PolyMarker Deletions

Design KASP markers for deletions

PolyMarker includes a tool to design genome-specific primers to amplify an arbitrary sequence.

polymarker_capillary.rb --help
Usage: polymarker_deletions.rb [options]
    -r, --reference FILE             Fasta file with the assembly
    -m, --sequences FILE             Fasta file with the sequences to amplify. the format must be Chromosome:start-end. Chromosome
    should match the names to the entries in the fasta files as it is used as main target
    -o, --output_folder FOLDER       Path to a folder where the outputs are going to be stored
    -g, --genomes_count INT          Number of genomes (default 3, for hexaploid)
    -A, --allow_non_specific         If used, semi-specific and non-specific primers will be produced
    -d, --database PREFIX            Path to the blast database. Only used if the aligner is blast. The default is the name of the contigs file without extension.
    -a <sep>,<index>|nrgene|first_two|embl|morex|scaffold,
        --arm_selection              Function to decide the chromome arm

Example

Reference genome 7B_amplicon_test_reference.fa:

>chr7A
CTTCCTGCTGCTGAACTTTCTGAGGATCACCGCCCTGATCGGAGACGCTGGCGACCTTTC
GACTCCTTGGCCTTGTGATGCTCTTGCCGACAAAGATACCTAGCTTCATAGCAGTAAAGA
AACCTATGGCGATGAACAGAGGCCTCACCACCCAATGGAAGTCCTCGAGGTGCTCATACT
TCTTCACAACCCCATCGCACTTGTCGAAGCTCACCACCTCCACCTCTGAAGGGTCGGCCA
AGCAGCTGCACACTTCGCCAAGGCTGAATTTGCCAGGGAAGGTCACAACAAGGCATCCAT
TCGAGAGTATCTGTGAAATACGGCCTGTGGCAAACACTCCGCCTCTCTTTCGCGGCCATT
CAAACCGGGGGCTTGATGTGTTAGTTCTCAGCCTCACAAATTGTCCCACGCAGTAGGCTT
CGGCCATTTGCAGATCTGAATATTCCCCCTTCCAGAGGGTGTCCATTCCTATTAAACCAA
CATACACGGTGCCATTACGGTCAATGCTATGAAGAATTCCGACCTGAGACCGCTTCTTGT
CTTCCTCCCTAAGCCTTACCCAGTCTCCGGCAGCGAAACCATAGGTCACCCTCTCCACTG
TCGAGGAATGGACCTTCAAAGGGTCATGTATTCCATGGACTCGTACAAGAATGTAGCTAT
CACGTTCTCCATCCTCCTCCATGCCAACTATGGTTCCATCAGGAATTTCCATTGTTTCAG
GAGTACAAGAGTTTTTAACCTTTCTCGAGCGGACCTTGTCACCAACTTGCAGCTTATCCT
TAAAGTGTGACCAATTAGTGTGACTTGGCACTACTGCCCTTGGGTTTTCAGAACTATCCC
AGCCATTGTTGTCATAATCCACATCTTTAGCACTGAAGAAATGAAAAAAGAAAACAAATT
TAGGAAAGAACTATGGTAAAATCACCCATCTGATGGCAGTTTAACCAAATACTGCAAGTT
GATAAGGCAGAAGTAGAAAGAGAGTTTTAGCATGTTACCTTTCAAATGCTTGCAAGATAT
CTGACATCAAGGGGCGATCCCGAAAGTCGTACTCAAAGCAGCTAGAAAGGACATTCTCAA
CCTCTGCAGGTAAGTTGTATGGGAATATCGGTTTCTCTTTCTTCAGGACAACCAACTGAT
AAATTTCATCTGGTGATTTGCCACGCCAAGGCTGAACGCCACTGAACATCTCAAGAATGC
TGCAGGCAAAGCCCCATGAATCTGTCTCGTAACTAATTGGACCTCTGATGTTTGGTTGCC
ATTGCTCTGGGGCCATGTAATTTGGAGTCCCAAGTCTTTGGATAAGCTCTGGGTTTGGCA
GAGAAAGTCCAAACAGCAAGGATGGAATCCCAAAATCCCCCAGCACAGCATGGTCATGGT
CATCAAGGAGAAAATTGCAAGGCTTGAGATTAAGAACAAATATTCCCCTGGAGTGTAGGT
CTAGCACACCACGCGCCAAATCGGCACCATATCTGCAAGTTATAAGAATTCATAAAAATA
TGTATTACTACTCAAGAAAATATCTTCTAAACAGAGCATCATAAGCATTCTCAAAAGTCA
ACAGGATAATGTGCCTGTACTAAGGTCATAAACTATATCTGCCAGTTAATCAATCAACAC
GTTCACTGGTAGAAAGTTGGAAACTGCATGCTTAGGTGACACTTCATCTAAAAAAAATGT
ATATTTGTCTCTGATAAATTGTATTCTTAAGATAAGCCGACGAACACTCAGAGTATTCAG
AACATCAAATATGTTCAAAACTGTGGAGACTTCCACAATACCTTAAAACATCCGACAAAG
GGAGCCTTCCACCTTTAAGCCGAGCCATCTTGTCCCCGATGGATCCTTCATAAAACTTCA
TCGCTATGCAAAGCTGAAAATCCATGAAACAAAAATTAAGCCACAACGCCATCTTTCATT
CACAGGTGACCCAACAGCAAGGCTTTGCCAGACAAAAACATACCCTCCCATTCTGCGTTG
AGATGCCATGTAGGAAGCAGACATTGCCCAGGCCCTGGCATTTGCCGAACACCTCGTCGA
ACCTCGCCGAGAACACCTGCAGCTGGTCGTCCCTGACCGGGTGCATCATCTTCACGGCG
>chr7B
GTGCACTTCTTGCTGCTGAACTTTCTGAGGATCGGCACCCTGATCGGAGACGCTGGCGAC
CTTTCGACTCCTTGGCCTTGTGATGCTCTTCCCGACGAACACACCTAGCTTCATAGCAGT
AAAGAAACCTATGGCGATGAACAGAGGTCTTACCGCCCAATGGAAGTCCTCGAGGTGCTC
ATACTTCTTCACAACCCCCTCGCACTTGTCGAAGCTCACCACCTCCACCTCAGAAGGGTC
GGCCAAGCAGCTGCACACTTCGCCAAGGCTCAACTTGCCAGGGAAGGTCACAACAAGGCA
TCCATTCGAGAGTATCTGTGAAATACGGCCTGTGGCAAACACCCCGCCTCTCTTTCGCTG
CCATTCGAACCGCGGGCTTGAAGTGTTGGTTCTCAGCCTCACAAATTGTCCCACACAGTA
GGCTTCGGCCATTTGCAGATCTGAATATTCCCCCTTCCAGAGGGTGTCCATTCCTATCAA
ACCAACATACACGGTGCCATTGCGGTCGATGCTATGAAGAATTCCGACCTCAGACCGCTT
CTTGTCTTCCTCCCTGAGCCTTACCCAGTATCCGGCAGCGAAACCATAGGTCACCCTCTC
CACTGTCGAGGAACGGACCTTCAAAGGGTCATGTATTGCATGGACTCGTACAAGAATGTT
GCTATCACGTTCTCCATCCTCCATGCCAACTATGGTCCCATCAGGGATTTCCATTGTTTC
AGGAGTACAAGAGTTTTTAACCTTTCTCGAGCGGACCTTGTCCCCAACTTGCAGCTTATC
CTTGAAGTGTGACCAGTTAGTGTGACTTGGTGCTACTGCCCTTGGGTTTTCAGAACTGTC
CCAGCCATTGTTGTCATAATCCACATCTTTAGCACTGAAGAAAGGAAAAAAAAACAAATT
TAGGGAAGTAACATGGTAAAATCACCCATCCAATGGCAGTTTAACCAAATAGTGCAAGTT
GATAAGGCAGAAGTAGAAAGAGAGTTTTAGCATGTTACCTTTCAAATGCTTGTAAGATAT
CTGACATCAAGGGGCGATCCCGAAAGTCATACTCGAAGCAGCTAGGAAGGACATTCTCAA
CCTCTGCAGGTAAGTTGTACGGGAATATCGGTTTCTCTTTCTTCAGGACAACCAACTGAT
AAATCTCGTCTGGTGATTTGCCACGCCAAGGCTGAACGCCAGTGAACATCTCAAGAATGC
TGCAGGCAAAGCCCCATGAATCTGTCTCGTAACTAATTGGACCTCTGATGTTTGGTTGCC
ACTGCTCTGGGGCCATGTAATTTGGAGTTCCAAGTCTTTGGATAAGCTCTGGGTTTGGCA
GCGACAGTCCAAACAGCAAGGATGGAATCCCAAAATCCCCCAGCACAGCATGGTCATGGT
CATCAAGGAGAAAATTGCAAGGTTTGAGATTAAGAACAAATATTCCCCTGGAGTGTAGGT
CTAGCACACCACGCGCCAAATCGGCGCCGTATCTGCAAATTACAGGAAATAATAAAAATA
TGTACCACTCAAGAAAACATCCTCTAAACAGAGCATCATAAGCATTCTCAAAAGTCAACC
AGGATAATGTGCCTGCACTAAGGTGATGAAGGATATCTGCCAGTTAATCAACCAACACGT
TCACTGGTAGAAAGTTGGAAACTGCATGCTTAGGTGACAATTCATCTAAAAAAACTGTAT
ATTTGCCTCTGGTAAACTGTATTCTTAAGATAAGCCGACAAACATTCAGTATTCAGAACA
TCAATATGTACAGAACTGTGGAGACTTCCACCATACCTTAAAACATCTGACAAAGGGAGC
CTTCCACCTTTAAGCCGAGCCATCTTGTCCCCGACGGATCCTTCGTAAAACTTCATCGCT
ATGCAAAGCTGAAAATCCATGAAACAAAAATTAAGCCACAGCACCATCTTTCATTCACAG
GTGACCCAACAGCAAGGCTATGCCAGACAGAGAACATACCCTCCCATTCTGCGTCGAGAT
GCCATGTAGGAAGCAGACATTGCCCAGGCCCTGGCACTTGCCGAACACCTCGTCGAACCT
CGCCGAGAACACCTGCAGCTGGTCGTCCCTGACCGGGTGCAGCATCTTCACAGCGACCTC
G
>chr7D
GTTGTGCACTTCTTGCTGCTGAACTTTCTGAGGATCGGCACCCTGATCAGAGACGCTGGC
GACCTTTCGACTCCTTGGCCTTGTGATGCTCTTCCCAACAAAGACACCTAGCTTCATAGC
AGTAAAGAAACCTATGGCAATGAACAGAGGCCTCACCGCCCAATGGAAGTCCTCGAGGTG
CTCATACTTCTTCACAACGCCCTCGCACTTGTCGAAGCTCACCACCTCCACCTCTGAAGG
GTCGGCCAAGCAGCTGCACACTTCGCCAAGGCTGAATTTGCCAGGGAAGGTCACAGCAAG
GCATCCATTCGAGAGTATCTGTGAAATACGGCCTATGGCGAACACTCCGCCTCTCTTTCG
CGGCCATTCAAACCGGGGGCTTGAAGTGTTAGTTCTGAGCCTCACAAATTGTCCCATGCA
GTAGGCTTCGGCCATTTGCAGATCTGAATATTCCCCCTTCCACAGGGTGTCCATTCCTAT
CAAACCAACATACACGGTGCCATTACGGTCAATGCTATGAAGAATTCCGACCTGAGACCG
CTTCTTGTCTTCCTCCCTAAGCCTTACCCAGTCTCCGGCAGCGAAACCATAGGTCACCCT
CTCCACTGCTGAGGAACGGACCTTCAGAGGGTCATGTATTCCATGGACTCGTACAAGAAT
GTAGCTATCGCGTTCTCCATCCTCCATGCCAACTATGGTTCCATCAGGGATTTCCATTGT
TTCAGGAGTACAAGAGTTTTTAACCTTTCTCGAGCGGACCTTGTCACCAACTTGCAGCTT
ATCCTTGAAGTGTGACCAATTAGTGTGACTTGGCACTACTGCCCTTGGGTTTTCAGAACT
ATCCCAGCCATTGTTGTCATAATCCACATCTTTAGCACTGAAGAAATGAAAAAAGAAAAC
AAATTTAGGGAAGAACCATAGTAAATCACCCATCCAATGGCAGTTTAACCAAATACTGCA
AGTTGATAAGGTAGAAGAAGAGAGTTTTAGCATGTTACCTTTCAAATGCTTGTAAGATAT
CTGACATCAAGGGGCGATCCCGAAAGTCGTACTCAAAGCAGCTAGAAAGGACATTCTCAA
CCTCTGCAGGTAAATTGTACGGGAATATCGGTTTCTCTTTCTTCAGGACAACCAACTGAT
AAATCTCGTCTGGTGATTTGCCACGCCAAGGCTGAACGCCACTGAACATCTCAAGAATGC
TGCAGGCAAAGCCCCATGAATCTGTCTCGTAACTAATTGGACCTCTGATGTTTGGTTGCC
ATTGCTCTGGGGCCATGTAATTTGGAGTCCCAAGTCTTTGGATAAGCTCTGGGTTTGGCA
GCGACAGCCCAAACAGCAAGGATGGAATCCCAAAATCCCCCAACACAGCATGGTCATGAT
CATCAAGGAGAAAATTACAAGGCTTGAGATTAAGAACAAATATTCCCCTGGAGTGTAGGT
CCAGCACACCACGTGCCAAATCAGCGCCATATCTGCAAGTTATAAGAATTCATAAAAATA
TGTACTACTCCAAACAGAGCATCATAAGCATTCTCAAAAGTCAACAGGATCATTTGCCTG
CACTAAGGTGATAAACTAGATCTGCCAGTTAATCAATCAACACGTTCACTGGTAGAAAGT
TGGAAACTGCATGCTTAGGTGACACTTCATCTAAAAAACTGTATATTTGCCTCTGTGGTA
AACTGTATTCTTAAGATAAGCCGACGAACATTCAGTATTCAGGACATCAAATATGTTCAA
AACTGTGGAGACTTCCACCATACCTTAAAACATCTGACAAAGGGAGCCTTCCACCTTTAA
GCCGAGCCATCTTGTCCCCGACGGATCCTTCATAGAACTTCATCACTATGCAAAGCTGAA
AATCCATGAAACAAAAATTAAGCCACAACGCCATCTTTCATTCACAGGCGACCCAACAGC
AAGGCTATGCCAGACAAAAACATACCCTCCCATTCTGCGTCGAGATGCCATGTAGGAAGC
AGACATTGCCCAGGCCCTGGCATTTGCCAAACACCTCGTCGAACCTCGCCGAGAACACCT
GCAGCTGGTCGTCCCTGACCGGGTGCAGCATCTTCACGGCGACCTCGTGGTACCGGTCAT
A

Make the plast database:

makeblastdb -in 7B_amplicon_test_reference.fa -dbtype nucl

Target region:

>chr7B:1000-1600
TTTCAAATGCTTGTAAGATATCTGACATCAAGGGGCGATCCCGAAAGTCATACTCGAAGC
AGCTAGGAAGGACATTCTCAACCTCTGCAGGTAAGTTGTACGGGAATATCGGTTTCTCTT
TCTTCAGGACAACCAACTGATAAATCTCGTCTGGTGATTTGCCACGCCAAGGCTGAACGC
CAGTGAACATCTCAAGAATGCTGCAGGCAAAGCCCCATGAATCTGTCTCGTAACTAATTG
GACCTCTGATGTTTGGTTGCCACTGCTCTGGGGCCATGTAATTTGGAGTTCCAAGTCTTT
GGATAAGCTCTGGGTTTGGCAGCGACAGTCCAAACAGCAAGGATGGAATCCCAAAATCCC
CCAGCACAGCATGGTCATGGTCATCAAGGAGAAAATTGCAAGGTTTGAGATTAAGAACAA
ATATTCCCCTGGAGTGTAGGTCTAGCACACCACGCGCCAAATCGGCGCCGTATCTGCAAA
TTACAGGAAATAATAAAAATATGTACCACTCAAGAAAACATCCTCTAAACAGAGCATCAT
AAGCATTCTCAAAAGTCAACCAGGATAATGTGCCTGCACTAAGGTGATGAAGGATATCTG
C

Execute Polymarker

polymarker_deletion.rb --reference 7B_amplicon_test_reference.fa --sequences 7B_amplicon_test.fa  --genomes_count 3  --output 7B_deletion_test_out --database 7B_amplicon_test_reference.fa