MP-Ref: Multiplex PCR Primer Design for Reference Genomes

Done Task link: 4d19a94c-c4af-49dd-a991-5acf1e1dce98 (remember for later use)

Coverage report

total size: 16569 bp, cov size: 16512, cov rate: 99.66%, miss size: 57, miss rate: 0.34%, download coverage report.

Chrom Start End Size (bp) CovSize (bp) CovRate (%) MissSize (bp) MissRate (%)
1 chrM 0 16569 16569 16512 99.66 57 0.34

Primer list

tube: 2, primers: 97, product size range (bp): 161 - 260, download primers.

Tube 1
ID TemplateID FpTm (°G) RpTm (°G) FpSize (bp) RpSize (bp) AmpSize (bp) AmpGC (%)
T1P1 chrM:30-160 59.16 59.89 26 25 208 47.60
T1P2 chrM:328-488 59.97 60.28 20 19 260 46.54
T1P3 chrM:646-806 60.02 59.46 23 24 228 45.61
T1P4 chrM:990-1150 60.82 59.54 21 22 253 43.87
T1P5 chrM:1335-1495 60.33 59.65 23 25 250 45.60
T1P6 chrM:1681-1841 59.25 59.83 20 20 241 40.25
T1P7 chrM:2048-2208 60.24 59.68 21 21 260 39.23
T1P8 chrM:2422-2582 59.95 60.00 25 25 254 44.88
T1P9 chrM:2759-2919 59.50 60.49 22 22 238 44.12
T1P10 chrM:3099-3259 59.76 59.62 22 21 254 43.31
T1P11 chrM:3446-3606 59.90 58.75 20 22 233 54.94
T1P12 chrM:3766-3926 59.89 59.18 23 22 249 48.59
T1P13 chrM:4141-4301 60.24 61.10 25 24 250 40.80
T1P14 chrM:4476-4636 60.29 60.03 22 22 248 44.76
T1P15 chrM:4787-4947 59.99 59.68 27 21 260 42.69
T1P16 chrM:5137-5297 59.71 59.64 24 24 252 48.41
T1P17 chrM:5510-5670 60.47 59.90 23 23 248 39.11
T1P18 chrM:5860-6020 59.76 60.29 22 22 232 49.57
T1P19 chrM:6196-6356 60.24 59.81 21 22 233 53.22
T1P20 chrM:6517-6677 60.19 59.52 24 24 245 45.31
T1P21 chrM:6852-7012 59.45 60.03 22 22 236 47.88
T1P22 chrM:7173-7333 60.08 60.23 23 22 259 42.47
T1P23 chrM:7487-7647 59.29 59.57 24 23 260 40.00
T1P24 chrM:7828-7988 60.61 59.71 22 23 230 50.43
T1P25 chrM:8152-8312 60.80 60.01 22 24 238 44.12
T1P26 chrM:8500-8660 60.26 60.50 20 24 258 41.47
T1P27 chrM:8846-9006 60.03 60.03 22 21 247 48.18
T1P28 chrM:9161-9321 60.05 59.56 20 20 258 48.84
T1P29 chrM:9523-9683 59.82 59.69 22 22 247 47.37
T1P30 chrM:9881-10041 60.03 59.15 21 22 256 36.33
T1P31 chrM:10234-10394 59.91 58.45 18 24 244 38.11
T1P32 chrM:10587-10747 60.13 59.49 24 25 241 46.06
T1P33 chrM:10938-11098 59.23 59.62 22 22 256 42.97
T1P34 chrM:11293-11453 59.83 59.54 20 20 251 43.82
T1P35 chrM:11636-11796 60.15 60.11 23 21 259 46.33
T1P36 chrM:11985-12145 59.89 59.90 24 23 242 43.39
T1P37 chrM:12339-12499 61.29 60.72 21 21 258 41.86
T1P38 chrM:12722-12882 60.11 60.41 26 25 251 42.63
T1P39 chrM:13053-13213 60.77 59.96 22 22 252 49.21
T1P40 chrM:13429-13589 59.89 60.50 26 26 248 43.95
T1P41 chrM:13759-13919 60.09 59.55 22 22 246 45.12
T1P42 chrM:14089-14249 59.75 59.62 20 22 240 42.08
T1P43 chrM:14425-14585 59.89 60.09 22 22 258 42.64
T1P44 chrM:14758-14918 59.61 60.09 25 22 259 47.10
T1P45 chrM:15112-15272 60.03 59.96 22 22 250 46.80
T1P46 chrM:15485-15645 60.03 59.18 22 24 246 47.15
T1P47 chrM:15799-15959 59.76 59.89 22 22 260 39.62
T1P48 chrM:16117-16277 59.83 59.48 20 22 237 45.15
T1P49 chrM:16454-16509 59.83 59.65 24 25 161 49.07
Tube 2
ID TemplateID FpTm (°G) RpTm (°G) FpSize (bp) RpSize (bp) AmpSize (bp) AmpGC (%)
T2P1 chrM:173-333 60.16 60.04 22 20 249 41.37
T2P2 chrM:489-649 59.57 59.70 23 24 236 47.88
T2P3 chrM:803-963 60.16 59.60 22 25 251 45.42
T2P4 chrM:1175-1335 60.12 59.82 20 20 227 49.34
T2P5 chrM:1520-1680 60.73 59.95 24 23 227 44.05
T2P6 chrM:1865-2025 59.82 60.27 23 23 256 43.36
T2P7 chrM:2234-2394 59.96 59.90 22 22 252 39.68
T2P8 chrM:2591-2751 60.58 59.89 19 22 234 45.30
T2P9 chrM:2918-3078 59.39 59.83 26 23 257 43.97
T2P10 chrM:3290-3450 60.08 59.44 23 22 257 45.14
T2P11 chrM:3608-3768 60.37 58.48 19 20 220 47.73
T2P12 chrM:3951-4111 59.06 59.50 22 22 249 45.38
T2P13 chrM:4321-4481 58.87 59.04 25 22 259 43.24
T2P14 chrM:4631-4791 59.46 59.89 24 22 257 38.13
T2P15 chrM:4942-5102 60.29 60.09 22 22 253 40.32
T2P16 chrM:5315-5475 61.31 60.18 22 25 256 42.58
T2P17 chrM:5685-5845 59.15 59.43 22 22 242 46.28
T2P18 chrM:6023-6183 60.21 59.95 23 23 235 48.09
T2P19 chrM:6356-6516 59.32 60.29 23 22 221 47.06
T2P20 chrM:6697-6857 59.83 60.24 22 21 245 42.86
T2P21 chrM:7011-7171 60.22 60.88 22 22 248 44.35
T2P22 chrM:7352-7482 60.73 59.77 22 24 206 45.63
T2P23 chrM:7670-7830 59.77 59.96 22 22 255 45.10
T2P24 chrM:7989-8149 60.90 59.51 22 22 223 47.53
T2P25 chrM:8329-8489 60.15 60.21 23 23 242 37.60
T2P26 chrM:8690-8850 59.71 60.27 24 23 260 42.31
T2P27 chrM:9005-9165 59.31 59.57 22 23 232 46.12
T2P28 chrM:9363-9523 60.04 59.19 21 19 239 47.28
T2P29 chrM:9712-9872 60.18 60.22 25 22 243 43.21
T2P30 chrM:10075-10235 58.44 58.55 28 28 260 37.69
T2P31 chrM:10408-10568 59.89 60.38 24 24 258 34.50
T2P32 chrM:10747-10907 60.01 59.77 24 25 257 39.30
T2P33 chrM:11126-11286 60.51 61.11 28 24 257 44.75
T2P34 chrM:11480-11640 59.72 59.46 23 23 228 44.74
T2P35 chrM:11824-11984 59.84 60.34 20 20 259 45.56
T2P36 chrM:12159-12319 60.43 60.24 24 25 260 40.00
T2P37 chrM:12526-12686 58.57 59.17 22 22 259 38.22
T2P38 chrM:12877-13037 59.68 59.82 20 20 250 52.40
T2P39 chrM:13232-13392 60.24 60.17 21 21 254 42.91
T2P40 chrM:13585-13745 59.18 59.43 23 22 240 49.17
T2P41 chrM:13930-14090 59.95 59.95 24 23 222 44.59
T2P42 chrM:14244-14404 60.22 60.91 26 24 254 48.03
T2P43 chrM:14594-14754 60.43 58.43 24 28 253 39.53
T2P44 chrM:14923-15083 60.16 59.89 22 22 260 47.69
T2P45 chrM:15292-15452 59.51 60.42 22 22 253 46.64
T2P46 chrM:15644-15804 59.83 58.96 23 25 244 44.26
T2P47 chrM:15961-16121 60.49 60.94 22 25 243 40.33
T2P48 chrM:16272-16432 60.16 60.23 22 22 255 50.59

Parameters

Target Database hg38.fa
Validation method ngs
Product Size Min Difference 0
Primer Min Size (bp) 18
Primer Opt Size (bp) 22
Primer Max Size (bp) 28
Primer Min GC (%) 32
Primer Opt GC (%) 50
Primer Max GC (%) 68
Primer Min Tm (°C) 58
Primer Opt Tm (°C) 60
Primer Max Tm (°C) 62
Product Min Size (bp) 160
Product Max Size (bp) 260
Product Min GC (%) 32
Product Opt GC (%) 50
Product Max GC (%) 68
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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