Nice way to view primers amplicon on hg19

Question

[sacha]

Hello,

I need to design primers for a large region of DNA, like 5000 bases. Then I have to create many primers with overlap! I can do it with primer3.

But now, I want a nice way to see all my amplicon aligned on my Sequence target.

Something like :

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
ppppxxxxxxxxxxxxxxxpppp 
                         pppxxxxxxxxxxxxxxxxxxxxxxxxxxpppppppppp
                                                                                    ppppppxxxxxxxxxxxxxxxxxxxxxxppppppppp

[sacha]

yes, but I prefere a Graphics user interface .

And I m not sure that primer3 generate multiple primer.

[Pierre Lindenbaum]

well it would be easy to transform the primer3 output to a BED file using awk. Can you please provide a output, and name your input sequence using something like "SEQUENCE_ID=chr17:10100-10653"

[sacha]

It looks great. But What do you use to render BED file?

My sequence is the Intro of GJB2 of human in hg19 : 13:20763739-20766928

Answer 1

[Pierre Lindenbaum]

Update: here is an example with XSLT+UCSC-Das-Server+awk generating a custom BED file for the UCSC:

	stylesheet=$(mktemp )
	cat << EOF > ${stylesheet}
	<xsl:stylesheet version="1.0" xmlns:xsl="http://www.w3.org/1999/XSL/Transform">
	<xsl:output method='text' />
	<xsl:template match="/">
	<xsl:text>SEQUENCE_TEMPLATE=</xsl:text>
	<xsl:value-of select="translate(normalize-space(/DASDNA/SEQUENCE/DNA/text()),' ','')"/>
	<xsl:text>
	SEQUENCE_ID=</xsl:text>
	<xsl:value-of select="concat(/DASDNA/SEQUENCE/@id,':',/DASDNA/SEQUENCE/@start,'-',/DASDNA/SEQUENCE/@stop)"/>
	<xsl:text>
	PRIMER_NUM_RETURN=100
	=
	</xsl:text>
	</xsl:template>
	</xsl:stylesheet>
	EOF
	curl "http://genome.ucsc.edu/cgi-bin/das/hg19/dna?segment=chr13%3A20763739%2C20766928" |\
	xsltproc --novalid ${stylesheet} - | primer3_core --default_version=1 | awk -F '=' '
	/^SEQUENCE_ID=/ {n=split($2,a,/[\:\-]/); chrom=a[1]; start=int(a[2])-1;end=int(a[3])}
	/^PRIMER_LEFT_[0-9]+=/ { n=split($2,a,/[,]/); p5s=int(a[1]);p5l=int(a[2]);};
	/^PRIMER_RIGHT_[0-9]+=/ { n=split($2,a,/[,]/); p3l=int(a[2]) ; p3s=int(a[1])+1 - p3l;
	pair_id++;
	pcr_start=start+p5s;
	pcr_end=start+p3s+p3l;
	printf("%s\t%d\t%d\tprimer_%s_%s_%s_%d\t500\t+\t%d\t%d\t100,100,100\t2\t%d,%d\t%d,%d\n",
	chrom,pcr_start,pcr_end,chrom,start,end,pair_id,pcr_start,pcr_end,p5l,p3l,0,p3s-p5s);
	};
	'
	rm ${stylesheet}

view raw biostar.sh hosted with ❤ by GitHub

	chr13 20764231 20764475 primer_chr13_20763738_20766928_1 500 + 20764231 20764475 100,100,100 2 20,20 0,224
	chr13 20764230 20764475 primer_chr13_20763738_20766928_2 500 + 20764230 20764475 100,100,100 2 20,20 0,225
	chr13 20764229 20764475 primer_chr13_20763738_20766928_3 500 + 20764229 20764475 100,100,100 2 20,20 0,226
	chr13 20763928 20764135 primer_chr13_20763738_20766928_4 500 + 20763928 20764135 100,100,100 2 20,20 0,187
	chr13 20764231 20764457 primer_chr13_20763738_20766928_5 500 + 20764231 20764457 100,100,100 2 20,20 0,206
	chr13 20764230 20764457 primer_chr13_20763738_20766928_6 500 + 20764230 20764457 100,100,100 2 20,20 0,207
	chr13 20764229 20764457 primer_chr13_20763738_20766928_7 500 + 20764229 20764457 100,100,100 2 20,20 0,208
	chr13 20763739 20763948 primer_chr13_20763738_20766928_8 500 + 20763739 20763948 100,100,100 2 20,20 0,189
	chr13 20764231 20764478 primer_chr13_20763738_20766928_9 500 + 20764231 20764478 100,100,100 2 20,20 0,227
	chr13 20764230 20764478 primer_chr13_20763738_20766928_10 500 + 20764230 20764478 100,100,100 2 20,20 0,228
	chr13 20764229 20764478 primer_chr13_20763738_20766928_11 500 + 20764229 20764478 100,100,100 2 20,20 0,229
	chr13 20764322 20764475 primer_chr13_20763738_20766928_12 500 + 20764322 20764475 100,100,100 2 20,20 0,133
	chr13 20764321 20764475 primer_chr13_20763738_20766928_13 500 + 20764321 20764475 100,100,100 2 20,20 0,134
	chr13 20764320 20764475 primer_chr13_20763738_20766928_14 500 + 20764320 20764475 100,100,100 2 20,20 0,135
	chr13 20764229 20764340 primer_chr13_20763738_20766928_15 500 + 20764229 20764340 100,100,100 2 20,20 0,91
	chr13 20764050 20764340 primer_chr13_20763738_20766928_16 500 + 20764050 20764340 100,100,100 2 20,20 0,270
	chr13 20764050 20764341 primer_chr13_20763738_20766928_17 500 + 20764050 20764341 100,100,100 2 20,20 0,271
	chr13 20764050 20764342 primer_chr13_20763738_20766928_18 500 + 20764050 20764342 100,100,100 2 20,20 0,272
	chr13 20764321 20764457 primer_chr13_20763738_20766928_19 500 + 20764321 20764457 100,100,100 2 20,20 0,116
	chr13 20764320 20764457 primer_chr13_20763738_20766928_20 500 + 20764320 20764457 100,100,100 2 20,20 0,117
	chr13 20764322 20764478 primer_chr13_20763738_20766928_21 500 + 20764322 20764478 100,100,100 2 20,20 0,136
	chr13 20764321 20764478 primer_chr13_20763738_20766928_22 500 + 20764321 20764478 100,100,100 2 20,20 0,137
	chr13 20764320 20764478 primer_chr13_20763738_20766928_23 500 + 20764320 20764478 100,100,100 2 20,20 0,138
	chr13 20764231 20764448 primer_chr13_20763738_20766928_24 500 + 20764231 20764448 100,100,100 2 20,20 0,197
	chr13 20764230 20764448 primer_chr13_20763738_20766928_25 500 + 20764230 20764448 100,100,100 2 20,20 0,198
	chr13 20764229 20764448 primer_chr13_20763738_20766928_26 500 + 20764229 20764448 100,100,100 2 20,20 0,199
	chr13 20763928 20764047 primer_chr13_20763738_20766928_27 500 + 20763928 20764047 100,100,100 2 20,20 0,99
	chr13 20764192 20764475 primer_chr13_20763738_20766928_28 500 + 20764192 20764475 100,100,100 2 20,20 0,263
	chr13 20763928 20764212 primer_chr13_20763738_20766928_29 500 + 20763928 20764212 100,100,100 2 20,20 0,264
	chr13 20764751 20765014 primer_chr13_20763738_20766928_30 500 + 20764751 20765014 100,100,100 2 20,20 0,243
	chr13 20764027 20764249 primer_chr13_20763738_20766928_31 500 + 20764027 20764249 100,100,100 2 20,20 0,202
	chr13 20764027 20764250 primer_chr13_20763738_20766928_32 500 + 20764027 20764250 100,100,100 2 20,20 0,203
	chr13 20764027 20764251 primer_chr13_20763738_20766928_33 500 + 20764027 20764251 100,100,100 2 20,20 0,204
	chr13 20764027 20764135 primer_chr13_20763738_20766928_34 500 + 20764027 20764135 100,100,100 2 20,20 0,88
	chr13 20765711 20765925 primer_chr13_20763738_20766928_35 500 + 20765711 20765925 100,100,100 2 20,20 0,194
	chr13 20764050 20764212 primer_chr13_20763738_20766928_36 500 + 20764050 20764212 100,100,100 2 20,20 0,142
	chr13 20764437 20764673 primer_chr13_20763738_20766928_37 500 + 20764437 20764673 100,100,100 2 20,20 0,216
	chr13 20764280 20764475 primer_chr13_20763738_20766928_38 500 + 20764280 20764475 100,100,100 2 20,20 0,175
	chr13 20764218 20764475 primer_chr13_20763738_20766928_39 500 + 20764218 20764475 100,100,100 2 20,20 0,237
	chr13 20764192 20764457 primer_chr13_20763738_20766928_40 500 + 20764192 20764457 100,100,100 2 20,20 0,245
	chr13 20764751 20764998 primer_chr13_20763738_20766928_41 500 + 20764751 20764998 100,100,100 2 20,20 0,227
	chr13 20765343 20765545 primer_chr13_20763738_20766928_42 500 + 20765343 20765545 100,100,100 2 20,20 0,182
	chr13 20765343 20765546 primer_chr13_20763738_20766928_43 500 + 20765343 20765546 100,100,100 2 20,20 0,183
	chr13 20764192 20764478 primer_chr13_20763738_20766928_44 500 + 20764192 20764478 100,100,100 2 20,20 0,266
	chr13 20765071 20765300 primer_chr13_20763738_20766928_45 500 + 20765071 20765300 100,100,100 2 20,20 0,209
	chr13 20765128 20765300 primer_chr13_20763738_20766928_46 500 + 20765128 20765300 100,100,100 2 20,20 0,152
	chr13 20764280 20764457 primer_chr13_20763738_20766928_47 500 + 20764280 20764457 100,100,100 2 20,20 0,157
	chr13 20764218 20764457 primer_chr13_20763738_20766928_48 500 + 20764218 20764457 100,100,100 2 20,20 0,219
	chr13 20763742 20763948 primer_chr13_20763738_20766928_49 500 + 20763742 20763948 100,100,100 2 20,20 0,186
	chr13 20764280 20764478 primer_chr13_20763738_20766928_50 500 + 20764280 20764478 100,100,100 2 20,20 0,178
	chr13 20764218 20764478 primer_chr13_20763738_20766928_51 500 + 20764218 20764478 100,100,100 2 20,20 0,240
	chr13 20764322 20764448 primer_chr13_20763738_20766928_52 500 + 20764322 20764448 100,100,100 2 20,20 0,106
	chr13 20764321 20764448 primer_chr13_20763738_20766928_53 500 + 20764321 20764448 100,100,100 2 20,20 0,107
	chr13 20764320 20764448 primer_chr13_20763738_20766928_54 500 + 20764320 20764448 100,100,100 2 20,20 0,108
	chr13 20765711 20765972 primer_chr13_20763738_20766928_55 500 + 20765711 20765972 100,100,100 2 20,20 0,241
	chr13 20765341 20765545 primer_chr13_20763738_20766928_56 500 + 20765341 20765545 100,100,100 2 20,20 0,184
	chr13 20765341 20765546 primer_chr13_20763738_20766928_57 500 + 20765341 20765546 100,100,100 2 20,20 0,185
	chr13 20764231 20764460 primer_chr13_20763738_20766928_58 500 + 20764231 20764460 100,100,100 2 20,20 0,209
	chr13 20764230 20764460 primer_chr13_20763738_20766928_59 500 + 20764230 20764460 100,100,100 2 20,20 0,210
	chr13 20764229 20764460 primer_chr13_20763738_20766928_60 500 + 20764229 20764460 100,100,100 2 20,20 0,211
	chr13 20764988 20765091 primer_chr13_20763738_20766928_61 500 + 20764988 20765091 100,100,100 2 20,20 0,83
	chr13 20765128 20765363 primer_chr13_20763738_20766928_62 500 + 20765128 20765363 100,100,100 2 20,20 0,215
	chr13 20764231 20764335 primer_chr13_20763738_20766928_63 500 + 20764231 20764335 100,100,100 2 20,20 0,84
	chr13 20764230 20764335 primer_chr13_20763738_20766928_64 500 + 20764230 20764335 100,100,100 2 20,20 0,85
	chr13 20764229 20764335 primer_chr13_20763738_20766928_65 500 + 20764229 20764335 100,100,100 2 20,20 0,86
	chr13 20764994 20765241 primer_chr13_20763738_20766928_66 500 + 20764994 20765241 100,100,100 2 20,20 0,227
	chr13 20764050 20764335 primer_chr13_20763738_20766928_67 500 + 20764050 20764335 100,100,100 2 20,20 0,265
	chr13 20764653 20764771 primer_chr13_20763738_20766928_68 500 + 20764653 20764771 100,100,100 2 20,20 0,98
	chr13 20764231 20764396 primer_chr13_20763738_20766928_69 500 + 20764231 20764396 100,100,100 2 20,20 0,145
	chr13 20764230 20764396 primer_chr13_20763738_20766928_70 500 + 20764230 20764396 100,100,100 2 20,20 0,146
	chr13 20764229 20764396 primer_chr13_20763738_20766928_71 500 + 20764229 20764396 100,100,100 2 20,20 0,147
	chr13 20764751 20765006 primer_chr13_20763738_20766928_72 500 + 20764751 20765006 100,100,100 2 20,20 0,235
	chr13 20765221 20765363 primer_chr13_20763738_20766928_73 500 + 20765221 20765363 100,100,100 2 20,20 0,122
	chr13 20764988 20765241 primer_chr13_20763738_20766928_74 500 + 20764988 20765241 100,100,100 2 20,20 0,233
	chr13 20764978 20765091 primer_chr13_20763738_20766928_75 500 + 20764978 20765091 100,100,100 2 20,20 0,93
	chr13 20764192 20764340 primer_chr13_20763738_20766928_76 500 + 20764192 20764340 100,100,100 2 20,20 0,128
	chr13 20764192 20764341 primer_chr13_20763738_20766928_77 500 + 20764192 20764341 100,100,100 2 20,20 0,129
	chr13 20764192 20764342 primer_chr13_20763738_20766928_78 500 + 20764192 20764342 100,100,100 2 20,20 0,130
	chr13 20763928 20764124 primer_chr13_20763738_20766928_79 500 + 20763928 20764124 100,100,100 2 20,20 0,176
	chr13 20763928 20764125 primer_chr13_20763738_20766928_80 500 + 20763928 20764125 100,100,100 2 20,20 0,177
	chr13 20764913 20765014 primer_chr13_20763738_20766928_81 500 + 20764913 20765014 100,100,100 2 20,20 0,81
	chr13 20765126 20765300 primer_chr13_20763738_20766928_82 500 + 20765126 20765300 100,100,100 2 20,20 0,154
	chr13 20765128 20765361 primer_chr13_20763738_20766928_83 500 + 20765128 20765361 100,100,100 2 20,20 0,213
	chr13 20765678 20765925 primer_chr13_20763738_20766928_84 500 + 20765678 20765925 100,100,100 2 20,20 0,227
	chr13 20764105 20764249 primer_chr13_20763738_20766928_85 500 + 20764105 20764249 100,100,100 2 20,20 0,124
	chr13 20764105 20764250 primer_chr13_20763738_20766928_86 500 + 20764105 20764250 100,100,100 2 20,20 0,125
	chr13 20764105 20764251 primer_chr13_20763738_20766928_87 500 + 20764105 20764251 100,100,100 2 20,20 0,126
	chr13 20764104 20764249 primer_chr13_20763738_20766928_88 500 + 20764104 20764249 100,100,100 2 20,20 0,125
	chr13 20764104 20764250 primer_chr13_20763738_20766928_89 500 + 20764104 20764250 100,100,100 2 20,20 0,126
	chr13 20764104 20764251 primer_chr13_20763738_20766928_90 500 + 20764104 20764251 100,100,100 2 20,20 0,127
	chr13 20764231 20764397 primer_chr13_20763738_20766928_91 500 + 20764231 20764397 100,100,100 2 20,20 0,146
	chr13 20764230 20764397 primer_chr13_20763738_20766928_92 500 + 20764230 20764397 100,100,100 2 20,20 0,147
	chr13 20764229 20764397 primer_chr13_20763738_20766928_93 500 + 20764229 20764397 100,100,100 2 20,20 0,148
	chr13 20763928 20764178 primer_chr13_20763738_20766928_94 500 + 20763928 20764178 100,100,100 2 20,20 0,230
	chr13 20764428 20764673 primer_chr13_20763738_20766928_95 500 + 20764428 20764673 100,100,100 2 20,20 0,225
	chr13 20765905 20766051 primer_chr13_20763738_20766928_96 500 + 20765905 20766051 100,100,100 2 20,20 0,126
	chr13 20764218 20764340 primer_chr13_20763738_20766928_97 500 + 20764218 20764340 100,100,100 2 20,20 0,102
	chr13 20764218 20764341 primer_chr13_20763738_20766928_98 500 + 20764218 20764341 100,100,100 2 20,20 0,103
	chr13 20764218 20764342 primer_chr13_20763738_20766928_99 500 + 20764218 20764342 100,100,100 2 20,20 0,104
	chr13 20765010 20765300 primer_chr13_20763738_20766928_100 500 + 20765010 20765300 100,100,100 2 20,20 0,270

view raw output.bed hosted with ❤ by GitHub

Answer 2

[Biomonika (Noolean)]

Use Geneious. It uses primer3 for primer design, nicely visualizes everything and trial version can be downloaded for free.

Answer 3

[seidel]

You could easily view this information using either the UCSC genome browser or IGV from Broad. At UCSC you can select the human genome and upload a BED file (or various other formats, there are some nice renderings allowing you to draw a thin line connecting the primers, color them, etc.) to view the results as a track. If you want to share the results with others, there's a way to do that as well. With IGV you can simply drop your BED file onto the IGV window and see it as a track.