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cfljam/Designing CAPS Markers.ipynb

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iPython Notebook describing basic use of the Python PCR Design Tools
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using_PCR_design_python_tools_1.ipynb
{
"metadata": {
"name": "using_PCR_design_python_tools_1"
},
"nbformat": 2,
"worksheets": [
{
"cells": [
{
"cell_type": "markdown",
"source": [
"#Bulk Design of PCR Primer Sets Using Python Tools",
"",
"####John McCallum john.mccallum@plantandfood.co.nz",
"",
"",
"This Notebook describes use of the python and shell tools for bulk design of PCR marker assays from the Unix command-line.",
"",
"",
"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",
"outputs": []
},
{
"cell_type": "markdown",
"source": [
"---",
"Learn about iPython at http://ipython.org. The only conventions needed to understand the following are ",
"",
"+ ! denotes a shell command",
"+ % denotes an iPython magic command -like *run*"
]
},
{
"cell_type": "markdown",
"source": [
"----",
"**Input Files**",
"",
"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",
"",
"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, deafulting to colon (but an alternate delimiter e.g. '_' can be specified)"
]
},
{
"cell_type": "code",
"collapsed": false,
"input": [
"!head -n5 test_data/targets"
],
"language": "python",
"outputs": [
{
"output_type": "stream",
"stream": "stdout",
"text": [
"JR844712:SAMTOOLS:SNP:310",
"JR844712:SAMTOOLS:SNP:353",
"JR844712:SAMTOOLS:SNP:470",
"JR844712:SAMTOOLS:SNP:572",
"JR844712:SAMTOOLS:SNP:573"
]
}
],
"prompt_number": 2
},
{
"cell_type": "markdown",
"source": [
"The input gff3 file describing the polymorphisms must follow the more restrictive GVF convention for des desribed at http://www.sequenceontology.org/resources/gvf.html",
"",
"Most importantly ",
"the reference and variant sequence must be given in field 9 using the attributes 'Reference_seq' and 'Variant_seq'.",
"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.",
"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",
"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",
"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",
"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",
"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",
"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",
"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",
"outputs": [
{
"output_type": "stream",
"stream": "stdout",
"text": [
"usage: design_HRM_primers.py [-h] -i IN_FILE -g GFF_FILE -T TARGET_FILE [-u]",
" [-n MAX_PRIMERS] [-p PROD_MIN_SIZE]",
" [-P PROD_MAX_SIZE] [-l OPT_PRIMER_LENGTH]",
" [-m MAX_TM_DIFF] [-t OPTIMUM_TM]",
" [-G OPT_GC_PERCENT] [-x MAXPOLYX] [-c GC_CLAMP]",
" [-d TARGET_DELIM]",
"",
"Primer set design and melt prediction parameters",
"",
"optional arguments:",
" -h, --help show this help message and exit",
" -i IN_FILE input sequence file, required",
" -g GFF_FILE input gff file with SNP and indels, required",
" -T TARGET_FILE input target SNP file, required",
" -u do uMelt prediction, optional",
" -n MAX_PRIMERS maximum number of primer pairs to return, default=5",
" -p PROD_MIN_SIZE minimum product size",
" -P PROD_MAX_SIZE maximum product size",
" -l OPT_PRIMER_LENGTH optimum primer length",
" -m MAX_TM_DIFF maximum tm difference between primers",
" -t OPTIMUM_TM optimum Tm for primers, recommend range 59 to 61",
" -G OPT_GC_PERCENT optimum GC percentage of primers",
" -x MAXPOLYX maximum polyx, recommend less than 4",
" -c GC_CLAMP number of C/Gs at end, recommend 2",
" -d TARGET_DELIM variant indentifier delimiter, used to separate",
" sequence ID from rest"
]
}
],
"prompt_number": 7
},
{
"cell_type": "code",
"collapsed": false,
"input": [
"ls test_data/targets*"
],
"language": "python",
"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",
"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",
"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",
"JR844712:SAMTOOLS:SNP:310"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 243 G A TGCTGTACTTAATAGCGCCG CACATTTCCAAGGTCACCAG 0 0 0",
"JR844712:SAMTOOLS:SNP:353 134 T C CTGGTGACCTTGGAAATGTG GATCATCAGGATCAGCATGG 0 0 0",
"JR844712:SAMTOOLS:SNP:470 270 C T AGTTGTCCATGCTGATCCTG CACATACAGATTGGCCTTGC 0 0"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 0",
"JR844712:SAMTOOLS:SNP:572 187 T C CATGCGGTATCATTGGACTC CACATACAGATTGGCCTTGC 0 0 0",
"JR844712:SAMTOOLS:SNP:573"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 187 G A CATGCGGTATCATTGGACTC CACATACAGATTGGCCTTGC 0 0 0",
"JR845107:SAMTOOLS:SNP:546 211 C T TCGAAACAAGCACTGGGTAG TAAATCCCTCAGTCCAAGCC 0 0 0",
"JR845347:SAMTOOLS:SNP:410"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 280 G A GAGGTTTATCTGGACTGCCC GCAGCCAAGTATTCATCTGG 0 0 0",
"JR845347:SAMTOOLS:SNP:418 226 G A AGGAGGCAGAAGAACGAAAG TCCATCCTTCAAGGTGTACG 0 0 0",
"JR845763:SAMTOOLS:SNP:164 297 C T GAAGAAGAAGAGAATGCGGC CCACAGTTTCTGGACACAGG 0 0 0",
"JR848350:SAMTOOLS:SNP:642"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 181 C T TCTCTTCTCCAGCCTCCTTC TACGACAGAAGAGGCGACAC 0 0 0",
"JR849045:SAMTOOLS:SNP:259 120 C G TCTGCTCTTAAGGCTTCATCTC TTGGACACAATCAAGGAAGC 0 0 0",
"JR849045:SAMTOOLS:SNP:448 185 C T GCCCAGCCTCTTCAGAGTAG TTTGAGATTGCTCCATCAGC 0 0 0",
"JR849045:SAMTOOLS:SNP:484"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 134 G A TAATTGAGCTGCCGATTCAC TTTGAGATTGCTCCATCAGC 0 0 0",
"JR849045:SAMTOOLS:SNP:620 223 A G GACATGGCATGAGGTTTGAG ATGGGTCAATCAGGAGAAGG 0 0 0",
"JR849805:SAMTOOLS:SNP:135 169 G A GCTTTCTTTGCATTTATTGCC TTCAGTCTTGCCATCTTTGC 0 0 0",
"JR850172:SAMTOOLS:SNP:101"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 230 G A CCATGGCAACTAAGAAAGCC CGAGCTTAAACGATGCTTTG 0 0 0",
"JR852189:SAMTOOLS:SNP:58 196 C A CAAATTCTCTCAATTCTAAGCCC TCCAACTCGTTGAGCGTATC 0 0 0",
"JR852189:SAMTOOLS:SNP:64 196 A C CAAATTCTCTCAATTCTAAGCCC TCCAACTCGTTGAGCGTATC 0 0 0",
"JR852189:SAMTOOLS:SNP:253"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 208 C T GGCCGATACTTGAAGAAAGG ACTCCACCCAGCAATTTACC 0 0 0",
"JR852189:SAMTOOLS:SNP:447 144 T C ATCAATCCCGTGCTTCTACC CCAAACAGCGTTACATGGAC 0 0 0",
"JR852189:SAMTOOLS:SNP:537 209 C T ATCAATCCCGTGCTTCTACC GAAGTACAAATTCCATCCAGAGC 0 0 0",
"JR854385:SAMTOOLS:SNP:128"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 182 G A AGAATGGCTTCCAAAGTTCTG AGAGGGAAGGAGGGAGAGAG 0 0 0",
"JR854385:SAMTOOLS:SNP:171 182 C T AGAATGGCTTCCAAAGTTCTG AGAGGGAAGGAGGGAGAGAG 0 0 0",
"JR854446:SAMTOOLS:SNP:166 222 G A AGAGGAAGTAAATGAACACGGAG GCTGCTGCTGTTGCTAACTC 0 0 0",
"JR854446:SAMTOOLS:SNP:207"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 198 T G CTGTCCTTAATGACTCCTCGG CTGGATTGTGCAGATGGAAC 0 0 0",
"JR854792:SAMTOOLS:SNP:228 221 G T TAAACAACAATGGCGGAGAC AGAAGCATCTTCCCAGAAGG 0 0 0",
"JR857827:SAMTOOLS:SNP:56 232 T C TAATAATCGGCCATGGCTTC ACCTTTGGAATTTCTGGCAC 0 0 0",
"JR857827:SAMTOOLS:SNP:99"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 192 T A TCTTCTTCGCCTTTGCTTTC ACCTTTGGAATTTCTGGCAC 0 0 0",
"JR863117:SAMTOOLS:SNP:120 167 G A TTCACATTCATGGCCGTAAG TGTAGGAACTGTAGGCGTGG 0 0 0",
"k31_439587:SAMTOOLS:SNP:187 215 G T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0",
"k31_439587:SAMTOOLS:SNP:212"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 215 C T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0",
"k31_439587:SAMTOOLS:SNP:265 215 C T GTGGCTTTCCACACAAACAC TGTCGAAGATGAAGTTTCCG 0 0 0",
"k31_439587:SAMTOOLS:SNP:342 169 T G TGTTCCGACTCAGATCAAGC GGAGAAGCTGAAGACCCTTG 0 0 0",
"k31_439587:SAMTOOLS:SNP:537 134 A G ATTCCCAGACAGAACAAGGG ACCATCCCTGAATCCATAGG 0 0 0",
"k31_439587:SAMTOOLS:SNP:557"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 250 A C ATTCCCAGACAGAACAAGGG ACGTAGTCATCGCCAACAAC 0 0 0",
"k31_439587:SAMTOOLS:SNP:598 250 T C ATTCCCAGACAGAACAAGGG ACGTAGTCATCGCCAACAAC 0 0 0",
"k31_565787:SAMTOOLS:SNP:109 298 C T CCTGGATTGGAATCTTCTGG AGAACCTCCTCTTCGTCAGC 0 0 0",
"k31_565787:SAMTOOLS:SNP:154 219 C T GGCACAAACGCTGTCACTAC AGAACCTCCTCTTCGTCAGC 0 0 0",
"k31_565787:SAMTOOLS:SNP:268"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 183 G A CTAGTAAGGTCGGCAAAGGC AGAACCTCCTCTTCGTCAGC 0 0 0",
"k31_714243:SAMTOOLS:SNP:151 257 C A TCTGGGAAGAGTATCGGACC TTCTTGCAGAAGTGTCGAGG 0 0 0",
"k31_714243:SAMTOOLS:SNP:342 289 T C GGTACTGGACTTCAGGTGGG CATCATCATAGGAGGAGCCC 0 0 0",
"k31_754967:SAMTOOLS:SNP:904"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 233 C T CCCAATCATCCAATCTCAAG AGTATCCAAACGGGTCAAGG 0 0 0",
"k31_754967:SAMTOOLS:SNP:910 233 T C CCCAATCATCCAATCTCAAG AGTATCCAAACGGGTCAAGG 0 0 0",
"k33_381591:SAMTOOLS:SNP:123"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 292 T C TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0",
"k33_381591:SAMTOOLS:SNP:169 292 A T TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0",
"k33_381591:SAMTOOLS:SNP:239 292 T C TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0",
"k33_381591:SAMTOOLS:SNP:240 292 C A TACATGGCATGGGAGATGAC CAATCACCGACAGTCAAACC 0 0 0",
"k35_208328:SAMTOOLS:SNP:272"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 281 A C GAAAGTGCTCACAGGAGCTG ATTTCTGGACCTCAAATGGC 0 0 0",
"k35_208328:SAMTOOLS:SNP:443 227 A G AAAGAAAGTGGTGGAGGTGG ATTTCTGGACCTCAAATGGC 0 0 0",
"k35_208328:SAMTOOLS:SNP:505 235 C T TACCTGCGTCACCGTTAGAG TTCAACTTTGGCAGGCTATG 0 0 0",
"k35_653504:SAMTOOLS:SNP:85 233 C T GGAAATGGCTGAATTGTTCTG ATATTTGGCAGTTTGGGAGC 0 0 0",
"k35_653504:SAMTOOLS:SNP:208"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 196 A G TACGCAGAAACAAGACCGAC ATATTTGGCAGTTTGGGAGC 0 0 0",
"k35_653504:SAMTOOLS:SNP:216 196 A G TACGCAGAAACAAGACCGAC ATATTTGGCAGTTTGGGAGC 0 0 0",
"k35_653504:SAMTOOLS:SNP:469 230 C A ATCTCCGATCTGGTCAAACC CAGCTTCCTCTTCTCATCCC 0 0 0",
"k39_566848:SAMTOOLS:SNP:1781"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 99 T G GTGATGGTTGAGCCAGTGTC CCCACGTTAGCCTCTTTAGC 0 0 0",
"k41_520516:SAMTOOLS:SNP:1229 216 C T CCAATCAAGCAGCCAATTAC TAAGGCAAGATAGTGGTGCG 0 0 0",
"k41_520516:SAMTOOLS:SNP:1246 216 C T CCAATCAAGCAGCCAATTAC TAAGGCAAGATAGTGGTGCG 0 0 0",
"k43_486433:SAMTOOLS:SNP:1177"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 286 A C GTAGGAAGCTTTGTCCTGGC TCTGAGGAAGAATTGGAGGG 0 0 0",
"k43_486433:SAMTOOLS:SNP:1180 286 A G GTAGGAAGCTTTGTCCTGGC TCTGAGGAAGAATTGGAGGG 0 0 0",
"k43_486433:SAMTOOLS:SNP:1762 153 A G ATAATTGCAGAGACATGGCG AGCAGTCTGACGTTCCTGAG 0 0 0",
"k45_231452:SAMTOOLS:SNP:679"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 155 A G TGGGAAGAATAGGATGGAGC AATGGCAGAGTGGAAGATCC 0 0 0",
"k45_463949:SAMTOOLS:SNP:142 221 T C AACTTGGGTTTGTCGCTCTC ATCCATCCGCCTGAAGTAAC 0 0 0",
"k49_172131:SAMTOOLS:SNP:45"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 184 A G CATAACATACTTACATACACACCGC CTGTTTAGCTTGCCCACATC 0 0 0",
"k49_172131:SAMTOOLS:SNP:328 271 C T TGTGGGCAAGCTAAACAGAC GGCGTCTGCTTCCAATAAAC 0 0 0",
"k49_172131:SAMTOOLS:SNP:337 271 C T TGTGGGCAAGCTAAACAGAC GGCGTCTGCTTCCAATAAAC 0 0 0",
"k49_172131:SAMTOOLS:SNP:685"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 205 A G CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0",
"k49_172131:SAMTOOLS:SNP:719 205 A C CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0",
"k49_172131:SAMTOOLS:SNP:766 205 A T CAGCCAGAAACACAGCATTC ATGTCTGCTGAAACCACGTC 0 0 0",
"k49_198840:SAMTOOLS:SNP:439"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 270 A G TCATAGACGAAGCAAGACGG CCTCTCTCCTCTCCGACATC 0 0 0",
"k49_198840:SAMTOOLS:SNP:442 270 A G TCATAGACGAAGCAAGACGG CCTCTCTCCTCTCCGACATC 0 0 0",
"k61_221473:SAMTOOLS:SNP:1250 269 T C CACGACCAAATGCAAAGAAC ATGAGAAGGATTTGATGGGC 0 0 0",
"k61_221473:SAMTOOLS:SNP:1328"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 269 C T CACGACCAAATGCAAAGAAC ATGAGAAGGATTTGATGGGC 0 0 0",
"k63_198617:SAMTOOLS:SNP:81 111 C T GACTTTATTCAAAGCAAACACTGAC TTGTATAACCCAGGGCCTTC 0 0 0",
"k65_172796:SAMTOOLS:SNP:467"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 255 C T AGTTCATAGCCACGCTGTTG ATTGCTCGGATGAGGGATAC 0 0 0",
"k65_172796:SAMTOOLS:SNP:533 173 C T AGTGACATTCGGCCTATTCC TTGGTGTAGCCAACGCTTAG 0 0 0",
"k65_172796:SAMTOOLS:SNP:665 209 T C TCCAAAGCGCATACTACAGC GGACCATGAACACAGCAAAG 0 0 0",
"k65_173050:SAMTOOLS:SNP:116"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 205 A G TAACGGGTACCTCCTGGAAC CTAGCTCTTCCGGGTTTGAG 0 0 0",
"k65_173050:SAMTOOLS:SNP:355 99 T C CATCGGTTGTCTTCCATGAG AGTAGCCTTCAGCAACGACC 0 0 0",
"k65_173050:SAMTOOLS:SNP:361 99 C T CATCGGTTGTCTTCCATGAG AGTAGCCTTCAGCAACGACC 0 0 0",
"k65_175163:SAMTOOLS:SNP:87"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 278 G A CTAAATCAACAAGCTCCCTCG CCGTTTCACTATTCGTCACC 0 0 0",
"k65_175163:SAMTOOLS:SNP:137 284 G A AGTCAGCTTCGGGTGTACG ACACGTCTTGGTCCAAATCC 0 0 0",
"k65_175163:SAMTOOLS:SNP:179 265 C T GCACAACTGGGACCTCAAC ATCCATTCCCATTCGTATCC 0 0 0",
"k65_175750:SAMTOOLS:SNP:883"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 213 A T GAATGATGCTCATGTCGGTC TTTCTGAGCTGGTGTCTTGG 0 0 0",
"k65_175750:SAMTOOLS:SNP:1135 216 T C CAGTTTGATGTGAACCCGAG GCCTTTCAATGAAGTGGACC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1169 216 A G CAGTTTGATGTGAACCCGAG GCCTTTCAATGAAGTGGACC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1216 186 T C GGTCCACTTCATTGAAAGGC TCTTCTCAATGCGACCTTTG 0 0 0",
"k65_175750:SAMTOOLS:SNP:1243"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 186 C T GGTCCACTTCATTGAAAGGC TCTTCTCAATGCGACCTTTG 0 0 0",
"k65_175750:SAMTOOLS:SNP:1357 149 A G CCACTGTTCTTTGTTGTCGC TTCTGCCAGTCTTCTCATGG 0 0 0",
"k65_175750:SAMTOOLS:SNP:1573 253 T C GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1633 253 G A GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1687"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 253 A G GCCAAGGAAGCCTACATTTC CACACAATCCAAGTTGAGCC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1813 109 C G GGCTCAACTTGGATTGTGTG TGCCTTGTGCAAGTAACTCC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1915 193 A G GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1933 193 G A GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0",
"k65_175750:SAMTOOLS:SNP:1942"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 193 G A GGCTCAACTTGGATTGTGTG TTATGTGCATGTGGTAGGGC 0 0 0",
"k65_175750:SAMTOOLS:SNP:2152 297 A C TAGAGCTTCTGGAAGCCACC TTGAGATCGAGCATGGAGAG 0 0 0",
"k65_175750:SAMTOOLS:SNP:2242 179 G C GTCCCTAGACACCTGGAAGC ATGCTGTGCACTTGAGGTTC 0 0 0",
"k65_175750:SAMTOOLS:SNP:2257 179 C T GTCCCTAGACACCTGGAAGC ATGCTGTGCACTTGAGGTTC 0 0 0",
"k65_175750:SAMTOOLS:SNP:2329"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 119 C T CAAGCTGAAAGAAGAAGCCC ATGCTGTGCACTTGAGGTTC 0 0 0",
"k65_175750:SAMTOOLS:SNP:2513 132 A G TCAGCCTTCGAAATGCATAG TCCACCTGTCTTCACCTCAG 0 0 0",
"k65_175750:SAMTOOLS:SNP:2725 256 G T TGAGGTGAAGACAGGTGGAG TCGCAGACCCACTTTGATAC 0 0 0",
"k65_175750:SAMTOOLS:SNP:2741 256 G A TGAGGTGAAGACAGGTGGAG TCGCAGACCCACTTTGATAC 0 0 0",
"k67_154235:SAMTOOLS:SNP:606"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 260 C T ACCATGCCTTCTCCGACTAC AGCATAGGTACAAGGGCCAG 0 0 0",
"k67_154235:SAMTOOLS:SNP:662 260 T C ACCATGCCTTCTCCGACTAC AGCATAGGTACAAGGGCCAG 0 0 0",
"k67_155965:SAMTOOLS:SNP:54 124 T C GTAGGCAACGAAGTCATCCC AGGGAAACCGCAGTAGACAC 0 0 0",
"k67_155965:SAMTOOLS:SNP:122"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 124 A G GTAGGCAACGAAGTCATCCC AGGGAAACCGCAGTAGACAC 0 0 0",
"k67_156400:SAMTOOLS:SNP:158 183 A C TACTGCGTTAACTGGCTTCG CGGTGAGAACAGGACTGAAG 0 0 0",
"k67_61933:SAMTOOLS:SNP:50"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 106 A G CTGCAACGATATTTGACTTGG ATCTGCAGCCTATCGTGAAG 0 0 0",
"k69_134502:SAMTOOLS:SNP:574 153 A C CTCTTTACGTGCCTCGTCAG CGAAACCCGAATAGAAGCTC 0 0 0",
"k69_155926:SAMTOOLS:SNP:90"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 138 C T TCTCAATCTCTCCCTTTCCC GCTCAAGGCAATAAAGCTCC 0 0 0",
"k69_155926:SAMTOOLS:SNP:909 271 C T TCTCTACGATTCGATGGTGC GCAGCATTCACGTCTTTAGG 0 0 0",
"k69_168089:SAMTOOLS:SNP:683"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 289 T C AGGTCCTCAGGAATTTCAGG CTTGGAGCAAATCAAACAGC 0 0 0",
"k69_170272:SAMTOOLS:SNP:117"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 108 G A GATAGTAATGCCACGTTCCG TTTGACCGACTCTCTTGTGG 0 0 0",
"k69_176262:SAMTOOLS:SNP:636 273 A G TGCTTCAAGACCACCAGAAG GGGAGGTTCCGTTAATTGTG 0 0 0",
"k69_176262:SAMTOOLS:SNP:717"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 273 A G TGCTTCAAGACCACCAGAAG GGGAGGTTCCGTTAATTGTG 0 0 0",
"k69_176262:SAMTOOLS:SNP:756 209 G A TCTGGAGACACTCCTGCATC TCGAGATGGCTTTGTTATGG 0 0 0",
"k69_176262:SAMTOOLS:SNP:777 209 T C TCTGGAGACACTCCTGCATC TCGAGATGGCTTTGTTATGG 0 0 0",
"k69_176262:SAMTOOLS:SNP:903"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 276 A T ATACCAAAGTTCCAGCACCC ATGGATATCGGATTCATGGG 0 0 0",
"k69_176262:SAMTOOLS:SNP:1101 217 T A GGAGGTAGCACAAGCAGTTG AGATTGACAAGGTGCGAGC 0 0 0",
"k69_176262:SAMTOOLS:SNP:1107 217 G A GGAGGTAGCACAAGCAGTTG AGATTGACAAGGTGCGAGC 0 0 0",
"k69_176262:SAMTOOLS:SNP:1188 273 A G TTGTTATGGCGTATGGGATG CGTAATTTCAGCAGTGAGGG 0 0 0",
"k69_176262:SAMTOOLS:SNP:1392"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 248 C T CCCTCACTGCTGAAATTACG GAAATCCGCCAGACCTACTC 0 0 0",
"k69_176262:SAMTOOLS:SNP:1401 248 T C CCCTCACTGCTGAAATTACG GAAATCCGCCAGACCTACTC 0 0 0",
"k69_177402:SAMTOOLS:SNP:232"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 289 C T GGCATGAAAGTGGCTAGTTG CAAGTGGATGTGGTAGTGGC 0 0 0",
"k69_198297:SAMTOOLS:SNP:352 180 G A GATGGTGCAAACAACGAATC TGAAATTGCAAAGAGGAACG 0 0 0",
"k69_198297:SAMTOOLS:SNP:434 180 C T GATGGTGCAAACAACGAATC TGAAATTGCAAAGAGGAACG 0 0 0",
"k69_224731:SAMTOOLS:SNP:927"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 206 A G TTCTGGAAACAAGACCCTCC CACAAATTTATGGTCTGCCG 0 0 0",
"k69_224731:SAMTOOLS:SNP:1323 284 A G TCTTCAGACACCCAGTTTGC ATCTGCTGCTCGTATGATCG 0 0 0",
"k69_224731:SAMTOOLS:SNP:1350 284 A G TCTTCAGACACCCAGTTTGC ATCTGCTGCTCGTATGATCG 0 0 0",
"k69_224731:SAMTOOLS:SNP:1551 205 G A TCATACGAGCAGCAGATTCC CGACACACTCCCAATCTACG 0 0 0",
"k69_244399:SAMTOOLS:SNP:62"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 171 C G TCCTCTCATTCTCTAGGCCAC GTTGACGATGACGTGGATTC 0 0 0",
"k69_244399:SAMTOOLS:SNP:168 230 G T GAACGAAACGAGCGGTAGTAG ATTTCGTTCTCAATCCCTGC 0 0 0",
"k69_244399:SAMTOOLS:SNP:375 249 C T GTTGGAAGAGCTGAGGAAGC GTGTTCGTCCAACCACTTTG 0 0 0",
"k69_281357:SAMTOOLS:SNP:1352"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 276 A G CTATCATTTCCATTGCCACG CAGGCCCATCTTCCTATCTC 0 0 0",
"k69_321427:SAMTOOLS:SNP:160 148 T C AATGCGGCTCTAATCACCTC AAAGGCCGATTTCCTGTTC 0 0 0",
"k69_321427:SAMTOOLS:SNP:214"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 212 G C AAAGAGAACAGGAAATCGGC CGGTAAAGAATTGGCAAGTG 0 0 0",
"k69_321427:SAMTOOLS:SNP:246 212 C A AAAGAGAACAGGAAATCGGC CGGTAAAGAATTGGCAAGTG 0 0 0",
"k69_321838:SAMTOOLS:SNP:256"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 251 C T CTAGTTTCACAGCTGCTCCG GCTTGAACCAGCTCTCTTCC 0 0 0",
"k69_321838:SAMTOOLS:SNP:266 251 C G CTAGTTTCACAGCTGCTCCG GCTTGAACCAGCTCTCTTCC 0 0 0",
"k69_321838:SAMTOOLS:SNP:337"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 273 G A CAAACGCAGCCTAAGTCAAG GCTTAGGTTTGGGAAGATGC 0 0 0",
"k69_323478:SAMTOOLS:SNP:869"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 277 C A ATTCAATGCAAGGCATCAAG AGTGGTGGTATGTTTGCGAC 0 0 0",
"k69_323478:SAMTOOLS:SNP:932 277 G T ATTCAATGCAAGGCATCAAG AGTGGTGGTATGTTTGCGAC 0 0 0",
"k69_323478:SAMTOOLS:SNP:1080"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 287 T G AGCTAAGTGAACATCCATGCC TCCGTTTCCGGATTCAAC 0 0 0",
"k69_323478:SAMTOOLS:SNP:1156 287 C T AGCTAAGTGAACATCCATGCC TCCGTTTCCGGATTCAAC 0 0 0",
"k69_323478:SAMTOOLS:SNP:1479"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 214 A G AAATAATTGAAAGACCCGCC CGAATTCATTTGCTCTACGG 0 0 0",
"k69_323478:SAMTOOLS:SNP:1504 214 C T AAATAATTGAAAGACCCGCC CGAATTCATTTGCTCTACGG 0 0 0",
"k69_323478:SAMTOOLS:SNP:1743"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 159 A C AGCATATTGGGAAGAGACGG GTTCAATAAACGCGCATCAG 0 0 0",
"k69_323478:SAMTOOLS:SNP:1866 230 T C AGCATATTGGGAAGAGACGG TCCCTTATTATTGCGTTCGTC 0 0 0",
"k69_323478:SAMTOOLS:SNP:1932"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 212 G T GATGCGCGTTTATTGAACTG TAGACCTTTGCCTTGTTGGG 0 0 0",
"k69_323609:SAMTOOLS:SNP:119"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 99 C T CGGTTAAAGAAGAGCAAGCC GCATCATCGTCATCATCCTC 0 0 0",
"k69_324482:SAMTOOLS:SNP:1050"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 165 C T TAGCGTCTCTATGCCACCAC GGATGAGATGGCTGTTGTTG 0 0 0",
"k69_324482:SAMTOOLS:SNP:1707 226 G T AGCTCGACAGCAAGAACATC TTAGAGATCGGGTCCTACCG 0 0 0",
"k69_324482:SAMTOOLS:SNP:1710"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 226 G A AGCTCGACAGCAAGAACATC TTAGAGATCGGGTCCTACCG 0 0 0",
"k69_324482:SAMTOOLS:SNP:1937 239 T G ACGCTTTCCACTAAGTTCGG TGTTACCGCTCTCTGTGCTC 0 0 0",
"k69_324482:SAMTOOLS:SNP:1956 273 C G ACGCTTTCCACTAAGTTCGG CAATGTGGAGCAGTTCCTTG 0 0 0",
"k69_324482:SAMTOOLS:SNP:2172 221 C T TGCTTCTTCAGGGATGTCAG CTATCCATGTGGCGGTGTAG 0 0 0",
"k69_324482:SAMTOOLS:SNP:2277"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 154 G A CTACACCGCCACATGGATAG TTCTCTTCTTGGATGGGAGG 0 0 0",
"k69_324482:SAMTOOLS:SNP:2307 154 T A CTACACCGCCACATGGATAG TTCTCTTCTTGGATGGGAGG 0 0 0",
"k69_324482:SAMTOOLS:SNP:2367 255 G A CTACACCGCCACATGGATAG CCATATGGAGAGGTGTTCCC 0 0 0",
"k69_324482:SAMTOOLS:SNP:2396 255 C T CTACACCGCCACATGGATAG CCATATGGAGAGGTGTTCCC 0 0 0",
"k69_324482:SAMTOOLS:SNP:2598"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 289 A G ATCACCTCCCATCCAAGAAG GTCTAGCGCAATTGTATGCC 0 0 0",
"k69_324568:SAMTOOLS:SNP:229 292 T C AGCACCCATTGATTGATGTC TCCTTCCCTGTCTCCAAATC 0 0 0",
"k69_324934:SAMTOOLS:SNP:248 297 A G ACGCAACAATGAAACCAATC TTTGTTAGCAATCCCGACAC 0 0 0",
"k69_324942:SAMTOOLS:SNP:458"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 243 G T CACCTGCAGCTATGATGGTC TGTTACACCAACTGGATGGG 0 0 0",
"k69_324942:SAMTOOLS:SNP:719 288 A G TGTACAATGGTGGGTGGAAG GCCTCCTTCCAGAGAGTGAC 0 0 0",
"k69_324942:SAMTOOLS:SNP:791 288 A T TGTACAATGGTGGGTGGAAG GCCTCCTTCCAGAGAGTGAC 0 0 0",
"k69_324942:SAMTOOLS:SNP:818 191 C G CAAATCGACCATAGCCAGTG GCCTCCTTCCAGAGAGTGAC 0 0 0",
"k69_324942:SAMTOOLS:SNP:983"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 242 G A GTCACTCTCTGGAAGGAGGC CAAACATACATCCGAATCGC 0 0 0",
"k69_50501:SAMTOOLS:SNP:444 283 T C TGTCAGGGAAGAACGAAGAG CTGGTTTCCGGTAGAAGAGG 0 0 0",
"k69_93535:SAMTOOLS:SNP:661 210 A G CAGGATGGGTTGTTTCACAC ATCTCGGACACCTGAACTCC 0 0 0",
"k69_93535:SAMTOOLS:SNP:887"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 270 A G GACATTGTTGCTGATGAGGC CCTATGAAGCTTCCCTGTCC 0 0 0",
"k69_93535:SAMTOOLS:SNP:1141 229 C T TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 0 0 0",
"k69_93535:SAMTOOLS:SNP:1147 229 C G TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 0 0 0",
"k69_93535:SAMTOOLS:SNP:1336 285 G A GGTTTGTTTGAAGAAACGGG ACCCAGGTTTCAACTCCTTG 0 0 0",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 259 T G CAAATCCAAACATGTTATCTTCTTC TTTCATAAACTCGTCCGGTG 0 0 0",
"k69_98089:SAMTOOLS:SNP:550 228 G A ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 0 0 0",
"k69_98089:SAMTOOLS:SNP:625 228 A G ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 0 0 0"
]
}
],
"prompt_number": 10
},
{
"cell_type": "markdown",
"source": [
"-----",
"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",
" See https://www.dna.utah.edu/umelt/umelt.html ",
"",
"First form a short-list of targets"
]
},
{
"cell_type": "code",
"collapsed": false,
"input": [
"!tail -n 5 test_data/targets > HRM_targets",
"!cat HRM_targets"
],
"language": "python",
"outputs": [
{
"output_type": "stream",
"stream": "stdout",
"text": [
"k69_93535:SAMTOOLS:SNP:1147",
"k69_93535:SAMTOOLS:SNP:1336",
"k69_98089:SAMTOOLS:SNP:30",
"k69_98089:SAMTOOLS:SNP:550",
"k69_98089:SAMTOOLS:SNP:625"
]
}
],
"prompt_number": 15
},
{
"cell_type": "markdown",
"source": [
"Now run the design tool specifying melt prediction with the -u (umelt) flag",
"",
"**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",
"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",
"k69_93535:SAMTOOLS:SNP:1147"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 229 C G TGCTAATGACAACGAGGAGG TGTTTGGTATCGTTTCACCC 87.2 87.25 0.05",
"k69_93535:SAMTOOLS:SNP:1147"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 137 C G GGACAGGGAAGCTTCATAGG TGTTTGGTATCGTTTCACCC 85.75 85.75 0.0",
"k69_93535:SAMTOOLS:SNP:1147"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 258 C G GGACAGGGAAGCTTCATAGG TCAGCTCCAATCGTAAGAGG 87.8 87.8 0.0",
"k69_93535:SAMTOOLS:SNP:1147"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 257 C G TGCTAATGACAACGAGGAGG TTTGCAATCTCCTCCAATTTC 87.25 87.25 0.0",
"k69_93535:SAMTOOLS:SNP:1147"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 259 C G GGACAGGGAAGCTTCATAGG CTCAGCTCCAATCGTAAGAGG 87.8 87.8 0.0",
"k69_93535:SAMTOOLS:SNP:1336"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 285 G A GGTTTGTTTGAAGAAACGGG ACCCAGGTTTCAACTCCTTG 89.15 88.8 0.35",
"k69_93535:SAMTOOLS:SNP:1336"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 259 G A AACAAGATTTGTGTCGGGTG ACCCAGGTTTCAACTCCTTG 89.2 88.8 0.4",
"k69_93535:SAMTOOLS:SNP:1336"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 153 G A TTAGAAGGAACGCGAGAAGG ACCCAGGTTTCAACTCCTTG 88.8 88.65 0.15",
"k69_93535:SAMTOOLS:SNP:1336"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 289 G A GGTTTGTTTGAAGAAACGGG AGCAACCCAGGTTTCAACTC 89.25 89.05 0.2",
"k69_93535:SAMTOOLS:SNP:1336"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 263 G A AACAAGATTTGTGTCGGGTG AGCAACCCAGGTTTCAACTC 89.25 89.05 0.2",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 259 T G CAAATCCAAACATGTTATCTTCTTC TTTCATAAACTCGTCCGGTG 93.7 93.7 0.0",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 257 T G CAAATCCAAACATGTTATCTTCTTC TCATAAACTCGTCCGGTGAC 93.7 93.7 0.0",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 177 T G CAAATCCAAACATGTTATCTTCTTC TTTATGTCGCTGTAGTTTGCG 92.25 92.25 0.0",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 289 T G CAAATCCAAACATGTTATCTTCTTC ATCAGAAAGCGTGGAATACG 93.7 93.7 0.0",
"k69_98089:SAMTOOLS:SNP:30"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 284 T G CAAATCCAAACATGTTATCTTCTTC AAAGCGTGGAATACGCATC 93.7 93.7 0.0",
"k69_98089:SAMTOOLS:SNP:550"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 228 G A ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 85.55 85.65 0.1",
"k69_98089:SAMTOOLS:SNP:550"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 151 G A AAAGAACGGGATGATAACGG GGCCGAATATACATACAACGTC 83.85 83.7 0.15",
"k69_98089:SAMTOOLS:SNP:550"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 227 G A ATGGAGAAGGTCGAGGTCAG GGCCGAATATACATACAACGTC 85.4 85.4 0.0",
"k69_98089:SAMTOOLS:SNP:550"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 229 G A GAATGGAGAAGGTCGAGGTC GGCCGAATATACATACAACGTC 85.4 85.4 0.0",
"k69_98089:SAMTOOLS:SNP:550"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 154 G A AAAGAACGGGATGATAACGG AACGGCCGAATATACATACAAC 84.25 84.0 0.25",
"k69_98089:SAMTOOLS:SNP:625"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 228 A G ATGGAGAAGGTCGAGGTCAG CGGCCGAATATACATACAACG 85.55 85.85 0.3",
"k69_98089:SAMTOOLS:SNP:625"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 151 A G AAAGAACGGGATGATAACGG GGCCGAATATACATACAACGTC 83.85 84.2 0.35",
"k69_98089:SAMTOOLS:SNP:625"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 227 A G ATGGAGAAGGTCGAGGTCAG GGCCGAATATACATACAACGTC 85.4 85.8 0.4",
"k69_98089:SAMTOOLS:SNP:625"
]
},
{
"output_type": "stream",
"stream": "stdout",
"text": [
" 229 A G GAATGGAGAAGGTCGAGGTC GGCCGAATATACATACAACGTC 85.4 85.8 0.4",
"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",
"source": [
"Inspection of the output shows us that ",
"+ 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. ",
"+ 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"
]
}
]
}
]
}
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