iPython Notebook describing basic use of the Python PCR Design Tools

Designing CAPS Markers.ipynb

{
 "metadata": {
  "name": "Designing CAPS Markers"
 }, 
 "nbformat": 2, 
 "worksheets": [
  {
   "cells": [
    {
     "cell_type": "markdown", 
     "source": [
      "#CAPS Marker Design", 
      "", 
      "Using the Python tools at https://github.com/cfljam/galaxy-pcr-markers", 
      "", 
      "CAPS (cleaved amplified polymorphic sequences) are simple and robust genetic markers. We can screen for polymorphisms that condition restriction polymorphisms by passing a multifasta file of reference sequences and a gff3 file."
     ]
    }, 
    {
     "cell_type": "code", 
     "collapsed": false, 
     "input": [
      "!python find_CAPS.py test_data/targets.fasta test_data/targets.gff > CAPS.out"
     ], 
     "language": "python", 
     "outputs": [], 
     "prompt_number": 16
    }, 
    {
     "cell_type": "markdown", 
     "source": [
      "Now we have some hits we can design primers. "
     ]
    }, 
    {
     "cell_type": "code", 
     "collapsed": false, 
     "input": [
      "!head CAPS.out"
     ], 
     "language": "python", 
     "outputs": [
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        "JR844712\t686\t688\tJR844712:SAMTOOLS:SNP:686\tHaeIII\treference", 
        "JR844712\t759\t761\tJR844712:SAMTOOLS:SNP:759\tDdeI\treference", 
        "JR845107\t546\t548\tJR845107:SAMTOOLS:SNP:546\tRsaI\treference", 
        "JR845347\t636\t638\tJR845347:SAMTOOLS:SNP:636\tAluI\tvariant", 
        "JR845763\t63\t65\tJR845763:SAMTOOLS:SNP:63\tHinfI\treference", 
        "JR848350\t517\t519\tJR848350:SAMTOOLS:SNP:517\tAluI\tvariant", 
        "JR848350\t517\t519\tJR848350:SAMTOOLS:SNP:517\tDdeI\tvariant", 
        "JR848350\t642\t644\tJR848350:SAMTOOLS:SNP:642\tAluI\tvariant", 
        "JR848350\t642\t644\tJR848350:SAMTOOLS:SNP:642\tPvuII\tvariant", 
        "JR848350\t762\t764\tJR848350:SAMTOOLS:SNP:762\tAluI\tvariant"
       ]
      }
     ], 
     "prompt_number": 17
    }, 
    {
     "cell_type": "markdown", 
     "source": [
      "In this case we will just design assays to Taq1 polymorphisms."
     ]
    }, 
    {
     "cell_type": "code", 
     "collapsed": false, 
     "input": [
      "!awk '/TaqI/ {print $4}' CAPS.out > Taq1CAPS.out", 
      "!head Taq1CAPS.out"
     ], 
     "language": "python", 
     "outputs": [
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        "k41_520516:SAMTOOLS:SNP:1229", 
        "k49_198840:SAMTOOLS:SNP:223", 
        "k49_198840:SAMTOOLS:SNP:634", 
        "k65_175750:SAMTOOLS:SNP:1216", 
        "k65_175750:SAMTOOLS:SNP:1243", 
        "k65_175750:SAMTOOLS:SNP:1813", 
        "k65_175750:SAMTOOLS:SNP:2257", 
        "k69_176262:SAMTOOLS:SNP:1401", 
        "k69_177402:SAMTOOLS:SNP:139", 
        "k69_244399:SAMTOOLS:SNP:546"
       ]
      }
     ], 
     "prompt_number": 24
    }, 
    {
     "cell_type": "markdown", 
     "source": [
      "Now we can pass these targets to the design tool, specifying in this case a product size range of 80-150 bp and requesting just the best set (n=1)"
     ]
    }, 
    {
     "cell_type": "code", 
     "collapsed": false, 
     "input": [
      "%run design_HRM_primers.py -i test_data/targets.fasta -g test_data/targets.gff -T Taq1CAPS.out -p 80 -P 150 -n 1"
     ], 
     "language": "python", 
     "outputs": [
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        "SNP_Target_ID Position Ref_base Variant_base PRIMER_LEFT_SEQUENCE PRIMER_RIGHT_SEQUENCE ref_melt_Tm var_melt_Tm Tm_difference", 
        "k41_520516:SAMTOOLS:SNP:1229 149 C T GATTCATCACTCTCCTCGTTG TGCGGATATTGATGTTGATG 0 0 0", 
        "k49_198840:SAMTOOLS:SNP:223"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 88 A G CAGTAGTGGTAGCCAAGCCC GATGGTTAATGCTTGGGAGG 0 0 0", 
        "k49_198840:SAMTOOLS:SNP:634 128 C G GATGTCGGAGAGGAGAGAGG TGAGAACCCAAACCCTAACC 0 0 0", 
        "k65_175750:SAMTOOLS:SNP:1216 116 T C GGTCCACTTCATTGAAAGGC GCGACAACAAAGAACAGTGG 0 0 0", 
        "k65_175750:SAMTOOLS:SNP:1243"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 116 C T GGTCCACTTCATTGAAAGGC GCGACAACAAAGAACAGTGG 0 0 0", 
        "k65_175750:SAMTOOLS:SNP:1813 109 C G GGCTCAACTTGGATTGTGTG TGCCTTGTGCAAGTAACTCC 0 0 0", 
        "k65_175750:SAMTOOLS:SNP:2257 79 C T GTCCCTAGACACCTGGAAGC GGGCTTCTTCTTTCAGCTTG 0 0 0", 
        "k69_176262:SAMTOOLS:SNP:1401"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 86 T C CCCTCACTGCTGAAATTACG GAGCGGGATCGGTTTAATAG 0 0 0", 
        "k69_177402:SAMTOOLS:SNP:139"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 138 T G GGCATGAAAGTGGCTAGTTG CAGGAAATCTCATGTTTGTCG 0 0 0", 
        "k69_244399:SAMTOOLS:SNP:546 149 T A GTTGGACGAACACAAAGCTG CATTCTTGCATTCTCTGCATC 0 0 0", 
        "k69_323478:SAMTOOLS:SNP:869"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 136 C A TGCACCAAGAACTTCAATCG TGATGAAGTTGCATTACGGG 0 0 0", 
        "k69_324482:SAMTOOLS:SNP:1194 137 A G AGAAATGGGTGGGTTCATTC ACCATTCATGGATCACTTCG 0 0 0", 
        "k69_324482:SAMTOOLS:SNP:2172"
       ]
      }, 
      {
       "output_type": "stream", 
       "stream": "stdout", 
       "text": [
        " 142 C T ACATCAGCAAGGAGAACACG CTATCCATGTGGCGGTGTAG 0 0 0", 
        "k69_93535:SAMTOOLS:SNP:1147 137 C G GGACAGGGAAGCTTCATAGG TGTTTGGTATCGTTTCACCC 0 0 0"
       ]
      }
     ], 
     "prompt_number": 29
    }
   ]
  }
 ]
}

using_PCR_design_python_tools_1.ipynb