to design KASP primers, making them as specific as possible.
##Installation
gem install bio-polyploid-tools
You need to have in your $PATH
the following programs:
The code has been developed on ruby 2.1.0, but it should work on 1.9.3 and above.
#PolyMarker
To run poolymerker with the CSS wheat contigs, you need to unzip the reference file from ensembl.
polymarker.rb --contigs Triticum_aestivum.IWGSC2.25.dna.genome.fa --marker_list snp_list.csv --output output_folder
The snp_list
file must follow the convention ID,Chromosome,SEQUENCE
with the SNP inside the sequence in the format [A/T]. As a reference, look at test/data/short_primer_design_test.csv
If you want to use the web interface, visit the PolyMarker webservice at TGAC
The available command line arguments are:
Usage: polymarker.rb [options]
-c, --contigs FILE File with contigs to use as database
-m, --marker_list FILE File with the list of markers to search from
-g, --genomes_count INT Number of genomes (default 3, for hexaploid)
-s, --snp_list FILE File with the list of snps to search from, requires --reference to get the sequence using a position
-t, --mutant_list FILE File with the list of positions with mutation and the mutation line.
requires --reference to get the sequence using a position
-r, --reference FILE Fasta file with the sequence for the markers (to complement --snp_list)
-o, --output FOLDER Output folder
-e, --exonerate_model MODEL Model to be used in exonerate to search for the contigs
-i, --min_identity INT Minimum identity to consider a hit (default 90)
-a, --arm_selection arm_selection_embl|arm_selection_morex|arm_selection_first_two
Function to decide the chromome arm
-p, --primer_3_preferences FILE file with preferences to be sent to primer3
-v, --variation_free_region INT If present, avoid generating the common primer if there are homoeologous SNPs within the specified distance (not tested)
-x, --extract_found_contigs If present, save in a separate file the contigs with matches. Useful to debug.
-P, --primers_to_order If present, saves a file named primers_to_order which contains the KASP tails
###Custom reference sequences.
By default, the contigs and pseudomolecules from ensembl are used. However, it is possible to use a custom reference. To define the chromosome where each contig belongs the argument arm_selection
is used. The defailt uses ids like: IWGSC_CSS_1AL_scaff_110
, where the third field, separated by underscores is used. A simple way to add costum references is to rename the fasta file to follow that convention. Another way is to use the option --arm_selection arm_selection_first_two
, where only the first two characters in each contig is used as identifier, useful when pseudomolecules are named after the chromosomes (ie: ">1A" in the fasta file).
If your contigs follow a different convention, in the file polymarker.rb
it is possible to define new parsers, by adding at the begining, with the rest of the parsers a new lambda like:
arm_selection_functions[:arm_selection_embl] = lambda do | contig_name|
arr = contig_name.split('_')
ret = "U"
ret = arr[2][0,2] if arr.size >= 3
ret = "3B" if arr.size == 2 and arr[0] == "v443"
ret = arr[0][0,2] if arr.size == 1
return ret
end
The function should return a 2 character string, when the first is the chromosome number and the second the chromosome group. The symbol in the hash is the name to be used in the argument --arm_selection
. If you want your parser to be added to the distribution, feel free to fork and make a pull request.
##Release Notes
###0.7.3
- FEATURE:
polymarker.rb
Added to the flag--arm_selection
the optionscaffold
, which now supports a scaffold specific primer. - FEATURE:
snp_position_to_polymarker
Added the option--mutant_list
to prepare files for PolyMarker from files with the following columnsID,Allele_1,position,Allele_1,target_chromosome
.
###0.7.2
- FEATURE: Added a flag
min_identity
to set the minimum identity to consider a hit. The default is 90
###0.7.1
- BUGFIX: Now the parser for
arm_selection_embl
works with the mixture of contigs and pseudomolecules - DOC: Added documentation on how to use custom references.
###0.7.0
- Added flag
genomes_count
for number of genomes, to be used on tetraploids, etc.
###0.6.1
- polymarker.rb now validates that all the files exist.
- BUGFIX: A reference was required even when it was not used to generate contigs.
#Notes
-
If the SNP is in a gap in the alignment to the chromosomes, it is ignored.
-
BUG: Blocks with NNNs are picked and treated as semi-specific.
-
BUG: If the name of the reference have space, the ID is not chopped. ">gene_1 (G12A)" shouls be treated as ">gene_1".
-
TODO: Add a parameter file to configure the alignments.
-
TODO: Produce primers for products of different sizes. This can probably be done with the primer_3_preferences option, but hasn't been tested.