31:T1645,* In 1999, [Stephensen et al](https://doi.org/10.1016/S0168-1702%2899%2900017-9) aligned all the known *Coronavirus* genus RNA-dependent RNA polymerase (RdRp) genes and designed primers for universal RT-PCR. This region of the genome is highly conserved, so is well-suited for building phylogenetic trees. These primers were based on the sequences of 11 known coronaviruses. * In 2003, [Drosten et al](https://doi.org/10.1056/NEJMoa030747) and [Ksiazek et al](https://doi.org/10.1056/NEJMoa030781) identified SARS using two pairs of primers that were "previously designed to conserved regions of open reading frame 1b to achieve broad reactivity with the genus coronavirus", by alignment of at least 12 known coronaviruses. The primers were provided by the CDC, through the WHO's SARS etiology network. The primer pairs were nested, to increase the chances of amplifying the intended section of the genome. * note: the inner reverse primer has a mismatch at the 3' end against SARS-like coronavirus genomes, which may alter the primer binding. * In 2005, [Moës et al](https://doi.org/10.1186/1471-2334-5-6) improved on the work of [Stephensen et al](https://doi.org/10.1016/S0168-1702%2899%2900017-9) and designed a new set of primers that could also detect HCoV-NL63, based on alignment of 14 coronaviruses. * In 2006, [Chu et al](https://doi.org/10.1099/vir.0.82203-0) adapted the work of Watanabe et al and published a set of nested primers with a new, hemi-nested reverse primer. * note: the text says Poon et al, cited Woo et al, but both Poon et al and Woo et al used the Watanabe/CDC primers. * In 2007, [de Souza Luna et al](https://doi.org/10.1128/JCM.02426-06) designed a new set of pan-coronavirus primers. * In 2007, [Hasoksuz et al](https://doi.org/10.1128/JVI.02361-06) modified the work of [Ksiazek et al](https://doi.org/10.1056/NEJMoa030781) and published a new set of pan-coronavirus primers. * note: the authors found that "the original universal primer pair targeted to this region from Ksiazek et al. was not optimal in RT-PCR with some CoV samples from wild ruminants". * In 2010, [Drexler et al](https://doi.org/10.1128/JVI.00650-10) used the primers of [de Souza Luna et al](https://doi.org/10.1128/JCM.02426-06) to amplify RdRp sequences, plus an extra set of forward primers that bind further upstream, increasing the length of the PCR product. * In 2010, [Watanabe et al](https://doi.org/10.3201/eid1608.100208) used the CDC inner primers (but cited [de Souza Luna et al](https://doi.org/10.1128/JCM.02426-06)) and designed a set of more specific nested primers. * In 2010, [Quan et al](https://doi.org/10.1128/mBio.00208-10) designed consensus primer sequences against the RdRp genes of all the coronaviruses known at that time. * note: the published reverse primer sequence has a typo - a duplicated "`CNA`"; citing works all use the correct sequence. * In 2011, [Chu et al](https://doi.org/10.1128/JVI.05838-11) published a new set of nested primers. * From 2009-2020 the PREDICT project used both the Quan primers and two nested pairs of primers modified from the Watanabe/CDC primers, in separate tests, to increase the chances of detecting novel coronaviruses. The first modified pair was designed to target a broad range of coronaviruses in bats, the second modified pair was designed to target a broad range of coronaviruses in other hosts. * note: the modified Watanabe/CDC reverse primer has the same mismatch at the 3' end with SARS-like coronavirus genomes, and also has one less nucleotide - a missing "`G`" - in the middle of one of the reverse primers, which may alter the primer binding. * In 2013, [Geldenhuys et al](https://10.1089/vbz.2012.1101) extended the work of Woo et al and published a new set of alpha- and betacoronavirus–specific primers. * note: primers available "on request" * In 2013, [Lelli et al](https://doi.org/10.3390/v5112679) published a new set of primers designed from amplifying a short RdRp sequence. * note: they also amplified RdRp using the primers of Watanabe et al (citing Dominguez et al), and for the samples where that failed they used a new forward primer with the previous reverse primer. * In 2014, [Woo et al](https://doi.org/10.1128/JVI.02351-13) published a new set of primers designed by multiple alignments of the nucleotide sequences of available RdRp genes of known coronaviruses. * In 2016, [Boniotti et al](https://doi.org/10.3201/eid2201.150544) published a new set of primers designed from 41 alpha, beta, gamma, and delta coronavirus sequences. * In 2016, [Huang et al](https://doi.org/10.1371/journal.ppat.1005883) published a new set of primers designed from a highly conserved region of the RdRp. * In 2018, [Geldenhuys et al](https://doi.org/10.1371/journal.pone.0194527) adapted their earlier work and published a new set of primers. * In 2018, [Hu et al](https://doi.org/10.1016/j.jviromet.2018.02.021) modified the work of [Hasoksuz et al](https://doi.org/10.1128/JVI.02361-06) and published a new set of pan-coronavirus primers. * note: the original version of figure 1 showed the wrong primer; the amended figure still seems to show incorrect sequences? * In 2021, in recognition of the many coronavirus genomes which have been discovered, [Holbrook et al](https://doi.org/10.3390/v13040599) designed a new set of primers by aligning all the known coronavirus genomes. These nested primer pairs are similar to the previous versions but have more degeneracy, to increase their ability to amplify RdRp genes from a wider range of targets. * note: in the methods section of the paper there's a typo - a missing "`T`" - in one of the primers; the sequences in Table S1 are correct.32:T51a,displayTargetSequence = { const container = html`

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  if (matches) {
    const forward = matches.forward;
    const reverse = matches.reverse;

    const parts = [
      targetSequenceUppercase.substring(
        forward.position - 250,
        forward.position
      ),
      targetSequenceUppercase.substring(
        forward.position,
        forward.position + forward.match.length
      ),
      targetSequenceUppercase.substring(
        forward.position + forward.match.length,
        reverse.position
      ),
      targetSequenceUppercase.substring(
        reverse.position,
        reverse.position + reverse.match.length
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      targetSequenceUppercase.substring(
        reverse.position + reverse.match.length,
        reverse.position + reverse.match.length + 250
      )
    ];

    container.append(parts[0]);
    container.append(
      html`${parts[1]}`
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    container.append(
      html`${parts[2]}`
    );
    container.append(
      html`${parts[3]}`
    );
    container.append(parts[4]);
  } else {
    container.append(target.sequence);
  }
  return container;
}33:T283d,// https://doi.org/10.1371/journal.pone.0236971

primers = [
  // https://doi.org/10.3390/v13040599
  {
    label: "HOLBROOK OUTER",
    forward: ["GGTTGGGAYTAYCCHAARTGYGA"], // GGTGGGAYTAYCCHAARTGYGA (note: missing T in methods section)
    reverse: ["CCRTCATCAGAHARWATCAT", "CCRTCATCACTHARWATCAT"]
  },
  {
    label: "HOLBROOK INNER",
    forward: ["GAYTAYCCHAARTGTGAYAGA", "GAYTAYCCHAARTGTGAYMGH"],
    reverse: ["CCRTCATCAGAHARWATCAT", "CCRTCATCACTHARWATCAT"]
  },
  // https://doi.org/10.1093/ve/vex012
  {
    label: "PREDICT MODIFIED WATANABE OUTER",
    forward: ["GGTTGGGAYTAYCCHAARTGTGA"], // CoV-FWD3
    reverse: ["CCATCATCASWYRGAATCATCATA"] // CoV-RVS3 CCATCATCASWYRAATCATCATA (note: added extra G to allow alignment)
  },
  {
    label: "PREDICT MODIFIED WATANABE INNER BAT",
    forward: ["GAYTAYCCHAARTGTGAYAGAGC"], // CoV-FWD4/Bat,
    reverse: ["CCATCATCASWYRGAATCATCATA"] // CoV-RVS3 CCATCATCASWYRAATCATCATA (note: added extra G to allow alignment)
  },
  // https://doi.org/10.1101/2020.06.19.158717
  {
    label: "PREDICT MODIFIED WATANABE INNER OTHER",
    forward: ["GAYTAYCCHAARTGTGAUMGWGC"], // CoV-FWD4/Other
    reverse: ["CCATCATCASWYRGAATCATCATA"] // CoV-RVS3 CCATCATCASWYRAATCATCATA (note: added extra G to allow alignment)
  },
  // https://doi.org/10.1128/JVI.79.2.884-895.2005
  // {
  //   label: "WOO",
  //   forward: ["GGTTGGGACTATCCTAAGTGTGA"],
  //   reverse: ["CCATCATCAGATAGAATCATCATA"]
  // },
  // https://doi.org/10.3201/eid2004.131769
  {
    label: "WOO", // 441bp
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // same as IN-6
    reverse: ["ACCATCATCNGANARDATCATNA"] // like IN-7 but with more degeneracy
  },
  // https://doi.org/10.3201/eid1608.100208
  // {
  //   label: "WATANABE",
  //   forward: ["GGTTGGGACTATCCTAAGTGTGA"],
  //   reverse: ["CCATCATCAGATAGAATCATCATA"]
  // },
  // {
  //   label: "WATANABE INNER",
  //   forward: ["TGATTTCTGCAATGATACTTGGTTC", "ACTTGATGATCTGTAACAACAATCG"],
  //   reverse: ["TACAACCTACGCTGCAACTC", "ATGAGTGTGCACAAGTGCTTAG"]
  // },
  // 10.1128/JVI.79.4.2001-2009.2005
  {
    label: "POON OUTER", // 1306bp
    forward: ["AYAACCAAGATCTTAATGG"],
    reverse: ["TGCTTAGAACCCAAAATCAT"]
  },
  {
    label: "POON INNER", // 440bp // note: "Forward and reverse primers (IN-6 and IN-7) were communicated through the World Health Organization's SARS etiology network by colleagues from the Centers for Disease Control and Prevention.", like in DROSTEN
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // IN-6
    reverse: ["CCATCATCAGATAGAATCATCATA"] // IN-7
  },
  // 10.1056/NEJMoa030781, 10.1056/NEJMoa030747
  {
    label: "CDC OUTER",
    forward: ["GGGTTGGGACTATCCTAAGTGTGA"], // IN-2
    reverse: ["TAACACACAACICCATCATCA"] // IN-4
  },
  {
    label: "CDC INNER / WATANABE OUTER",
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // IN-6
    reverse: ["CCATCATCAGATAGAATCATCATA"] // IN-7
  },
  // 10.1186/1471-2334-5-6
  {
    label: "MOES",
    forward: ["ACWCARHTVAAYYTNAARTAYGC"],
    reverse: ["TCRCAYTTDGGRTARTCCCA"]
  },
  // 10.1086/428138
  {
    label: "ESPER",
    forward: ["GCGCAAAATAATGAATTAATGCC"],
    reverse: ["GACGCACCACCATATGAATCCTG"]
  },
  // 10.1016/j.jviromet.2004.07.008
  {
    label: "ADACHI",
    forward: ["TGATGGGTTGGGACTATCCTAAATGTGA"], // CORO1
    reverse: ["GTAGTTGCATCACCGGAAGTTGTGCCACC"] // CORO2
  },
  // 10.1007/s00705-006-0840-x
  {
    label: "ESCUTENAIRE",
    forward: ["TGATGATGSNGTTGTNTGYTAYAA"], // 11-FW
    reverse: ["GCATWGTRTGYTGNGARCARAATTC"] // 13-RV
  },
  // 10.1016/S0168-1702(99)00017-9
  {
    label: "STEPHENSON",
    forward: ["ACTCARWTRAATYTNAAATAYGC"],
    reverse: ["TCACAYTTWGGATARTCCCA"]
  },
  // https://doi.org/10.1128/mBio.00208-10
  {
    label: "QUAN OUTER",
    forward: ["CGTTGGIACWAAYBTVCCWYTICARBTRGG"], // CoV-FWD1
    reverse: ["GGTCATKATAGCRTCAVMASWWGCNACATG"] // CoV-RVS1 "GGTCATKATAGCRTCAVMASWWGCNACNACATG" (note: removed repeated CNA)
  },
  {
    label: "QUAN INNER",
    forward: ["GGCWCCWCCHGGNGARCAATT"], // CoV-FWD2
    reverse: ["GGWAWCCCCAYTGYTGWAYRTC"] // CoV-RVS2
  },
  // https://doi.org/10.1128/JCM.02426-06
  {
    label: "DE SOUZA LUNA OUTER",
    forward: ["CTTATGGGTTGGGATTATCCTAAGTGTGA", "CTTATGGGTTGGGATTATCCCAAATGTGA"], // PCS
    reverse: [
      "CACACAACACCTTCATCAGATAGAATCATCA" // PCNAs
    ]
  },
  {
    label: "DE SOUZA LUNA INNER",
    forward: ["TTATGGGTTGGGATTATC", "TGATGGGATGGGACTATC"], // PC2S2
    reverse: [
      "TCATCACTCAGAATCATCA",
      "TCATCAGAAAGAATCATCA",
      "TCGTCGGACAAGATCATCA"
    ] // PC2As1
  },
  // 10.1371/journal.pone.0194527
  {
    label: "GELDENHUYS OUTER",
    forward: ["ATGGGHTGGGAYTAYCCHAARTGYGA"],
    reverse: ["CCRTCATCWGAIARDATCATCAT", "CATCRTCASDIARDATCATCAT"]
  },
  {
    label: "GELDENHUYS INNER",
    forward: ["TYTAYNTNAARCCWGGTGG", "TAYGTIAARCCWGGHGGIAC"],
    reverse: ["CCRTCATCWGAIARDATCATCAT", "CATCRTCASDIARDATCATCAT"]
  },
  // 10.3390/v5112679
  {
    label: "DELLI",
    forward: ["CDCAYGARTTYTGYTCNCARC"],
    reverse: ["RHGGRTANGCRTCWATDGC"]
  },
  // 10.1371/journal.ppat.1005883
  {
    label: "HUANG OUTER", // (299bp)
    forward: ["TGTTATTGGAACCACGAAGTTYTAYGGNGGNTG"], // panCoVs-OF
    reverse: ["TAGTAGCATCTCCGCTGCTAGTNCCNCCNGGYTT"] // panCoVs-OR
  },
  {
    label: "HUANG INNER", // (228bp)
    forward: ["GTTTTGTTCTTATGGGTTGGGATTAYCCNAARTGYGA"], // panCoVs-IF
    reverse: ["TAGTAGCATCTCCGCTGCTAGTNCCNCCNGGYTT"] // panCoVs-OR
  },
  // {
  //   label: "HUANG SEQUENCING OUTER",
  //   forward: ["TGTTATTGGAACCACGAAG"], // panCorona-seq-1F
  //   reverse: ["GCATCTCCGCTGCTAGT"] // panCorona-seq-R
  // },
  // {
  //   label: "HUANG SEQUENCING INNER",
  //   forward: ["CTTATGGGTTGGGATTA"], // panCorona-seq-2F
  //   reverse: ["GCATCTCCGCTGCTAGT"] // panCorona-seq-R
  // },
  // 10.1128/JVI.02361-06
  // 10.1007/978-1-59745-181-9_5
  {
    label: "HASOKSUZ", // (452bp)
    forward: ["GGGDTGGGAYTAYCCHAARTGYGA"], // IN-2deg
    reverse: ["TARCAVACAACISYRTCRTCA"] // IN-4deg
  },
  // 10.1016/j.jviromet.2018.02.021
  {
    label: "HU", // (668bp)
    forward: [
      "AARTTYTAYGGHGGYTGG" // panCoV-F2
      // "TGGGDTGGGAYTAYCCHAART" // panCoV-F1?
    ],
    reverse: ["GARCARAATTCATGHGGDCC"] // panCoV-R1
  },
  // https://doi.org/10.3201/eid1911.131172
  {
    label: "NESTED CII-PAN-COV-III OUTER",
    forward: ["ACWGTTCARGGICCWCCIGG"],
    reverse: ["GGCAGCTGWGCWGGRTCICCNACRTA"]
  },
  {
    label: "NESTED CII-PAN-COV-III INNER",
    forward: ["GTTCARGGGCCWCCGGGNAC"],
    reverse: ["AGCTGWGCWGGRTCGCCIACRTANAC"]
  },
  // https://doi.org/10.1128/JVI.00650-10
  // http://hdl.handle.net/10362/90337
  {
    label: "DREXLER EXTENDED",
    forward: [
      "CTTCTTCTTTGCTCAGGATGGCAATGCTGC", // SP3080
      "ATACTTTGATTGTTACGATGGTGGCTG", // SP3195
      "CTATAACTCAAATGAATCTTAAGTATGC", // SP3374
      "TTCTTTGCACAGAAGGGTGATGC", // GrISP1
      "CTTTGCACAAAAAGGTGATGCWGC" // GrISP2
    ],
    reverse: [] // TODO
  },
  // {
  //   label: "DREXLER COMBINED OUTER",
  //   forward: [
  //     "CTTCTTCTTTGCTCAGGATGGCAATGCTGC",
  //     "TTCTTTGCACAGAAGGGTGATGC",
  //     "CTTTGCACAAAAAGGTGATGCWGC"
  //   ],
  //   reverse: [
  //     "TAGAAWCCACCATTDGARTAMACAAC",
  //     "TTGCAMARACACTARTACYTCTCACT",
  //     "CCACCCGTACAATGAACAACTTC"
  //   ]
  // },
  // {
  //   label: "DREXLER COMBINED INNER",
  //   forward: [
  //     "CTTCTTCTTTGCTCAGGATGGCAATGCTGC",
  //     "TTCTTTGCACAGAAGGGTGATGC",
  //     "CTTTGCACAAAAAGGTGATGCWGC"
  //   ],
  //   reverse: [
  //     "GAMCCYAAWAYCATSGCAGAGATCAT", // note: missing "T" and incorrect round number in Table 3 of Simoes theses
  //     "GAACCMAGWATCATGGCDGAGATCAT",
  //     "CTTTGCACCYAGTATCATCGCTG"
  //   ]
  // },
  // {
  //   label: "DREXLER BETACOV OUTER",
  //   forward: ["CTTCTTCTTTGCTCAGGATGGCAATGCTGC"],
  //   reverse: ["TAGAAWCCACCATTDGARTAMACAAC"]
  // },
  // {
  //   label: "DREXLER BETACOV INNER",
  //   forward: ["CTTCTTCTTTGCTCAGGATGGCAATGCTGC"],
  //   reverse: ["GAMCCYAAWAYCATSGCAGAGATCAT"] // note: missing "T" and incorrect round number in Table 3 of Simoes theses
  // },
  // {
  //   label: "DREXLER ALPHACOV G1 OUTER",
  //   forward: ["TTCTTTGCACAGAAGGGTGATGC"],
  //   reverse: ["TTGCAMARACACTARTACYTCTCACT"]
  // },
  // {
  //   label: "DREXLER ALPHACOV G1 INNER",
  //   forward: ["TTCTTTGCACAGAAGGGTGATGC"],
  //   reverse: ["GAACCMAGWATCATGGCDGAGATCAT"]
  // },
  // {
  //   label: "DREXLER ALPHACOV G2 OUTER",
  //   forward: ["TTCTTTGCACAGAAGGGTGATGC"],
  //   reverse: ["TAGAAWCCACCATTDGARTAMACAAC"]
  // },
  // {
  //   label: "DREXLER ALPHACOV G2 INNER",
  //   forward: ["TTCTTTGCACAGAAGGGTGATGC"],
  //   reverse: ["GAMCCYAAWAYCATSGCAGAGATCAT"]
  // },
  // {
  //   label: "DREXLER STURNIRACOV OUTER",
  //   forward: ["CTTTGCACAAAAAGGTGATGCWGC"],
  //   reverse: ["CCACCCGTACAATGAACAACTTC"]
  // },
  // {
  //   label: "DREXLER STURNIRACOV INNER",
  //   forward: ["CTTTGCACAAAAAGGTGATGCWGC"],
  //   reverse: ["CTTTGCACCYAGTATCATCGCTG"]
  // },
  // 10.2807/1560-7917.ES.2020.25.21.2000880
  {
    label: "CORMAN SARS-CoV-2",
    forward: ["GTGARATGGTCATGTGTGGCGG"],
    reverse: ["CARATGTTAAASACACTATTAGCATA"]
  },
  // 10.3201/eid2201.150544
  {
    label: "BONIOTTI",
    forward: ["GGNTGGGAYTAYCCNAARTGYGA"], // CoV_8F, same as LELLI PHYLO
    reverse: ["TGYTGNGARCARAAYTCRTG"] // CoV_10R
  },
  // 10.3390/v5112679
  {
    label: "LELLI", // 180bp
    forward: ["CDCAYGARTTYTGYTCNCARC"], // IZS-CoV forward
    reverse: ["RHGGRTANGCRTCWATDGC"] // IZS-CoV reverse
  },
  {
    label: "LELLI PHYLO", // 760bp // note: used when 440bp amplification using Watanabe failed, in 6 samples
    forward: ["GGNTGGGAYTAYCCNAARTGYGA"],
    reverse: ["RHGGRTANGCRTCWATDGC"] // IZS-CoV reverse
  },
  // 10.1099/vir.0.82203-0
  {
    label: "CHU 2006 1", // 440bp
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // POON
    reverse: ["CCATCATCAGATAGAATCATCAT"] // POON
  },
  {
    label: "CHU 2006 2", // 435bp, for samples that were negative in the first assay
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // POON
    reverse: ["ATCAGATAGAATCATCATAGAGA"]
  },
  // 10.1128/JVI.05838-11
  {
    label: "CHU 2011 OUTER", // 602bp
    forward: ["GGKTGGGAYTAYCCKAARTG"],
    reverse: ["TGYTGTSWRCARAAYTCRTG"]
  },
  {
    label: "CHU 2011 INNER", // 400bp
    forward: ["GGTTGGGACTATCCTAAGTGTGA"], // IN-6
    reverse: ["CCATCATCAGATAGAATCATCAT"] // IN-7
  }
]34:[]
2f:{"title":"Pan-coronavirus primer analysis","cells":[{"id":0,"value":"# Pan-coronavirus primer analysis","pinned":false,"hidden":false,"mode":"md"},{"id":444,"value":"Analyse pan-coronavirus primer pairs by aligning them with *Coronavirus* reference genome sequences.","pinned":false,"hidden":false,"mode":"md"},{"id":534,"value":"## Background","pinned":false,"hidden":false,"mode":"md"},{"id":549,"value":"For several years researchers have used RT-PCR to detect coronaviruses in animal samples - particularly in resource-poor locations where next-generation sequencing isn't available - using primer pairs designed to amplify as many known and novel coronaviruses as possible.","pinned":false,"hidden":false,"mode":"md"},{"id":559,"value":"$31","pinned":false,"hidden":false,"mode":"md"},{"id":31,"value":"## Coronaviridae genome reference sequences","pinned":false,"hidden":false,"mode":"md"},{"id":654,"value":"viewof term = Inputs.text({\n  value:\n    \"Coronaviridae[Organism] AND srcdb_refseq[PROP] NOT wgs[PROP] NOT cellular organisms[ORGN] NOT AC_000001:AC_999999[PACC]\",\n  submit: true,\n  width: \"100%\"\n})","pinned":false,"hidden":false,"mode":"ojs"},{"id":651,"value":"Choose one of the ${targets.length} *Coronaviridae* genome reference sequences from GenBank:","pinned":false,"hidden":false,"mode":"md"},{"id":36,"value":"viewof selectedTarget = Inputs.select(targets, {\n  format: (item) => item.title,\n  label: \"Target\",\n  width: \"100%\"\n})","pinned":false,"hidden":false,"mode":"ojs"},{"id":15,"value":"## Pan-coronavirus primer pairs","pinned":false,"hidden":false,"mode":"md"},{"id":468,"value":"Choose a pair of primers designed for pan-coronavirus amplification.","pinned":false,"hidden":false,"mode":"md"},{"id":239,"value":"viewof selectedPrimer = Inputs.select(primers, {\n  format: (item) => item.label,\n  label: \"Primer pair\",\n  width: \"100%\"\n})","pinned":false,"hidden":false,"mode":"ojs"},{"id":157,"value":"html`\n\n\n\n\n\n\n\n\n\n\n\nforward \n${primer.forward.join(\"\\n\")}
\n${displayNucleotides(nucleotides.forward).join(\"\\n\")}
\n
reverse \n${primer.reverse.join(\"\\n\")}
\n${displayNucleotides(nucleotides.reverse).join(\"\\n\")}
\n
`","pinned":false,"hidden":false,"mode":"ojs"},{"id":595,"value":"The pair of primers with the best match to the target sequence produces a ${matches.size}bp product:","pinned":false,"hidden":false,"mode":"md"},{"id":474,"value":"\n  \n    \n      \n      \n      \n      \n    \n  \n\n  \n    \n    \n    \n    \n  \n  \n    \n    \n    \n    \n  \n\ndirection matching sequence score position
forward ${matches.forward.match} ${matches.forward.score} ${matches.forward.position}
reverse ${matches.reverse.match} ${matches.reverse.score} ${matches.reverse.position}
","pinned":false,"hidden":false,"mode":"html"},{"id":100,"value":"html`${target.definition}
`","pinned":false,"hidden":false,"mode":"ojs"},{"id":75,"value":"$32","pinned":false,"hidden":false,"mode":"ojs"},{"id":494,"value":"## Primers and sources","pinned":false,"hidden":false,"mode":"md"},{"id":233,"value":"$33","pinned":false,"hidden":false,"mode":"ojs"},{"id":744,"value":"Or analyse a custom pair of primers:","pinned":false,"hidden":false,"mode":"md"},{"id":743,"value":"viewof forward = Inputs.text({ width: \"100%\", label: \"forward\" })","pinned":false,"hidden":false,"mode":"ojs"},{"id":750,"value":"viewof reverse = Inputs.text({ width: \"100%\", label: \"reverse\" })","pinned":false,"hidden":false,"mode":"ojs"},{"id":607,"value":"## Calculations","pinned":false,"hidden":false,"mode":"md"},{"id":755,"value":"primer = {\n  if (forward && reverse) {\n    return {\n      label: \"CUSTOM\",\n      forward: [forward.replace(/\\s+/g, \"\")],\n      reverse: [reverse.replace(/\\s+/g, \"\")]\n    };\n  }\n\n  return selectedPrimer;\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":335,"value":"targetSequenceUppercase = target.sequence.toUpperCase()","pinned":false,"hidden":false,"mode":"ojs"},{"id":126,"value":"findMatches = (sequence, primers, direction) => {\n  const matches = [];\n\n  // const isReversed = direction === \"reverse\";\n\n  for (let i = 0; i < sequence.length; i++) {\n    for (const primer of primers) {\n      let score = 100;\n      let match = \"\";\n\n      for (let j = 0; j < primer.length; j++) {\n        let matched = undefined;\n\n        const targetChar = sequence[i + j];\n\n        const chars = primer[j];\n\n        for (const char of chars) {\n          if (targetChar === char) {\n            matched = targetChar;\n            break;\n          }\n        }\n\n        if (matched) {\n          score = score + 1;\n          match += matched;\n        } else {\n          score = score - 2;\n          match += \"-\";\n        }\n      }\n\n      if (score > 50) {\n        matches.push({\n          // match: isReversed ? match.split(\"\").reverse().join(\"\") : match,\n          match,\n          score,\n          position: i\n        });\n      }\n    }\n  }\n\n  matches.sort((a, b) => b.score - a.score);\n\n  return matches[0];\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":339,"value":"matches = {\n  const forward = findMatches(targetSequenceUppercase, nucleotides.forward);\n  const reverse = findMatches(targetSequenceUppercase, nucleotides.reverse);\n  const size = reverse.position + reverse.match.length - forward.position;\n\n  return { forward, reverse, size };\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":349,"value":"displayNucleotides = (items) =>\n  items.map((nucleotides) =>\n    nucleotides.map((item) => \"[\" + item.join(\"/\") + \"]\").join(\"\")\n  )","pinned":false,"hidden":false,"mode":"ojs"},{"id":81,"value":"buildNucleotides = (primerSequences, direction) => {\n  return primerSequences.map((primerSequence) => {\n    const nucleotides = [];\n\n    const sequence = [...primerSequence];\n\n    if (direction === \"reverse\") {\n      sequence.reverse();\n    }\n\n    for (let i = 0; i < sequence.length; i++) {\n      const nucleotide = sequence[i];\n      \n      let item = variants[nucleotide] || [nucleotide];\n      \n      if (direction === \"reverse\") {\n        item = item.map((character) => reversed[character]);\n      }\n      \n      nucleotides.push(item);\n    }\n\n    return nucleotides;\n  });\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":361,"value":"nucleotides = {\n  return {\n    forward: buildNucleotides(primer.forward, \"forward\"),\n    reverse: buildNucleotides(primer.reverse, \"reverse\")\n  };\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":308,"value":"reversed = {\n  return {\n    A: \"T\",\n    T: \"A\",\n    C: \"G\",\n    G: \"C\"\n  };\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":292,"value":"variants = {\n  // https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=BlastHelp\n\n  return {\n    K: [\"G\", \"T\"],\n    M: [\"A\", \"C\"],\n    B: [\"G\", \"T\", \"C\"],\n    V: [\"G\", \"C\", \"A\"],\n    S: [\"G\", \"C\"],\n    W: [\"A\", \"T\"],\n    D: [\"G\", \"A\", \"T\"],\n    Y: [\"T\", \"C\"],\n    R: [\"G\", \"A\"],\n    H: [\"A\", \"C\", \"T\"],\n    N: [\"A\", \"C\", \"T\", \"G\"],\n    I: [\"C\", \"A\"] // == M\n    // https://patentimages.storage.googleapis.com/36/6b/7f/dfacc9ea7d56a9/US20100035227A1.pdf\n    // I: [\"U\", \"C\", \"A\"]\n    // G: [\"U\", \"C\"],\n    // U: [\"U\", \"C\"],\n  };\n}","pinned":true,"hidden":false,"mode":"ojs"},{"id":262,"value":"## Sequence data from GenBank","pinned":false,"hidden":false,"mode":"md"},{"id":689,"value":"fetching ? html`` : html`✅ loaded data`","pinned":false,"hidden":false,"mode":"ojs"},{"id":682,"value":"mutable fetching = true","pinned":false,"hidden":false,"mode":"ojs"},{"id":184,"value":"esearch = {\n  if (!term) return;\n\n  mutable fetching = true;\n\n  const params = new URLSearchParams({\n    db: \"nucleotide\",\n    retmode: \"json\",\n    usehistory: \"y\",\n    term,\n    retmax: 0\n  });\n\n  return d3.json(\n    \"https://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?\" + params\n  );\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":832,"value":"esummary = {\n  if (!esearch?.esearchresult?.count) return;\n\n  const params = new URLSearchParams({\n    db: \"nucleotide\",\n    retmode: \"xml\",\n    query_key: esearch.esearchresult.querykey,\n    webenv: esearch.esearchresult.webenv,\n    retmode: \"json\",\n    retmax: 100\n  });\n\n  const data = await d3.json(\n    \"https://eutils.ncbi.nlm.nih.gov/entrez/eutils/esummary.fcgi?\" + params\n  );\n\n  mutable fetching = false;\n\n  return data;\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":190,"value":"target = {\n  if (!selectedTarget) return;\n\n  const params = new URLSearchParams({\n    db: \"nuccore\",\n    retmode: \"text\",\n    rettype: \"fasta\",\n    id: selectedTarget.id\n  });\n\n  const fasta = await d3.text(\n    \"https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?\" + params\n  );\n\n  const sequence = fasta.split(\"\\n\").slice(1).join(\"\");\n\n  mutable fetching = false;\n\n  return {\n    definition: selectedTarget.title,\n    sequence\n  };\n}","pinned":false,"hidden":false,"mode":"ojs"},{"id":207,"value":"targets = {\n  const targets = [];\n\n  for (const uid of esummary.result.uids) {\n    const item = esummary.result[uid];\n\n    targets.push({\n      id: item.uid,\n      title: item.title\n    });\n  }\n\n  targets.sort((a, b) => a.title.localeCompare(b.title));\n\n  return targets;\n}","pinned":false,"hidden":false,"mode":"ojs"}],"files":"$W34","stdlib":"1","titling":"auto"}

direction	matching sequence	score	position
forward	`${matches.forward.match}`	${matches.forward.score}	${matches.forward.position}
reverse	`${matches.reverse.match}`	${matches.reverse.score}	${matches.reverse.position}