MP-Ref: Multiplex PCR Primer Design for Reference Genomes

Done Task link: 7074b1f8-8737-4c5b-81e5-4fc8efb12059 (remember for later use)

Coverage report

total size: 19050 bp, cov size: 19050, cov rate: 100.00%, miss size: 0, miss rate: 0.00%, download coverage report.

Chrom Start End Size (bp) CovSize (bp) CovRate (%) MissSize (bp) MissRate (%)
1 chr13 32889611 32889809 198 198 100.00 0 0.00
2 chr13 32890553 32890669 116 116 100.00 0 0.00
3 chr13 32893208 32893467 259 259 100.00 0 0.00
4 chr13 32899207 32899326 119 119 100.00 0 0.00
5 chr13 32900232 32900292 60 60 100.00 0 0.00
6 chr13 32900373 32900424 51 51 100.00 0 0.00
7 chr13 32900630 32900755 125 125 100.00 0 0.00
8 chr13 32903574 32903634 60 60 100.00 0 0.00
9 chr13 32905050 32905172 122 122 100.00 0 0.00
10 chr13 32906403 32907529 1126 1126 100.00 0 0.00
11 chr13 32910396 32915338 4942 4942 100.00 0 0.00
12 chr13 32918689 32918795 106 106 100.00 0 0.00
13 chr13 32920958 32921038 80 80 100.00 0 0.00
14 chr13 32928992 32929430 438 438 100.00 0 0.00
15 chr13 32930559 32930751 192 192 100.00 0 0.00
16 chr13 32931873 32932071 198 198 100.00 0 0.00
17 chr13 32936654 32936835 181 181 100.00 0 0.00
18 chr13 32937310 32937675 365 365 100.00 0 0.00
19 chr13 32944533 32944699 166 166 100.00 0 0.00
20 chr13 32945087 32945242 155 155 100.00 0 0.00
21 chr13 32950801 32950933 132 132 100.00 0 0.00
22 chr13 32953448 32953657 209 209 100.00 0 0.00
23 chr13 32953881 32954055 174 174 100.00 0 0.00
24 chr13 32954138 32954287 149 149 100.00 0 0.00
25 chr13 32968820 32969075 255 255 100.00 0 0.00
26 chr13 32971029 32971186 157 157 100.00 0 0.00
27 chr13 32972293 32973814 1521 1521 100.00 0 0.00
28 chr17 41196306 41197824 1518 1518 100.00 0 0.00
29 chr17 41199654 41199725 71 71 100.00 0 0.00
30 chr17 41201132 41201216 84 84 100.00 0 0.00
31 chr17 41203074 41203139 65 65 100.00 0 0.00
32 chr17 41209063 41209157 94 94 100.00 0 0.00
33 chr17 41215344 41215395 51 51 100.00 0 0.00
34 chr17 41215885 41215973 88 88 100.00 0 0.00
35 chr17 41219619 41219717 98 98 100.00 0 0.00
36 chr17 41222939 41223260 321 321 100.00 0 0.00
37 chr17 41226342 41226543 201 201 100.00 0 0.00
38 chr17 41228499 41228636 137 137 100.00 0 0.00
39 chr17 41231345 41231421 76 76 100.00 0 0.00
40 chr17 41234415 41234597 182 182 100.00 0 0.00
41 chr17 41242955 41243054 99 99 100.00 0 0.00
42 chr17 41243446 41246882 3436 3436 100.00 0 0.00
43 chr17 41247857 41247944 87 87 100.00 0 0.00
44 chr17 41249255 41249311 56 56 100.00 0 0.00
45 chr17 41251786 41251902 116 116 100.00 0 0.00
46 chr17 41256133 41256283 150 150 100.00 0 0.00
47 chr17 41256879 41256978 99 99 100.00 0 0.00
48 chr17 41258467 41258555 88 88 100.00 0 0.00
49 chr17 41267737 41267801 64 64 100.00 0 0.00
50 chr17 41276028 41276137 109 109 100.00 0 0.00
51 chr17 41277282 41277386 104 104 100.00 0 0.00

Primer list

tube: 2, primers: 128, product size range (bp): 160 - 280, download primers.

Tube 1
ID TemplateID FpTm (°G) RpTm (°G) FpSize (bp) RpSize (bp) AmpSize (bp) AmpGC (%)
T1P1 chr13:32889611-32889809 60.70 58.70 16 18 267 64.79
T1P2 chr13:32893208-32893407 60.43 59.59 24 25 280 35.71
T1P3 chr13:32899207-32899326 58.23 60.03 29 22 272 30.51
T1P4 chr13:32900379-32900424 60.19 59.90 24 22 276 32.25
T1P5 chr13:32903574-32903634 59.71 60.01 24 24 273 28.57
T1P6 chr13:32906403-32906563 59.83 60.14 26 28 280 29.29
T1P7 chr13:32906722-32906882 59.34 60.02 24 23 273 38.46
T1P8 chr13:32907082-32907242 59.71 60.49 23 22 255 38.82
T1P9 chr13:32907449-32907529 59.77 58.18 22 25 279 31.54
T1P10 chr13:32910592-32910752 60.13 60.51 29 28 280 36.43
T1P11 chr13:32910982-32911142 59.88 59.89 26 26 251 31.87
T1P12 chr13:32911321-32911481 60.08 60.02 23 23 239 34.31
T1P13 chr13:32911651-32911811 60.15 60.10 28 27 262 32.44
T1P14 chr13:32912005-32912165 60.55 60.48 22 22 257 40.86
T1P15 chr13:32912380-32912540 60.01 60.22 32 26 280 27.14
T1P16 chr13:32912708-32912868 61.24 59.72 22 27 274 31.02
T1P17 chr13:32913084-32913244 60.97 60.78 22 23 279 40.50
T1P18 chr13:32913421-32913581 59.93 60.67 29 28 266 31.95
T1P19 chr13:32913735-32913895 59.89 59.77 27 22 266 32.33
T1P20 chr13:32914065-32914225 59.37 59.22 22 23 256 33.98
T1P21 chr13:32914416-32914576 59.43 59.65 25 25 279 35.84
T1P22 chr13:32914797-32914957 59.77 60.08 24 23 267 33.71
T1P23 chr13:32915189-32915338 59.83 60.52 26 23 212 37.26
T1P24 chr13:32920958-32921038 59.63 59.37 28 29 261 28.35
T1P25 chr13:32929166-32929326 58.60 59.44 23 22 279 33.69
T1P26 chr13:32930559-32930751 60.30 60.30 25 28 280 38.93
T1P27 chr13:32932053-32932071 60.88 60.79 22 23 268 35.82
T1P28 chr13:32937310-32937470 59.35 60.08 24 23 280 36.07
T1P29 chr13:32944533-32944669 59.89 59.94 26 26 258 36.05
T1P30 chr13:32945087-32945242 60.74 58.49 24 29 277 34.30
T1P31 chr13:32953448-32953627 58.12 58.39 27 28 262 35.88
T1P32 chr13:32953881-32954055 60.35 59.05 25 25 267 33.33
T1P33 chr13:32968820-32969015 59.23 60.76 32 22 279 36.92
T1P34 chr13:32971029-32971186 59.34 59.52 24 23 256 37.50
T1P35 chr13:32972478-32972638 59.89 60.22 22 22 275 39.27
T1P36 chr13:32972840-32973000 60.09 59.90 23 23 277 35.74
T1P37 chr13:32973188-32973348 59.63 58.96 23 24 272 39.34
T1P38 chr13:32973556-32973716 60.44 59.71 24 24 265 30.19
T1P39 chr17:41196306-41196466 60.55 60.34 20 20 276 41.30
T1P40 chr17:41196698-41196858 59.88 60.46 27 23 263 34.98
T1P41 chr17:41197026-41197186 60.16 60.03 22 22 244 48.77
T1P42 chr17:41197393-41197553 60.13 60.23 19 22 280 41.07
T1P43 chr17:41197740-41197824 60.47 59.23 20 22 204 52.94
T1P44 chr17:41201132-41201216 59.16 59.96 22 22 207 46.38
T1P45 chr17:41209063-41209157 60.22 59.77 22 22 199 46.23
T1P46 chr17:41215885-41215973 60.10 60.03 21 21 277 38.99
T1P47 chr17:41222939-41223099 59.31 60.43 28 24 272 44.12
T1P48 chr17:41226342-41226543 59.36 61.19 24 22 273 43.96
T1P49 chr17:41231345-41231421 59.50 59.76 22 22 223 41.70
T1P50 chr17:41242955-41243054 60.10 59.84 21 22 196 44.90
T1P51 chr17:41243623-41243783 60.16 60.24 22 21 278 41.37
T1P52 chr17:41243934-41244094 60.46 60.08 23 23 249 43.78
T1P53 chr17:41244246-41244406 59.29 59.61 27 25 267 37.08
T1P54 chr17:41244581-41244741 60.30 60.25 25 24 275 37.82
T1P55 chr17:41244910-41245070 60.41 60.40 20 20 263 41.06
T1P56 chr17:41245287-41245447 59.16 59.70 24 22 278 38.13
T1P57 chr17:41245671-41245831 60.32 60.27 27 27 257 36.58
T1P58 chr17:41246036-41246196 59.83 59.69 22 21 257 39.30
T1P59 chr17:41246436-41246596 59.65 60.28 23 19 280 42.86
T1P60 chr17:41246757-41246882 59.90 59.96 21 22 279 38.71
T1P61 chr17:41249255-41249311 59.89 60.08 24 23 257 33.07
T1P62 chr17:41256879-41256978 60.21 60.15 23 23 180 37.78
T1P63 chr17:41258467-41258555 61.02 61.42 22 22 238 33.61
T1P64 chr17:41276028-41276137 59.34 59.46 27 27 277 28.52
Tube 2
ID TemplateID FpTm (°G) RpTm (°G) FpSize (bp) RpSize (bp) AmpSize (bp) AmpGC (%)
T2P1 chr13:32890553-32890669 60.04 60.04 21 21 276 36.23
T2P2 chr13:32893402-32893467 60.04 60.03 20 22 224 39.29
T2P3 chr13:32900232-32900292 58.05 60.89 30 26 272 27.94
T2P4 chr13:32900630-32900755 59.90 59.45 22 23 277 37.18
T2P5 chr13:32905050-32905172 59.22 60.75 26 22 260 32.69
T2P6 chr13:32906564-32906724 59.34 60.49 24 22 280 31.79
T2P7 chr13:32906926-32907086 60.05 61.35 26 24 268 37.31
T2P8 chr13:32907238-32907398 60.49 60.51 22 21 268 35.82
T2P9 chr13:32910396-32910556 59.98 60.21 29 23 272 32.35
T2P10 chr13:32910779-32910939 60.06 59.83 25 24 280 33.21
T2P11 chr13:32911150-32911310 59.59 60.50 24 24 243 35.39
T2P12 chr13:32911488-32911648 60.10 60.10 21 21 222 35.14
T2P13 chr13:32911842-32912002 59.75 60.12 20 20 234 38.89
T2P14 chr13:32912175-32912335 60.16 60.19 22 24 273 31.14
T2P15 chr13:32912537-32912697 58.91 58.77 22 22 275 34.18
T2P16 chr13:32912905-32913065 59.83 59.77 25 24 277 35.74
T2P17 chr13:32913242-32913402 59.96 59.64 22 22 255 34.90
T2P18 chr13:32913580-32913740 60.42 59.68 27 30 248 31.45
T2P19 chr13:32913908-32914068 60.80 59.84 27 29 238 34.87
T2P20 chr13:32914238-32914398 59.71 60.22 22 22 275 34.91
T2P21 chr13:32914628-32914788 60.22 60.18 22 25 277 35.38
T2P22 chr13:32914982-32915142 60.04 60.04 28 28 280 33.21
T2P23 chr13:32918689-32918795 59.64 60.52 32 30 280 26.79
T2P24 chr13:32928992-32929152 60.96 60.59 23 23 280 35.00
T2P25 chr13:32929358-32929430 60.27 60.26 23 24 240 34.17
T2P26 chr13:32931873-32932041 58.92 60.46 31 28 274 33.94
T2P27 chr13:32936654-32936835 59.83 60.17 27 26 277 36.82
T2P28 chr13:32937508-32937668 59.83 59.95 24 25 229 43.23
T2P29 chr13:32944666-32944699 59.90 59.90 21 20 261 42.91
T2P30 chr13:32950801-32950933 59.77 59.59 24 24 277 37.55
T2P31 chr13:32953632-32953657 60.33 59.58 23 23 278 32.73
T2P32 chr13:32954138-32954287 59.78 59.98 30 30 278 32.37
T2P33 chr13:32969010-32969075 59.83 60.22 22 22 275 38.91
T2P34 chr13:32972293-32972453 58.91 61.63 29 25 261 37.93
T2P35 chr13:32972685-32972845 60.47 60.34 25 26 278 38.13
T2P36 chr13:32973031-32973191 60.21 60.36 27 19 280 36.79
T2P37 chr13:32973379-32973539 59.27 60.64 32 27 274 31.02
T2P38 chr13:32973745-32973814 59.42 59.83 25 25 251 31.87
T2P39 chr17:41196517-41196677 59.46 60.22 23 22 254 35.04
T2P40 chr17:41196869-41197029 59.88 61.56 32 22 269 47.21
T2P41 chr17:41197183-41197343 60.08 59.15 23 23 273 44.32
T2P42 chr17:41197571-41197731 59.57 59.96 22 22 252 52.38
T2P43 chr17:41199654-41199725 60.21 59.96 23 23 160 51.25
T2P44 chr17:41203074-41203139 60.62 58.77 20 22 278 44.24
T2P45 chr17:41215344-41215395 59.96 60.09 22 22 264 39.77
T2P46 chr17:41219619-41219717 59.11 60.03 22 22 278 43.88
T2P47 chr17:41223125-41223260 59.71 58.94 24 25 266 45.86
T2P48 chr17:41228499-41228636 59.41 59.32 24 26 224 36.61
T2P49 chr17:41234415-41234597 59.72 60.43 26 27 275 42.55
T2P50 chr17:41243446-41243606 60.31 59.64 21 22 236 42.80
T2P51 chr17:41243778-41243938 60.42 61.16 22 22 269 41.64
T2P52 chr17:41244091-41244251 59.78 59.83 23 25 280 36.79
T2P53 chr17:41244401-41244561 59.95 60.21 24 23 277 35.02
T2P54 chr17:41244753-41244913 59.83 59.62 21 21 280 40.36
T2P55 chr17:41245115-41245275 60.24 60.56 25 22 275 40.73
T2P56 chr17:41245505-41245665 59.54 60.08 24 23 280 40.71
T2P57 chr17:41245860-41246020 60.27 60.88 23 22 278 37.41
T2P58 chr17:41246229-41246389 60.01 59.89 25 26 280 38.57
T2P59 chr17:41246596-41246756 59.95 60.89 23 22 280 42.86
T2P60 chr17:41247857-41247944 61.02 60.59 22 23 266 39.10
T2P61 chr17:41251786-41251902 60.10 60.19 21 20 279 39.43
T2P62 chr17:41256133-41256283 61.79 61.09 20 28 278 34.89
T2P63 chr17:41267737-41267801 60.79 60.39 23 23 254 37.40
T2P64 chr17:41277282-41277386 59.90 60.71 22 21 237 58.23

Parameters

Target Database hg19.fa
Validation method ngs
Product Size Min Difference 0
Primer Min Size (bp) 16
Primer Opt Size (bp) 22
Primer Max Size (bp) 32
Primer Min GC (%) 20
Primer Opt GC (%) 50
Primer Max GC (%) 82
Primer Min Tm (°C) 58
Primer Opt Tm (°C) 60
Primer Max Tm (°C) 62
Product Min Size (bp) 160
Product Max Size (bp) 280
Product Min GC (%) 25
Product Opt GC (%) 50
Product Max GC (%) 78
Dimer ΔG (kcal/mol) -3.50
Hairpin Score 5
Non-specific Amplicon Tm (°C) 47
Non-specific Amplicon Min Size (bp) 0
Non-specific Amplicon Max Size (bp) 300
Min Off-targets 0
Max Off-targets 10
Minimum Diffierent Bases 50
SNP Skip Size 9
InDel Skip Size 15
Background Database 1 -
Background Database 2 -
Min Amplicon Count on Background Genomes 0
Max Amplicon Count on Background Genomes 0
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