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Designing HRM primer sets
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| { | |
| "metadata": { | |
| "name": "using_PCR_design_python_tools_1" | |
| }, | |
| "nbformat": 3, | |
| "nbformat_minor": 0, | |
| "worksheets": [ | |
| { | |
| "cells": [ | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "#Bulk Design of HRM Primer Sets Using Python Tools\n", | |
| "\n", | |
| "####John McCallum john.mccallum@plantandfood.co.nz\n", | |
| "\n", | |
| "\n", | |
| "This Notebook describes use of the python and shell tools for bulk design of PCR marker assays from the Unix command-line.\n", | |
| "\n", | |
| "\n", | |
| "These are available at https://github.com/cfljam/galaxy-pcr-markers and can be most easily obtained by installing Git and cloning:" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": true, | |
| "input": [ | |
| "git clone https://github.com/cfljam/galaxy-pcr-markers.git" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [] | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "---\n", | |
| "Learn about iPython at http://ipython.org. The only conventions needed to understand the following are \n", | |
| "\n", | |
| "+ ! denotes a shell command\n", | |
| "+ % denotes an iPython magic command -like *run*" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "ls" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "\u001b[0m\u001b[01;32mdesign_HRM_primers.py\u001b[0m* example.int parse_cmh.py run_p3.py \u001b[01;34mtest_data\u001b[0m/ umelt_service.py~\r\n", | |
| "example.for example.rev plot_allele_freqs.R run_p3.pyc umelt_service.py umelt_service.pyc\r\n" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 3 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "----\n", | |
| "**Input Files**\n", | |
| "\n", | |
| "We require a multi-fasta file containing reference sequence, a GFF3 file describing features of interest for design and masking in these sequences, and a list of targets\n", | |
| "\n", | |
| "The targets file is a list that uniquely identifies the feature of interest, where the reference contig or chromosome ID is the first portion of the string, delimited from remaining content by a delimiter, defaulting to colon (but an alternate delimiter e.g. '_' can be specified)" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "!head -n5 test_data/targets" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "JR844712:SAMTOOLS:SNP:310\n", | |
| "JR844712:SAMTOOLS:SNP:353\n", | |
| "JR844712:SAMTOOLS:SNP:470\n", | |
| "JR844712:SAMTOOLS:SNP:572\n", | |
| "JR844712:SAMTOOLS:SNP:573" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 2 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "The input gff3 file describing the polymorphisms must follow the more restrictive GVF convention desribed at http://www.sequenceontology.org/resources/gvf.html\n", | |
| "\n", | |
| "Most importantly \n", | |
| "the reference and variant sequence must be given in field 9 using the attributes 'Reference_seq' and 'Variant_seq'.\n", | |
| "The feature identifier in field 9 must match that in the target list. In this case we use the simple approach of concatenating the first 4 fields of the gff file.\n", | |
| "An alternative convention, used in Tassel would be to delimit location from contig by underscore i.e. S1239_567" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "!head -n5 test_data/targets.gff" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "JR844712\tSAMTOOLS\tINDEL\t4\t5\t11.8\t.\t.\tID=JR844712:SAMTOOLS:INDEL:4;Variant_seq=CCGTTTG;Reference_seq=C;INDEL;DP=28;VDB=0.0024;AF1=1;AC1=24;DP4=0,0,10,0;MQ=6;FQ=-37\n", | |
| "JR844712\tSAMTOOLS\tSNP\t310\t311\t13.7\t.\t.\tID=JR844712:SAMTOOLS:SNP:310;Variant_seq=A;Reference_seq=G;DP=7968;VDB=0.0206;AF1=1;AC1=24;DP4=1447,1382,2647,2159;MQ=0;FQ=-44.7;PV4=0.00096,0,0.027,0.0074\n", | |
| "JR844712\tSAMTOOLS\tSNP\t353\t354\t9.92\t.\t.\tID=JR844712:SAMTOOLS:SNP:353;Variant_seq=C;Reference_seq=T;DP=7344;VDB=0.0358;AF1=1;AC1=24;DP4=1357,962,2115,1590;MQ=0;FQ=-45.4;PV4=0.28,0.073,0.15,1\n", | |
| "JR844712\tSAMTOOLS\tSNP\t470\t471\t74.4\t.\t.\tID=JR844712:SAMTOOLS:SNP:470;Variant_seq=T;Reference_seq=C;DP=6915;VDB=0.0378;AF1=1;AC1=24;DP4=953,915,2521,2207;MQ=0;FQ=-46.7;PV4=0.095,0.38,1,0.19\n", | |
| "JR844712\tSAMTOOLS\tSNP\t572\t573\t31.7\t.\t.\tID=JR844712:SAMTOOLS:SNP:572;Variant_seq=C;Reference_seq=T;DP=6564;VDB=0.0379;AF1=1;AC1=24;DP4=984,960,1901,1816;MQ=0;FQ=-41.9;PV4=0.72,0,6.6e-15,1" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 3 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "The design tool allows specifying most of the possible Primer 3 tags as command-line options (and could be trivially modified to pass any others as entries in a Python dict)" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "%run design_HRM_primers.py -h" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "usage: design_HRM_primers.py [-h] -i IN_FILE -g GFF_FILE -T TARGET_FILE [-u]\n", | |
| " [-n MAX_PRIMERS] [-p PROD_MIN_SIZE]\n", | |
| " [-P PROD_MAX_SIZE] [-l OPT_PRIMER_LENGTH]\n", | |
| " [-m MAX_TM_DIFF] [-t OPTIMUM_TM]\n", | |
| " [-G OPT_GC_PERCENT] [-x MAXPOLYX] [-c GC_CLAMP]\n", | |
| " [-d TARGET_DELIM]\n", | |
| "\n", | |
| "Primer set design and melt prediction parameters\n", | |
| "\n", | |
| "optional arguments:\n", | |
| " -h, --help show this help message and exit\n", | |
| " -i IN_FILE input sequence file, required\n", | |
| " -g GFF_FILE input gff file with SNP and indels, required\n", | |
| " -T TARGET_FILE input target SNP file, required\n", | |
| " -u do uMelt prediction, optional\n", | |
| " -n MAX_PRIMERS maximum number of primer pairs to return, default=5\n", | |
| " -p PROD_MIN_SIZE minimum product size\n", | |
| " -P PROD_MAX_SIZE maximum product size\n", | |
| " -l OPT_PRIMER_LENGTH optimum primer length\n", | |
| " -m MAX_TM_DIFF maximum tm difference between primers\n", | |
| " -t OPTIMUM_TM optimum Tm for primers, recommend range 59 to 61\n", | |
| " -G OPT_GC_PERCENT optimum GC percentage of primers\n", | |
| " -x MAXPOLYX maximum polyx, recommend less than 4\n", | |
| " -c GC_CLAMP number of C/Gs at end, recommend 2\n", | |
| " -d TARGET_DELIM variant indentifier delimiter, used to separate\n", | |
| " sequence ID from rest" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 7 | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "ls test_dat" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "test_data/targets \u001b[0m\u001b[01;32mtest_data/targets.fasta\u001b[0m* test_data/targets.gff" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 6 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "For this first example we will just request a single primer set for each feature in our target set." | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "%run design_HRM_primers.py -i test_data/targets.fasta -g test_data/targets.gff -T test_data/targets -n 1" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "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\n", | |
| "JR844712:SAMTOOLS:SNP:310" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 243 G A TGCTGTACTTAATAGCGCCG CACATTTCCAAGGTCACCAG 0 0 0\n", | |
| "JR844712:SAMTOOLS:SNP:353 134 T C CTGGTGACCTTGGAAATGTG GATCATCAGGATCAGCATGG 0 0 0\n", | |
| "JR844712:SAMTOOLS:SNP:470 270 C T AGTTGTCCATGCTGATCCTG CACATACAGATTGGCCTTGC 0 0" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 0\n", | |
| "JR844712:SAMTOOLS:SNP:572 187 T C CATGCGGTATCATTGGACTC CACATACAGATTGGCCTTGC 0 0 0\n", | |
| "JR844712:SAMTOOLS:SNP:573" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 187 G A CATGCGGTATCATTGGACTC CACATACAGATTGGCCTTGC 0 0 0\n", | |
| "JR845107:SAMTOOLS:SNP:546 211 C T TCGAAACAAGCACTGGGTAG TAAATCCCTCAGTCCAAGCC 0 0 0\n", | |
| "JR845347:SAMTOOLS:SNP:410" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 280 G A GAGGTTTATCTGGACTGCCC GCAGCCAAGTATTCATCTGG 0 0 0\n", | |
| "JR845347:SAMTOOLS:SNP:418 226 G A AGGAGGCAGAAGAACGAAAG TCCATCCTTCAAGGTGTACG 0 0 0\n", | |
| "JR845763:SAMTOOLS:SNP:164 297 C T GAAGAAGAAGAGAATGCGGC CCACAGTTTCTGGACACAGG 0 0 0\n", | |
| "JR848350:SAMTOOLS:SNP:642" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 181 C T TCTCTTCTCCAGCCTCCTTC TACGACAGAAGAGGCGACAC 0 0 0\n", | |
| "JR849045:SAMTOOLS:SNP:259 120 C G TCTGCTCTTAAGGCTTCATCTC TTGGACACAATCAAGGAAGC 0 0 0\n", | |
| "JR849045:SAMTOOLS:SNP:448 185 C T GCCCAGCCTCTTCAGAGTAG TTTGAGATTGCTCCATCAGC 0 0 0\n", | |
| "JR849045:SAMTOOLS:SNP:484" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 134 G A TAATTGAGCTGCCGATTCAC TTTGAGATTGCTCCATCAGC 0 0 0\n", | |
| "JR849045:SAMTOOLS:SNP:620 223 A G GACATGGCATGAGGTTTGAG ATGGGTCAATCAGGAGAAGG 0 0 0\n", | |
| "JR849805:SAMTOOLS:SNP:135 169 G A GCTTTCTTTGCATTTATTGCC TTCAGTCTTGCCATCTTTGC 0 0 0\n", | |
| "JR850172:SAMTOOLS:SNP:101" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 230 G A CCATGGCAACTAAGAAAGCC CGAGCTTAAACGATGCTTTG 0 0 0\n", | |
| "JR852189:SAMTOOLS:SNP:58 196 C A CAAATTCTCTCAATTCTAAGCCC TCCAACTCGTTGAGCGTATC 0 0 0\n", | |
| "JR852189:SAMTOOLS:SNP:64 196 A C CAAATTCTCTCAATTCTAAGCCC TCCAACTCGTTGAGCGTATC 0 0 0\n", | |
| "JR852189:SAMTOOLS:SNP:253" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 208 C T GGCCGATACTTGAAGAAAGG ACTCCACCCAGCAATTTACC 0 0 0\n", | |
| "JR852189:SAMTOOLS:SNP:447 144 T C ATCAATCCCGTGCTTCTACC CCAAACAGCGTTACATGGAC 0 0 0\n", | |
| "JR852189:SAMTOOLS:SNP:537 209 C T ATCAATCCCGTGCTTCTACC GAAGTACAAATTCCATCCAGAGC 0 0 0\n", | |
| "JR854385:SAMTOOLS:SNP:128" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 182 G A AGAATGGCTTCCAAAGTTCTG AGAGGGAAGGAGGGAGAGAG 0 0 0\n", | |
| "JR854385:SAMTOOLS:SNP:171 182 C T AGAATGGCTTCCAAAGTTCTG AGAGGGAAGGAGGGAGAGAG 0 0 0\n", | |
| "JR854446:SAMTOOLS:SNP:166 222 G A AGAGGAAGTAAATGAACACGGAG GCTGCTGCTGTTGCTAACTC 0 0 0\n", | |
| "JR854446:SAMTOOLS:SNP:207" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 198 T G CTGTCCTTAATGACTCCTCGG CTGGATTGTGCAGATGGAAC 0 0 0\n", | |
| "JR854792:SAMTOOLS:SNP:228 221 G T TAAACAACAATGGCGGAGAC AGAAGCATCTTCCCAGAAGG 0 0 0\n", | |
| "JR857827:SAMTOOLS:SNP:56 232 T C TAATAATCGGCCATGGCTTC ACCTTTGGAATTTCTGGCAC 0 0 0\n", | |
| "JR857827:SAMTOOLS:SNP:99" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 192 T A TCTTCTTCGCCTTTGCTTTC ACCTTTGGAATTTCTGGCAC 0 0 0\n", | |
| "JR863117:SAMTOOLS:SNP:120 167 G A TTCACATTCATGGCCGTAAG TGTAGGAACTGTAGGCGTGG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:187 215 G T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:212" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 215 C T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:265 215 C T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:342 169 T G TGTTCCGACTCAGATCAAGC GGAGAAGCTGAAGACCCTTG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:537 134 A G ATTCCCAGACAGAACAAGGG ACCATCCCTGAATCCATAGG 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:557" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 250 A C ATTCCCAGACAGAACAAGGG ACGTAGTCATCGCCAACAAC 0 0 0\n", | |
| "k31_439587:SAMTOOLS:SNP:598 250 T C ATTCCCAGACAGAACAAGGG ACGTAGTCATCGCCAACAAC 0 0 0\n", | |
| "k31_565787:SAMTOOLS:SNP:109 298 C T CCTGGATTGGAATCTTCTGG AGAACCTCCTCTTCGTCAGC 0 0 0\n", | |
| "k31_565787:SAMTOOLS:SNP:154 219 C T GGCACAAACGCTGTCACTAC AGAACCTCCTCTTCGTCAGC 0 0 0\n", | |
| "k31_565787:SAMTOOLS:SNP:268" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 183 G A CTAGTAAGGTCGGCAAAGGC AGAACCTCCTCTTCGTCAGC 0 0 0\n", | |
| "k31_714243:SAMTOOLS:SNP:151 257 C A TCTGGGAAGAGTATCGGACC TTCTTGCAGAAGTGTCGAGG 0 0 0\n", | |
| "k31_714243:SAMTOOLS:SNP:342 289 T C GGTACTGGACTTCAGGTGGG CATCATCATAGGAGGAGCCC 0 0 0\n", | |
| "k31_754967:SAMTOOLS:SNP:904" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 233 C T CCCAATCATCCAATCTCAAG AGTATCCAAACGGGTCAAGG 0 0 0\n", | |
| "k31_754967:SAMTOOLS:SNP:910 233 T C CCCAATCATCCAATCTCAAG AGTATCCAAACGGGTCAAGG 0 0 0\n", | |
| "k33_381591:SAMTOOLS:SNP:123" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 292 T C TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0\n", | |
| "k33_381591:SAMTOOLS:SNP:169 292 A T TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0\n", | |
| "k33_381591:SAMTOOLS:SNP:239 292 T C TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0\n", | |
| "k33_381591:SAMTOOLS:SNP:240 292 C A TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0\n", | |
| "k35_208328:SAMTOOLS:SNP:272" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 281 A C GAAAGTGCTCACAGGAGCTG ATTTCTGGACCTCAAATGGC 0 0 0\n", | |
| "k35_208328:SAMTOOLS:SNP:443 227 A G AAAGAAAGTGGTGGAGGTGG ATTTCTGGACCTCAAATGGC 0 0 0\n", | |
| "k35_208328:SAMTOOLS:SNP:505 235 C T TACCTGCGTCACCGTTAGAG TTCAACTTTGGCAGGCTATG 0 0 0\n", | |
| "k35_653504:SAMTOOLS:SNP:85 233 C T GGAAATGGCTGAATTGTTCTG ATATTTGGCAGTTTGGGAGC 0 0 0\n", | |
| "k35_653504:SAMTOOLS:SNP:208" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 196 A G TACGCAGAAACAAGACCGAC ATATTTGGCAGTTTGGGAGC 0 0 0\n", | |
| "k35_653504:SAMTOOLS:SNP:216 196 A G TACGCAGAAACAAGACCGAC ATATTTGGCAGTTTGGGAGC 0 0 0\n", | |
| "k35_653504:SAMTOOLS:SNP:469 230 C A ATCTCCGATCTGGTCAAACC CAGCTTCCTCTTCTCATCCC 0 0 0\n", | |
| "k39_566848:SAMTOOLS:SNP:1781" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 99 T G GTGATGGTTGAGCCAGTGTC CCCACGTTAGCCTCTTTAGC 0 0 0\n", | |
| "k41_520516:SAMTOOLS:SNP:1229 216 C T CCAATCAAGCAGCCAATTAC TAAGGCAAGATAGTGGTGCG 0 0 0\n", | |
| "k41_520516:SAMTOOLS:SNP:1246 216 C T CCAATCAAGCAGCCAATTAC TAAGGCAAGATAGTGGTGCG 0 0 0\n", | |
| "k43_486433:SAMTOOLS:SNP:1177" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 286 A C GTAGGAAGCTTTGTCCTGGC TCTGAGGAAGAATTGGAGGG 0 0 0\n", | |
| "k43_486433:SAMTOOLS:SNP:1180 286 A G GTAGGAAGCTTTGTCCTGGC TCTGAGGAAGAATTGGAGGG 0 0 0\n", | |
| "k43_486433:SAMTOOLS:SNP:1762 153 A G ATAATTGCAGAGACATGGCG AGCAGTCTGACGTTCCTGAG 0 0 0\n", | |
| "k45_231452:SAMTOOLS:SNP:679" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 155 A G TGGGAAGAATAGGATGGAGC AATGGCAGAGTGGAAGATCC 0 0 0\n", | |
| "k45_463949:SAMTOOLS:SNP:142 221 T C AACTTGGGTTTGTCGCTCTC ATCCATCCGCCTGAAGTAAC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:45" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 184 A G CATAACATACTTACATACACACCGC CTGTTTAGCTTGCCCACATC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:328 271 C T TGTGGGCAAGCTAAACAGAC GGCGTCTGCTTCCAATAAAC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:337 271 C T TGTGGGCAAGCTAAACAGAC GGCGTCTGCTTCCAATAAAC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:685" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 205 A G CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:719 205 A C CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0\n", | |
| "k49_172131:SAMTOOLS:SNP:766 205 A T CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0\n", | |
| "k49_198840:SAMTOOLS:SNP:439" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 270 A G TCATAGACGAAGCAAGACGG CCTCTCTCCTCTCCGACATC 0 0 0\n", | |
| "k49_198840:SAMTOOLS:SNP:442 270 A G TCATAGACGAAGCAAGACGG CCTCTCTCCTCTCCGACATC 0 0 0\n", | |
| "k61_221473:SAMTOOLS:SNP:1250 269 T C CACGACCAAATGCAAAGAAC ATGAGAAGGATTTGATGGGC 0 0 0\n", | |
| "k61_221473:SAMTOOLS:SNP:1328" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 269 C T CACGACCAAATGCAAAGAAC ATGAGAAGGATTTGATGGGC 0 0 0\n", | |
| "k63_198617:SAMTOOLS:SNP:81 111 C T GACTTTATTCAAAGCAAACACTGAC TTGTATAACCCAGGGCCTTC 0 0 0\n", | |
| "k65_172796:SAMTOOLS:SNP:467" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 255 C T AGTTCATAGCCACGCTGTTG ATTGCTCGGATGAGGGATAC 0 0 0\n", | |
| "k65_172796:SAMTOOLS:SNP:533 173 C T AGTGACATTCGGCCTATTCC TTGGTGTAGCCAACGCTTAG 0 0 0\n", | |
| "k65_172796:SAMTOOLS:SNP:665 209 T C TCCAAAGCGCATACTACAGC GGACCATGAACACAGCAAAG 0 0 0\n", | |
| "k65_173050:SAMTOOLS:SNP:116" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 205 A G TAACGGGTACCTCCTGGAAC CTAGCTCTTCCGGGTTTGAG 0 0 0\n", | |
| "k65_173050:SAMTOOLS:SNP:355 99 T C CATCGGTTGTCTTCCATGAG AGTAGCCTTCAGCAACGACC 0 0 0\n", | |
| "k65_173050:SAMTOOLS:SNP:361 99 C T CATCGGTTGTCTTCCATGAG AGTAGCCTTCAGCAACGACC 0 0 0\n", | |
| "k65_175163:SAMTOOLS:SNP:87" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 278 G A CTAAATCAACAAGCTCCCTCG CCGTTTCACTATTCGTCACC 0 0 0\n", | |
| "k65_175163:SAMTOOLS:SNP:137 284 G A AGTCAGCTTCGGGTGTACG ACACGTCTTGGTCCAAATCC 0 0 0\n", | |
| "k65_175163:SAMTOOLS:SNP:179 265 C T GCACAACTGGGACCTCAAC ATCCATTCCCATTCGTATCC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:883" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 213 A T GAATGATGCTCATGTCGGTC TTTCTGAGCTGGTGTCTTGG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1135 216 T C CAGTTTGATGTGAACCCGAG GCCTTTCAATGAAGTGGACC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1169 216 A G CAGTTTGATGTGAACCCGAG GCCTTTCAATGAAGTGGACC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1216 186 T C GGTCCACTTCATTGAAAGGC TCTTCTCAATGCGACCTTTG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1243" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 186 C T GGTCCACTTCATTGAAAGGC TCTTCTCAATGCGACCTTTG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1357 149 A G CCACTGTTCTTTGTTGTCGC TTCTGCCAGTCTTCTCATGG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1573 253 T C GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1633 253 G A GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1687" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 253 A G GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1813 109 C G GGCTCAACTTGGATTGTGTG TGCCTTGTGCAAGTAACTCC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1915 193 A G GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1933 193 G A GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:1942" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 193 G A GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2152 297 A C TAGAGCTTCTGGAAGCCACC TTGAGATCGAGCATGGAGAG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2242 179 G C GTCCCTAGACACCTGGAAGC ATGCTGTGCACTTGAGGTTC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2257 179 C T GTCCCTAGACACCTGGAAGC ATGCTGTGCACTTGAGGTTC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2329" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 119 C T CAAGCTGAAAGAAGAAGCCC ATGCTGTGCACTTGAGGTTC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2513 132 A G TCAGCCTTCGAAATGCATAG TCCACCTGTCTTCACCTCAG 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2725 256 G T TGAGGTGAAGACAGGTGGAG TCGCAGACCCACTTTGATAC 0 0 0\n", | |
| "k65_175750:SAMTOOLS:SNP:2741 256 G A TGAGGTGAAGACAGGTGGAG TCGCAGACCCACTTTGATAC 0 0 0\n", | |
| "k67_154235:SAMTOOLS:SNP:606" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 260 C T ACCATGCCTTCTCCGACTAC AGCATAGGTACAAGGGCCAG 0 0 0\n", | |
| "k67_154235:SAMTOOLS:SNP:662 260 T C ACCATGCCTTCTCCGACTAC AGCATAGGTACAAGGGCCAG 0 0 0\n", | |
| "k67_155965:SAMTOOLS:SNP:54 124 T C GTAGGCAACGAAGTCATCCC AGGGAAACCGCAGTAGACAC 0 0 0\n", | |
| "k67_155965:SAMTOOLS:SNP:122" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 124 A G GTAGGCAACGAAGTCATCCC AGGGAAACCGCAGTAGACAC 0 0 0\n", | |
| "k67_156400:SAMTOOLS:SNP:158 183 A C TACTGCGTTAACTGGCTTCG CGGTGAGAACAGGACTGAAG 0 0 0\n", | |
| "k67_61933:SAMTOOLS:SNP:50" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 106 A G CTGCAACGATATTTGACTTGG ATCTGCAGCCTATCGTGAAG 0 0 0\n", | |
| "k69_134502:SAMTOOLS:SNP:574 153 A C CTCTTTACGTGCCTCGTCAG CGAAACCCGAATAGAAGCTC 0 0 0\n", | |
| "k69_155926:SAMTOOLS:SNP:90" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 138 C T TCTCAATCTCTCCCTTTCCC GCTCAAGGCAATAAAGCTCC 0 0 0\n", | |
| "k69_155926:SAMTOOLS:SNP:909 271 C T TCTCTACGATTCGATGGTGC GCAGCATTCACGTCTTTAGG 0 0 0\n", | |
| "k69_168089:SAMTOOLS:SNP:683" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 289 T C AGGTCCTCAGGAATTTCAGG CTTGGAGCAAATCAAACAGC 0 0 0\n", | |
| "k69_170272:SAMTOOLS:SNP:117" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 108 G A GATAGTAATGCCACGTTCCG TTTGACCGACTCTCTTGTGG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:636 273 A G TGCTTCAAGACCACCAGAAG GGGAGGTTCCGTTAATTGTG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:717" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 273 A G TGCTTCAAGACCACCAGAAG GGGAGGTTCCGTTAATTGTG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:756 209 G A TCTGGAGACACTCCTGCATC TCGAGATGGCTTTGTTATGG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:777 209 T C TCTGGAGACACTCCTGCATC TCGAGATGGCTTTGTTATGG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:903" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 276 A T ATACCAAAGTTCCAGCACCC ATGGATATCGGATTCATGGG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:1101 217 T A GGAGGTAGCACAAGCAGTTG AGATTGACAAGGTGCGAGC 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:1107 217 G A GGAGGTAGCACAAGCAGTTG AGATTGACAAGGTGCGAGC 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:1188 273 A G TTGTTATGGCGTATGGGATG CGTAATTTCAGCAGTGAGGG 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:1392" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 248 C T CCCTCACTGCTGAAATTACG GAAATCCGCCAGACCTACTC 0 0 0\n", | |
| "k69_176262:SAMTOOLS:SNP:1401 248 T C CCCTCACTGCTGAAATTACG GAAATCCGCCAGACCTACTC 0 0 0\n", | |
| "k69_177402:SAMTOOLS:SNP:232" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 289 C T GGCATGAAAGTGGCTAGTTG CAAGTGGATGTGGTAGTGGC 0 0 0\n", | |
| "k69_198297:SAMTOOLS:SNP:352 180 G A GATGGTGCAAACAACGAATC TGAAATTGCAAAGAGGAACG 0 0 0\n", | |
| "k69_198297:SAMTOOLS:SNP:434 180 C T GATGGTGCAAACAACGAATC TGAAATTGCAAAGAGGAACG 0 0 0\n", | |
| "k69_224731:SAMTOOLS:SNP:927" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 206 A G TTCTGGAAACAAGACCCTCC CACAAATTTATGGTCTGCCG 0 0 0\n", | |
| "k69_224731:SAMTOOLS:SNP:1323 284 A G TCTTCAGACACCCAGTTTGC ATCTGCTGCTCGTATGATCG 0 0 0\n", | |
| "k69_224731:SAMTOOLS:SNP:1350 284 A G TCTTCAGACACCCAGTTTGC ATCTGCTGCTCGTATGATCG 0 0 0\n", | |
| "k69_224731:SAMTOOLS:SNP:1551 205 G A TCATACGAGCAGCAGATTCC CGACACACTCCCAATCTACG 0 0 0\n", | |
| "k69_244399:SAMTOOLS:SNP:62" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 171 C G TCCTCTCATTCTCTAGGCCAC GTTGACGATGACGTGGATTC 0 0 0\n", | |
| "k69_244399:SAMTOOLS:SNP:168 230 G T GAACGAAACGAGCGGTAGTAG ATTTCGTTCTCAATCCCTGC 0 0 0\n", | |
| "k69_244399:SAMTOOLS:SNP:375 249 C T GTTGGAAGAGCTGAGGAAGC GTGTTCGTCCAACCACTTTG 0 0 0\n", | |
| "k69_281357:SAMTOOLS:SNP:1352" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 276 A G CTATCATTTCCATTGCCACG CAGGCCCATCTTCCTATCTC 0 0 0\n", | |
| "k69_321427:SAMTOOLS:SNP:160 148 T C AATGCGGCTCTAATCACCTC AAAGGCCGATTTCCTGTTC 0 0 0\n", | |
| "k69_321427:SAMTOOLS:SNP:214" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 212 G C AAAGAGAACAGGAAATCGGC CGGTAAAGAATTGGCAAGTG 0 0 0\n", | |
| "k69_321427:SAMTOOLS:SNP:246 212 C A AAAGAGAACAGGAAATCGGC CGGTAAAGAATTGGCAAGTG 0 0 0\n", | |
| "k69_321838:SAMTOOLS:SNP:256" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 251 C T CTAGTTTCACAGCTGCTCCG GCTTGAACCAGCTCTCTTCC 0 0 0\n", | |
| "k69_321838:SAMTOOLS:SNP:266 251 C G CTAGTTTCACAGCTGCTCCG GCTTGAACCAGCTCTCTTCC 0 0 0\n", | |
| "k69_321838:SAMTOOLS:SNP:337" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 273 G A CAAACGCAGCCTAAGTCAAG GCTTAGGTTTGGGAAGATGC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:869" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 277 C A ATTCAATGCAAGGCATCAAG AGTGGTGGTATGTTTGCGAC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:932 277 G T ATTCAATGCAAGGCATCAAG AGTGGTGGTATGTTTGCGAC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1080" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 287 T G AGCTAAGTGAACATCCATGCC TCCGTTTCCGGATTCAAC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1156 287 C T AGCTAAGTGAACATCCATGCC TCCGTTTCCGGATTCAAC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1479" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 214 A G AAATAATTGAAAGACCCGCC CGAATTCATTTGCTCTACGG 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1504 214 C T AAATAATTGAAAGACCCGCC CGAATTCATTTGCTCTACGG 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1743" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 159 A C AGCATATTGGGAAGAGACGG GTTCAATAAACGCGCATCAG 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1866 230 T C AGCATATTGGGAAGAGACGG TCCCTTATTATTGCGTTCGTC 0 0 0\n", | |
| "k69_323478:SAMTOOLS:SNP:1932" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 212 G T GATGCGCGTTTATTGAACTG TAGACCTTTGCCTTGTTGGG 0 0 0\n", | |
| "k69_323609:SAMTOOLS:SNP:119" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 99 C T CGGTTAAAGAAGAGCAAGCC GCATCATCGTCATCATCCTC 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:1050" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 165 C T TAGCGTCTCTATGCCACCAC GGATGAGATGGCTGTTGTTG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:1707 226 G T AGCTCGACAGCAAGAACATC TTAGAGATCGGGTCCTACCG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:1710" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 226 G A AGCTCGACAGCAAGAACATC TTAGAGATCGGGTCCTACCG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:1937 239 T G ACGCTTTCCACTAAGTTCGG TGTTACCGCTCTCTGTGCTC 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:1956 273 C G ACGCTTTCCACTAAGTTCGG CAATGTGGAGCAGTTCCTTG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2172 221 C T TGCTTCTTCAGGGATGTCAG CTATCCATGTGGCGGTGTAG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2277" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 154 G A CTACACCGCCACATGGATAG TTCTCTTCTTGGATGGGAGG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2307 154 T A CTACACCGCCACATGGATAG TTCTCTTCTTGGATGGGAGG 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2367 255 G A CTACACCGCCACATGGATAG CCATATGGAGAGGTGTTCCC 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2396 255 C T CTACACCGCCACATGGATAG CCATATGGAGAGGTGTTCCC 0 0 0\n", | |
| "k69_324482:SAMTOOLS:SNP:2598" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 289 A G ATCACCTCCCATCCAAGAAG GTCTAGCGCAATTGTATGCC 0 0 0\n", | |
| "k69_324568:SAMTOOLS:SNP:229 292 T C AGCACCCATTGATTGATGTC TCCTTCCCTGTCTCCAAATC 0 0 0\n", | |
| "k69_324934:SAMTOOLS:SNP:248 297 A G ACGCAACAATGAAACCAATC TTTGTTAGCAATCCCGACAC 0 0 0\n", | |
| "k69_324942:SAMTOOLS:SNP:458" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 243 G T CACCTGCAGCTATGATGGTC TGTTACACCAACTGGATGGG 0 0 0\n", | |
| "k69_324942:SAMTOOLS:SNP:719 288 A G TGTACAATGGTGGGTGGAAG GCCTCCTTCCAGAGAGTGAC 0 0 0\n", | |
| "k69_324942:SAMTOOLS:SNP:791 288 A T TGTACAATGGTGGGTGGAAG GCCTCCTTCCAGAGAGTGAC 0 0 0\n", | |
| "k69_324942:SAMTOOLS:SNP:818 191 C G CAAATCGACCATAGCCAGTG GCCTCCTTCCAGAGAGTGAC 0 0 0\n", | |
| "k69_324942:SAMTOOLS:SNP:983" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 242 G A GTCACTCTCTGGAAGGAGGC CAAACATACATCCGAATCGC 0 0 0\n", | |
| "k69_50501:SAMTOOLS:SNP:444 283 T C TGTCAGGGAAGAACGAAGAG CTGGTTTCCGGTAGAAGAGG 0 0 0\n", | |
| "k69_93535:SAMTOOLS:SNP:661 210 A G CAGGATGGGTTGTTTCACAC ATCTCGGACACCTGAACTCC 0 0 0\n", | |
| "k69_93535:SAMTOOLS:SNP:887" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 270 A G GACATTGTTGCTGATGAGGC CCTATGAAGCTTCCCTGTCC 0 0 0\n", | |
| "k69_93535:SAMTOOLS:SNP:1141 229 C T TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 0 0 0\n", | |
| "k69_93535:SAMTOOLS:SNP:1147 229 C G TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 0 0 0\n", | |
| "k69_93535:SAMTOOLS:SNP:1336 285 G A GGTTTGTTTGAAGAAACGGG ACCCAGGTTTCAACTCCTTG 0 0 0\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 259 T G CAAATCCAAACATGTTATCTTCTTC TTTCATAAACTCGTCCGGTG 0 0 0\n", | |
| "k69_98089:SAMTOOLS:SNP:550 228 G A ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 0 0 0\n", | |
| "k69_98089:SAMTOOLS:SNP:625 228 A G ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 0 0 0" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 10 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "-----\n", | |
| "Now we will design candidate primer pairs for several for a subset of the targets and request melt prediction from the Umelt web service provided by the Wittwer Lab at the University of Utah\n", | |
| " See https://www.dna.utah.edu/umelt/umelt.html \n", | |
| "\n", | |
| "First form a short-list of targets" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "!tail -n 5 test_data/targets > HRM_targets\n", | |
| "!cat HRM_targets" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "outputs": [ | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| "k69_93535:SAMTOOLS:SNP:1147\n", | |
| "k69_93535:SAMTOOLS:SNP:1336\n", | |
| "k69_98089:SAMTOOLS:SNP:30\n", | |
| "k69_98089:SAMTOOLS:SNP:550\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 15 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "Now run the design tool specifying melt prediction with the -u (umelt) flag\n", | |
| "\n", | |
| "**Note** that this design uses the default size range of 100-300 bp which may not be ideal" | |
| ] | |
| }, | |
| { | |
| "cell_type": "code", | |
| "collapsed": false, | |
| "input": [ | |
| "%run design_HRM_primers.py -i test_data/targets.fasta -g test_data/targets.gff -T HRM_targets -u" | |
| ], | |
| "language": "python", | |
| "metadata": {}, | |
| "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\n", | |
| "k69_93535:SAMTOOLS:SNP:1147" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 229 C G TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 87.2 87.25 0.05\n", | |
| "k69_93535:SAMTOOLS:SNP:1147" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 137 C G GGACAGGGAAGCTTCATAGG TGTTTGGTATCGTTTCACCC 85.75 85.75 0.0\n", | |
| "k69_93535:SAMTOOLS:SNP:1147" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 258 C G GGACAGGGAAGCTTCATAGG TCAGCTCCAATCGTAAGAGG 87.8 87.8 0.0\n", | |
| "k69_93535:SAMTOOLS:SNP:1147" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 257 C G TGCTAATGACAACGAGGAGG TTTGCAATCTCCTCCAATTTC 87.25 87.25 0.0\n", | |
| "k69_93535:SAMTOOLS:SNP:1147" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 259 C G GGACAGGGAAGCTTCATAGG CTCAGCTCCAATCGTAAGAGG 87.8 87.8 0.0\n", | |
| "k69_93535:SAMTOOLS:SNP:1336" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 285 G A GGTTTGTTTGAAGAAACGGG ACCCAGGTTTCAACTCCTTG 89.15 88.8 0.35\n", | |
| "k69_93535:SAMTOOLS:SNP:1336" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 259 G A AACAAGATTTGTGTCGGGTG ACCCAGGTTTCAACTCCTTG 89.2 88.8 0.4\n", | |
| "k69_93535:SAMTOOLS:SNP:1336" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 153 G A TTAGAAGGAACGCGAGAAGG ACCCAGGTTTCAACTCCTTG 88.8 88.65 0.15\n", | |
| "k69_93535:SAMTOOLS:SNP:1336" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 289 G A GGTTTGTTTGAAGAAACGGG AGCAACCCAGGTTTCAACTC 89.25 89.05 0.2\n", | |
| "k69_93535:SAMTOOLS:SNP:1336" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 263 G A AACAAGATTTGTGTCGGGTG AGCAACCCAGGTTTCAACTC 89.25 89.05 0.2\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 259 T G CAAATCCAAACATGTTATCTTCTTC TTTCATAAACTCGTCCGGTG 93.7 93.7 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 257 T G CAAATCCAAACATGTTATCTTCTTC TCATAAACTCGTCCGGTGAC 93.7 93.7 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 177 T G CAAATCCAAACATGTTATCTTCTTC TTTATGTCGCTGTAGTTTGCG 92.25 92.25 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 289 T G CAAATCCAAACATGTTATCTTCTTC ATCAGAAAGCGTGGAATACG 93.7 93.7 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:30" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 284 T G CAAATCCAAACATGTTATCTTCTTC AAAGCGTGGAATACGCATC 93.7 93.7 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:550" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 228 G A ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 85.55 85.65 0.1\n", | |
| "k69_98089:SAMTOOLS:SNP:550" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 151 G A AAAGAACGGGATGATAACGG GGCCGAATATACATACAACGTC 83.85 83.7 0.15\n", | |
| "k69_98089:SAMTOOLS:SNP:550" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 227 G A ATGGAGAAGGTCGAGGTCAG GGCCGAATATACATACAACGTC 85.4 85.4 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:550" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 229 G A GAATGGAGAAGGTCGAGGTC GGCCGAATATACATACAACGTC 85.4 85.4 0.0\n", | |
| "k69_98089:SAMTOOLS:SNP:550" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 154 G A AAAGAACGGGATGATAACGG AACGGCCGAATATACATACAAC 84.25 84.0 0.25\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 228 A G ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 85.55 85.85 0.3\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 151 A G AAAGAACGGGATGATAACGG GGCCGAATATACATACAACGTC 83.85 84.2 0.35\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 227 A G ATGGAGAAGGTCGAGGTCAG GGCCGAATATACATACAACGTC 85.4 85.8 0.4\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 229 A G GAATGGAGAAGGTCGAGGTC GGCCGAATATACATACAACGTC 85.4 85.8 0.4\n", | |
| "k69_98089:SAMTOOLS:SNP:625" | |
| ] | |
| }, | |
| { | |
| "output_type": "stream", | |
| "stream": "stdout", | |
| "text": [ | |
| " 150 A G AGAACGGGATGATAACGGTG CGGCCGAATATACATACAACG 83.95 84.2 0.25" | |
| ] | |
| } | |
| ], | |
| "prompt_number": 2 | |
| }, | |
| { | |
| "cell_type": "markdown", | |
| "metadata": {}, | |
| "source": [ | |
| "Inspection of the output shows us that \n", | |
| "+ while the SNP in k69_93535 at position 1147 is a poor prospect for HRM amplicon design, we can design one for that at position 1336 with a reasonable difference (0.4 deg C) between the precited melt temperatures of homozygotes. \n", | |
| "+ likewise for contig k69_98089 the SNP at position 625 is a good choice and that a predcted Tm difference of ~0.4 Degrees can be achieved" | |
| ] | |
| } | |
| ], | |
| "metadata": {} | |
| } | |
| ] | |
| } |
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