qPrimerDB - qPCR Primer Database

A resource for real-time quantitative PCR primers

Version: 1.2

Birds_and_Reptiles

Anas platyrhynchos
Gallus gallus
Meleagris gallopavo
Pelodiscus sinensis
Taeniopygia guttata

Fish

Astyanax mexicanus
Danio rerio
Gadus morhua
Gasterosteus aculeatus
Latimeria chalumnae
Lepisosteus oculatus
Oreochromis niloticus
Oryzias latipes
Petromyzon marinus
Poecilia formosa
Takifugu rubripes
Xiphophorus maculatus

Insecta

Acromyrmex echinatior
Aedes aegypti
Anopheles coluzzii
Anopheles stephensi
Apis dorsata
Apis florea
Apis mellifera
Athalia rosae
Bactrocera cucurbitae
Bactrocera dorsalis
Bombus terrestris
Bombyx mori
Camponotus floridanus
Cerapachys biroi
Ceratitis capitata
Chilo suppressalis
Danaus plexippus
Diaphorina citri
Drosophila ananassae
Drosophila erecta
Drosophila grimshawi
Drosophila melanogaster
Drosophila mojavensis
Drosophila sechellia
Drosophila simulans
Drosophila virilis
Drosophila willistoni
Drosophila yakuba
Fopius arisanus
Harpegnathos saltator
Heliconius melpomene
Linepithema humile
Macrocentrus cingulum
Megachile rotundata
Megaselia scalaris
Melitaea cinxia
Microplitis demolitor
Musca domestica
Nasonia vitripennis
Nilaparvata lugens
Pediculus humanus
Plutella xylostella
Pogonomyrmex barbatus
Rhodnius prolixus
Solenopsis invicta
Tribolium castaneum
Vollenhovia emeryi
Wasmannia auropunctata
Zootermopsis nevadensis

Mammalia

Ailuropoda melanoleuca
Bos taurus
Callithrix jacchus
Canis familiaris
Cavia porcellus
Chlorocebus sabaeus
Choloepus hoffmanni
Dasypus novemcinctus
Dipodomys ordii
Echinops telfairi
Equus caballus
Erinaceus europaeus
Felis catus
Gorilla gorilla
Homo sapiens
Ictidomys tridecemlineatus
Loxodonta africana
Macaca mulatta
Macropus eugenii
Microcebus murinus
Monodelphis domestica
Mus musculus
Myotis lucifugus
Nomascus leucogenys
Ornithorhynchus anatinus
Oryctolagus cuniculus
Otolemur garnettii
Ovis aries
Pan troglodytes
Papio anubis
Pongo abelii
Procavia capensis
Pteropus vampyrus
Rattus norvegicus
Sarcophilus harrisii
Sus scrofa
Tarsius syrichta
Tupaia belangeri
Tursiops truncatus
Vicugna pacos

Other_animals

Anolis carolinensis
Caenorhabditis briggsae
Caenorhabditis elegans
Ciona intestinalis
Ciona savignyi
Ixodes scapularis
Lottia gigantea
Mnemiopsis leidyi
Octopus bimaculoides
Sarcoptes scabiei
Strongylocentrotus purpuratus
Strongyloides ratti
Tetranychus urticae
Xenopus tropicalis

Fungi

Absidia glauca
Acidomyces richmondensis
Acidomyces richmondensis bfw
Acremonium chrysogenum atcc 11550
Agaricus bisporus var burnettii jb137 s8
Allomyces macrogynus atcc 38327
Alternaria alternata
Amanita muscaria koide bx008
Amphiamblys sp wsbs2006
Arthrobotrys oligospora atcc 24927
Arthroderma otae cbs 113480
Aschersonia aleyrodis rcef 2490
Ascochyta rabiei
Ascoidea rubescens dsm 1968
Ascosphaera apis arsef 7405
Ashbya gossypii
Aspergillus aculeatus atcc 16872
Aspergillus bombycis
Aspergillus brasiliensis cbs 101740
Aspergillus calidoustus
Aspergillus carbonarius item 5010
Aspergillus clavatus
Aspergillus cristatus
Aspergillus flavus
Aspergillus flavus af70
Aspergillus fumigatus
Aspergillus fumigatus var rp 2014
Aspergillus fumigatus z5
Aspergillus fumigatusa1163
Aspergillus glaucus cbs 516 65
Aspergillus lentulus
Aspergillus luchuensis
Aspergillus luchuensis cbs 106 47
Aspergillus nidulans
Aspergillus nidulans fgsc a4
Aspergillus niger
Aspergillus niger atcc 1015
Aspergillus niger gca 001515345
Aspergillus nomius nrrl 13137
Aspergillus ochraceoroseus
Aspergillus oryzae
Aspergillus oryzae 3 042
Aspergillus oryzae gca 002007945
Aspergillus parasiticus su 1
Aspergillus rambellii
Aspergillus ruber cbs 135680
Aspergillus sydowii cbs 593 65
Aspergillus terreus
Aspergillus udagawae
Aspergillus ustus
Aspergillus versicolor cbs 583 65
Aureobasidium melanogenum cbs 110374
Aureobasidium namibiae cbs 147 97
Aureobasidium pullulans exf 150
Aureobasidium subglaciale exf 2481
Baudoinia panamericana uamh 10762
Beauveria bassiana
Beauveria bassiana arsef 2860
Beauveria bassiana d1 5
Bipolaris maydis atcc 48331
Bipolaris maydis c5
Bipolaris oryzae atcc 44560
Bipolaris sorokiniana nd90pr
Bipolaris victoriae fi3
Bipolaris zeicola 26 r 13
Blastomyces dermatitidis atcc 18188
Blastomyces dermatitidis er 3
Blastomyces gilchristii slh14081
Blumeria graminis
Blumeria graminis f sp tritici 96224
Botrytis cinerea
Botrytis cinerea bcdw1
Botrytis cinerea t4
Brettanomyces bruxellensis awri1499
Byssochlamys spectabilis no 5
Candida albicans 12c
Candida albicans gc75
Candida albicans p34048
Candida albicans p37005
Candida albicans sc5314
Candida albicans sc5314 gca 000784655
Candida albicans wo 1
Candida dubliniensis cd36
Candida glabrata
Candida maltosa xu316
Candida orthopsilosis co 90 125
Candida tenuis atcc 10573
Candida tropicalis mya 3404
Capronia coronata cbs 617 96
Capronia epimyces cbs 606 96
Ceratocystis platani
Chaetomium globosum cbs 148 51
Chaetomium thermophilum var thermophilum dsm 1495
Cladophialophora bantiana cbs 173 52
Cladophialophora carrionii cbs 160 54
Cladophialophora immunda
Cladophialophora psammophila cbs 110553
Cladophialophora yegresii cbs 114405
Claviceps purpurea 20 1
Clavispora lusitaniae atcc 42720
Coccidioides immitis rmscc 2394
Coccidioides immitis rs
Coccidioides posadasii c735 delta sowgp
Coccidioides posadasii rmscc 3488
Coccidioides posadasii str silveira
Colletotrichum fioriniae pj7
Colletotrichum gloeosporioides
Colletotrichum gloeosporioides cg 14
Colletotrichum graminicola
Colletotrichum higginsianum
Colletotrichum orbiculare
Colletotrichum sublineola
Cordyceps militaris cm01
Cryptococcus neoformans
Cyphellophora europaea cbs 101466
Dactylellina haptotyla cbs 200 50
Debaryomyces hansenii cbs767
Diaporthe ampelina
Drechslerella stenobrocha 248
Emmonsia parva uamh 139
Endocarpon pusillum z07020
Eremothecium cymbalariae dbvpg 7215
Eremothecium gossypii fdag1
Erysiphe necator
Exophiala aquamarina cbs 119918
Exophiala dermatitidis nih ut8656
Exophiala mesophila
Exophiala oligosperma
Exophiala sideris
Exophiala spinifera
Exophiala xenobiotica
Fonsecaea pedrosoi cbs 271 37
Fusarium oxysporum f sp conglutinans race 2 54008
Fusarium oxysporum f sp cubense race 1
Fusarium oxysporum f sp lycopersici mn25
Fusarium oxysporum f sp melonis 26406
Fusarium oxysporum f sp pisi hdv247
Fusarium oxysporum f sp radicis lycopersici 26381
Fusarium oxysporum fo47
Fusarium oxysporum fosc 3 a
Glarea lozoyensis 74030
Kazachstania africana cbs 2517
Kazachstania naganishii cbs 8797
Kluyveromyces lactis
Komagataella phaffii cbs 7435
Kuraishia capsulata cbs 1993
Lachancea lanzarotensis
Lachancea thermotolerans cbs 6340
Lodderomyces elongisporus nrrl yb 4239
Macrophomina phaseolina ms6
Magnaporthe oryzae
Marssonina brunnea f sp multigermtubi mb m1
Metarhizium acridum cqma 102
Metarhizium album arsef 1941
Metarhizium anisopliae
Metarhizium anisopliae arsef 23
Metarhizium anisopliae arsef 549
Metarhizium anisopliae brip 53284
Metarhizium brunneum arsef 3297
Metarhizium guizhouense arsef 977
Metarhizium robertsii
Meyerozyma guilliermondii atcc 6260
Millerozyma farinosa cbs 7064
Oidiodendron maius zn
Penicillium brasilianum
Penicillium camemberti fm 013
Phialophora americana
Pseudogymnoascus sp vkm f 103
Rasamsonia emersonii cbs 393 64
Rhinocladiella mackenziei cbs 650 93
Saccharomyces arboricola h 6
Saccharomyces cerevisiae
Saccharomyces cerevisiae awri796
Saccharomyces cerevisiae cen pk113 7d
Saccharomyces cerevisiae ec1118
Saccharomyces cerevisiae fostersb
Saccharomyces cerevisiae fosterso
Saccharomyces cerevisiae kyokai no 7
Saccharomyces cerevisiae lalvin qa23
Saccharomyces cerevisiae p283
Saccharomyces cerevisiae r008
Saccharomyces cerevisiae rm11 1a
Saccharomyces cerevisiae vin13
Saccharomyces cerevisiae x saccharomyces kudriavzevii vin7
Saccharomyces cerevisiae yjm189
Saccharomyces cerevisiae yjm789
Saccharomyces kudriavzevii ifo 1802
Saccharomycetaceae sp ashbya aceri
Saitoella complicata nrrl y 17804
Scedosporium apiospermum
Scheffersomyces stipitis cbs 6054
Schizosaccharomyces pombe
Schizosaccharomyces pombe 972h
Schizosaccharomyces pombe EBC7
Sclerotinia borealis f 4128
Sclerotinia sclerotiorum
Setosphaeria turcica et28a
Sordaria macrospora
Spathaspora passalidarum nrrl y 27907
Sphaerulina musiva so2202
Sporothrix brasiliensis 5110
Sporothrix schenckii 1099 18
Sporothrix schenckii atcc 58251
Stachybotrys chartarum ibt 7711
Stachybotrys chlorohalonata ibt 40285
Talaromyces cellulolyticus
Talaromyces islandicus
Talaromyces marneffei atcc 18224
Talaromyces marneffei pm1
Talaromyces stipitatus atcc 10500
Tetrapisispora blattae cbs 6284
Tetrapisispora phaffii cbs 4417
Thermothelomyces thermophila atcc 42464
Thielavia terrestris nrrl 8126
Thielaviopsis punctulata
Torrubiella hemipterigena
Torulaspora delbrueckii
Trichoderma atroviride imi 206040
Trichoderma harzianum
Trichoderma reesei rut c 30
Trichophyton benhamiae cbs 112371
Trichophyton equinum cbs 127 97
Trichophyton interdigitale h6
Trichophyton rubrum cbs 202 88
Trichophyton rubrum d6
Trichophyton rubrum mr850
Trichophyton soudanense cbs 452 61
Trichophyton tonsurans cbs 112818
Trichophyton verrucosum hki 0517
Uncinocarpus reesii 1704
Ustilaginoidea virens
Vanderwaltozyma polyspora dsm 70294
Verruconis gallopava
Verticillium alfalfae vams 102
Wickerhamomyces ciferrii
Yarrowia lipolytica
Zygosaccharomyces bailii isa1307
Zygosaccharomyces rouxii
Zymoseptoria brevis
Zymoseptoria tritici

Microsporidia

Encephalitozoon cuniculi EC1
Encephalitozoon cuniculi EC2
Encephalitozoon cuniculi EC3
Enterocytozoon bieneusi H348
Nematocida parisii ERTm1
Nosema bombycis CQ1
Nosema ceranae BRL01
Spraguea lophii 42-110
Vittaforma corneae ATCC50505

Dicotyledons

Betula pendula

Eudicotyledons

Aethionema arabicum
Amaranthus hypochondriacus
Anacardium occidentale
Aquilegia coerulea
Arabidopsis halleri
Arabidopsis lyrata
Arabidopsis thaliana
Arachis duranensis
Arachis ipaensis
Barbarea vulgaris
Beta vulgaris
Boechera stricta
Brassica juncea
Brassica napus
Brassica nigra
Brassica oleracea capitata
Brassica oleracea italica
Brassica rapa
Calotropis gigantea
Camelina sativa
Camellia sinensis
Capsella grandiflora
Capsella rubella
Capsicum annuum Zunla
Capsicum annuum var glabriusculum
Cardamine hirsuta
Carica papaya
Castanea mollissima
Chenopodium quinoa
Cicer arietinum
Cicer reticulatum
Citrus clementina
Citrus sinensis
Coffea canephora
Cucumis melo
Cucumis sativus
Cucurbita moschata
Daucus carota
Eucalyptus camaldulensis
Eucalyptus grandis
Eutrema salsugineum
Fragaria nipponica
Fragaria nubicola
Fragaria orientalis
Fragaria vesca
Fragaria x ananassa
Glycine max
Gossypium arboreum BGI
Gossypium barbadense NAU
Gossypium hirsutum
Gossypium hirsutum BGI
Gossypium raimondii
Gossypium raimondii BGI
Helianthus annuus
Hevea brasiliensis
Hibiscus syriacus
Ipomoea batatas
Ipomoea trifida
Jatropha curcas
Kalanchoe fedtschenkoi
Kalanchoe laxiflora
Lactuca sativa
Levenworthia alabamica
Linum usitatissimum
Lotus japonicus
Malus domestica
Manihot esculenta
Medicago truncatula
Mimulus guttatus
Nicotiana benthamiana
Nicotiana sylvestris
Nicotiana tabacum
Nicotiana tomentosiformis
Olea europaea
Petunia axillaris
Petunia inflata
Phaseolus vulgaris
Populus deltoides
Populus tremula
Populus tremuloides
Populus trichocarpa
Prunus persica
Raphanus sativus
Ricinus communis
Rosa multiflora
Rubus occidentalis
Salix purpurea
Sesamum indicum
Sida fallax
Solanum lycopersicum
Solanum pennellii
Solanum tuberosum
Thellungiella parvula
Theobroma cacao
Trifolium pratense
Vigna angularis
Vigna radiata
Vigna unguiculata
Vitis vinifera

Monocotyledons

Aegilops tauschii
Ananas comosus
Asparagus officinalis
Brachypodium distachyon
Brachypodium hybridum
Brachypodium stacei
Elaeis guineensis
Hordeum vulgare
Leersia perrieri
Musa Itinerans
Musa acuminata
Oropetium thomaeum
Oryza barthii
Oryza brachyantha
Oryza glaberrima
Oryza glumaepatula
Oryza longistaminata
Oryza meridionalis
Oryza punctata
Oryza rufipogon
Oryza sativa indica
Oryza sativa japonica
Panicum hallii
Setaria italica
Setaria viridis
Sorghum bicolor
Spirodela polyrhiza
Triticum aestivum
Triticum urartu
Zea mays
Zostera marina
Zostera muelleri
Zoysia japonica
Zoysia matrella

Other_plants

Amborella trichopoda
Chlamydomonas reinhardtii
Cyanidioschyzon merolae
Dunaliella salina
Marchantia polymorpha
Micromonas
Micromonas pusilla
Ostreococcus lucimarinus
Physcomitrella patens
Pinus taeda
Pseudotsuga menziesii
Selaginella moellendorffii
Volvox carteri

Pipeline

The workflow used for gene-specific qPCR primer design

workflow
  • Step 1 Download the coding gene, mNRA and genome sequence for target species for which complete genome sequences have been assembled, annotated and published. It is recommended to retrieve above-mentioned sequences from integrative databases, such as NCBI (https://www.ncbi.nlm.nih.gov), Ensembl (http://www.ensembl.org), USCS (http://genome.ucsc.edu), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and Gramene (http://www.gramene.org). Researchers also could obtain required sequences from specific genome databases or prepared their own de novo assembled transcriptome sequence for gene-specific qPCR primer design. For each gene, only the longest coding/mRNA sequence for each gene was retained as representative coding/mNRA sequence, all duplicated coding/mNRA sequences will be removed.
  • Step 2 To design as many qPCR primers as possible for each gene, all sequences retained in Step 1 are shredded into a series of short, overlapping template fragments using BBMap (http://jgi.doe.gov/data-and-tools/bbtools) with the specified window size and step size currently set to 300 bp and 50 bp, respectively. The window and step size can be adjusted for organisms with complex genomes, but this will dramatically increase the computation time.
  • Step 3 Step 3 performs automatic primer design for all of the shredded template fragments using Primer3 with the following parameters: amplicon size 80–300 bp; amplicon GC content 40–60%, with an optimal GC content of 50%; primer length 18–28 nt, with an optimal length of 22 nt; and melting temperature (Tm) 58–64ºC, with an optimal Tm of 60ºC and maximum Tm difference per primer pair of less than 3ºC. For each gene, duplicated candidate primer pairs generated at neighboring windows are removed.

  • Step 4 The output file of Primer3 were transformed into ePCR input format using an in-house perl script. Then, ePCR was employed for the first-round of specificity check to all designed primer pairs. All primers with one mismatch or indel against any off-target sequence are filtered out. The remaining candidate primer pairs are used for the next round of specificity validation.

  • Step 5 Step 5 indexes the databases for the genome sequence and coding/mRNA sequence of the target organism based on the k-mer index algorithm using the thermodynamics-based gene-specificity checking program MFEprimer-2.0. By default, the k-mer is set to 9, which is sufficient for primer specificity checking; this value can be smaller if more stringent conditions are desired, but much more time will be needed for computation. The specificity of the qPCR primer pairs against genomic sequence and coding/mRNA sequence databases is then estimated, and related parameters such as the binding site in the sequence, the size and GC content of the amplicon, and the Tm and Gibbs free energy (ΔG) of the forward and reverse primers can be saved.

  • Step 6 The primer pairs for each gene are divided into three levels based on PPC and the binding stability of the binding site, ΔG, as follows:

           Standard for selection of level 1 primer pairs: the binding ability index, primer pair coverage (PPC) = 10. The primer pairs will be removed if their PPCs against any non-specific amplicons are higher than 1.

           Standard for selection of level 2 primer pairs: the binding stability index ΔG < −9 kcal/mol. The primer pairs will be filtered out if the ΔG of binding of either the forward or reverse primer to off-target sequences is less than −9 kcal/mo.

           Standard for selection of level 3 primer pairs: the binding stability index Gibbs free energy (ΔG) −11 kcal/mol. The primer pairs will be filtered out if the ΔG of binding of either the forward or reverse primer to off-target sequences is less than −11 kcal/mol

           In addition, all qualified primer pairs are further divided into two categories: the best primers and alternative primers. The former category includes only one best, unique primer pair with the lowest total ΔG of the pair, which is the gene-specific qPCR primer pair recommended for the detection of gene expression. The latter category includes all alternative gene-specific qPCR primer pairs.

  • Step 7 Step 7 carries out a BLAT search using each pair of primers against the genomic sequence. The number of exons spanned and the genomic position of each primer and amplicon can be determined to select primers spanning an exon-intron boundary, and this information can be used to avoid amplifying contaminating genomic DNA

  • After computation, our workflow generated a total of 51,091,785 gene-specific qPCR primer pairs, corresponding to 93.44% of the average coverage ratios per organism and an average of 15.33 primer pairs per gene. In human and mouse, more than 19 and 22 primer pairs per gene were obtained, with a coverage ratio of 78.83 and 90.32%, respectively, indicating that the results of our workflow are in accordance with those in MRPrimerW and PrimerBank. Moreover, it is more difficult to design qPCR primers for plants than for animals. Using the same workflow, the coverage ratios decreased from 93.80% in animals to 93.08% in plants, and the number of primer pairs per gene significantly dropped from 17.20 to 12.55 (permutation test P-value < 0.001). This reduction might have been due to the difficulty in designing qPCR primers for polyploid plants, such as oilseed rape, soybean (Glycine max), potato (Solanum tuberosum), and tomato (Solanum lycopersicum). Hence, our database will greatly facilitate qPCR experiments using organisms with complex genomes

Database implementation

  • qPrimerDB is implemented using PostgreSQL 9.6 (http://www.postgresql.org), PHP 5.5 (http://www.php.net), Apache Web Server 2.4 (http://www.apache.org), and BioPerl 1.6 (http://bioperl.org) on a Linux CentOS 6.8 operating system. Our database is supported by integration with the Chado database schema and Drupal, a popular Content Management System (CMS). There are four layers in the qPrimerDB architecture, as described for SilkPathDB. In the core layer, all qPrimerDB data are stored within the Chado schema and maintained using the PostgreSQL. The second layer governs communications between the core layer and the outer layers, as a configurable layer. The third layer invokes background programs to process and respond to queries, and the Drupal CMS is used for website content management. The outermost layer is a user interface layer that allows the users to directly interact with the database. To provide a friendly interface for users accessing the database from desktop and mobile devices, the Bootstrap framework (http://getboot strap.com) is employed. PHP and BioPerl scripts are used to generate primer index and search results pages by retrieving primer information for query genes and organisms provided by the user. The BLAST database is built with NCBI BLAST+ 2.6.0, with the multiple database search function. Finally, jQWidgets (http://www.jqwidgets.com) is used to organize and present the feature index, search, and BLAST results