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Yakazara-kureba mRNA kutevedzana -PacBio

De novofull-length transcriptome sequencing, inozivikanwawo seDe novoIso-Seq inotora zvakanakira PacBio sequencer mukureba kwekuverenga, iyo inogonesa kutevedzana kweakazara-kureba cDNA mamorekuru pasina chero mabreak.Izvi zvinodzivirira zvachose chero zvikanganiso zvinogadzirwa mumatanho egungano ezvinyorwa uye zvinovaka unigene seti ine isoform-level resolution.Iyi unigene seti inopa ruzivo rwakasimba rwemajini se "reference genome" pa transcriptome-level.Uye zvakare, kusanganisa nechizvarwa chinotevera chekuteedzera data, iyi sevhisi inopa iyo chaiyo quantification yeisoform-level kutaura.

Platform: PacBio Sequel II
Raibhurari: SMRT bhero raibhurari

  • :
  • Service Details

    Demo Results

    Nyaya Yokudzidza

    Basa Rinobatsira

    2

    ● Kuverenga zvakananga kwemorekuru wecDNA yakareba kubva pa3'- kumagumo kusvika pa5'- kumagumo

    ● Iso-form level resolution mukugadzirisa maitiro

    ● Zvinyorwa nekururama kwepamusoro uye kutendeseka

    ● Inoenderana zvakanyanya nemhando dzevaiours

    ● Huru hwekutevedzana hune 4 PacBio Sequel II sequencing mapuratifomu ane

    ● Yakanyanya ruzivo nepamusoro pe700 Pacbio-based RNA sequencing mapurojekiti

    ● BMKCloud-based result delivery: Customized data-mining inowanikwa pachikuva.

    ● Mushure mekutengesa masevhisi anoshanda kwemwedzi mitatu pakupedzwa kweprojekiti

    Sevhisi Zvinotsanangurwa

    Platform: PacBio Sequel II

    Sequencing raibhurari: Poly A- enriched mRNA raibhurari

    Inokurudzirwa kuburitsa data: 20 Gb/sample (Zvichienderana nemhando)

    FLNC (%): ≥75%

    *FLNC: Yakazara-kureba isiri-chimeric transcipts

    Bioinformatics inoongorora

    ● Raw data processing
     
    ● Kuzivikanwa kwezvinyorwa
     
    ● Kutevedzana kwemaitiro
     
    ● Kuenzana Kwemashoko
     
    ● Chiratidzo cheBasa

    hurefu hwakazara pacbio

    Sample Zvinodiwa uye Delivery

    Sample Zvinodiwa:

    Nucleotides:

    Conc.(ng/μl)

    Mari (μg)

    Kuchena

    Kuperera

    ≥ 120

    ≥ 0.6

    OD260/280=1.7-2.5

    OD260/230=0.5-2.5

    Yakaganhurirwa kana isina mapuroteni kana kusvibiswa kweDNA kunoratidzwa pagel.

    Kumiti: RIN≥7.5;

    Zvemhuka: RIN≥8.0;

    5.0≥ 28S/18S≥1.0;

    kukwirira kwakadzika kana kusina kwekutanga

    Tissue: Huremu (yakaoma):≥1 g
    *Kune tishu diki pane 5 mg, tinokurudzira kutumira flash yakaomeswa nechando (mumvura nitrogen) sampuli yetishu.

    Kumiswa kwesero:Nhamba yemasero = 3 × 1061 × 107
    * Isu tinokurudzira kutumira chando cell lysate.Kana iyo sero inoverenga idiki pane 5 × 105, flash yakaomeswa mu nitrogen yemvura inokurudzirwa, iyo inosarudzika kune micro extraction.

    Mienzaniso yeropa:Kuwanda≥1 mL

    Microorganism:Husa ≥ 1 g

    Yakakurudzirwa Sample Delivery

    Container:
    2 ml centrifuge chubhu (Tin foil haina kukurudzirwa)
    Sample labeling: Boka+ replicate eg A1, A2, A3;B1, B2, B3...

    Shipment:

    1. Dry-ice: Samples dzinoda kurongedzerwa mumabhegi uye kuvigwa muoma-aizi.
    2. RNAstable tubes: RNA samples inogona kuomeswa muRNA stabilization tube (eg RNAstable®) uye inotumirwa mukamuri yekupisa.

    Kuyerera Kwebasa Rebasa

    Muenzaniso QC

    Kuedza kugadzira

    sampuli yekuendesa

    Muenzaniso wekutumira

    Pilot experiment

    RNA kubviswa

    Kugadzirira Raibhurari

    Kuvakwa kweraibhurari

    Sequencing

    Sequencing

    Data analysis

    Data analysis

    Mushure mekutengesa Services

    Mushure mekutengesa masevhisi


  • Zvakapfuura:
  • Zvinotevera:

  • 1.FLNC kureba kugoverwa

    Hurefu hwehurefu hwakazara isiri-chimeric kuverenga(FLNC) inoratidza kureba kwecDNA mukuvaka raibhurari.FLNC kureba kugovera chiratidzo chakakosha mukuongorora mhando yekuvaka raibhurari.

    mRNA-FLNC-kuverenga-kureba-kugovera

    FLNC kuverenga kureba kugoverwa

    2.Pedzisa ORF nharaunda kureba kugoverwa

    Isu tinoshandisa TransDecoder kufanotaura mapuroteni ekukodha matunhu uye anoenderana amino acid kutevedzana kugadzira unigene seti, iyo ine yakazara isina-yedundant zvinyorwa zvinyorwa mumasampuli ese.

    mRNA-Yakazara-ORF-kureba-kugovera

    Zadzisa ORF kureba kwedunhu kugovera

    3.KEGG nzira yekuvandudza kuongorora

    Zvinyorwa zvakasiyana-siyana (DETs) zvinogona kuonekwa nekubatanidza NGS-based RNA sequencing data pane yakazara-yakareba zvinyorwa zvinyorwa zvinogadzirwa nePacBio sequencing data.Aya maDET anogona kuenderera mberi nekugadziridzwa kune akasiyana mashandiro ekuongorora, semuenzaniso KEGG nzira yekuvandudza ongororo.

    mRNA-DEG-KEGG-nzira-kupfumisa

    DET KEGG nzira yekupfumisa -Dot plot

    Nyaya yeBMK

    Mamiriro ekusimudzira ePopulus stem transcriptome

    Rakatsikiswa: Plant Biotechnology Journal, 2019

    Sequencing strategy:
    Sample collection:stem matunhu: apex, yekutanga internode (IN1), yechipiri internode (IN2), yechitatu internode (IN3), internode (IN4) uye internode (IN5) kubva kuNanlin895
    NGS-kutevedzana:RNA yevanhu gumi nevashanu vakaiswa semumwechete biological sample.Zvitatu zvebhayoloji zvinodzokororwa pamapoinzi ega ega akagadziriswa kutevedzana kweNGS
    TGS-kutevedzana:Matunhu akapatsanurwa kuita matunhu matatu, kureva apex, IN1-IN3 uye IN4-IN5.Dunhu rega rega rakagadziriswa PacBio kutevedzana nemhando ina dzemaraibhurari: 0-1 kb, 1-2 kb, 2-3 kb uye 3-10 kb.

    Mibairo yakakosha

    1. Huwandu hwe87150 hwakazara-hurefu hwakanyorwa hwakaonekwa, umo, 2081 novel isoforms uye 62058 novel imwe spliced ​​isoforms yakaonekwa.
    2.1187 lncRNA uye 356 fusion majini akaonekwa.
    3.Kubva pakukura kwekutanga kusvika pakukura kwechipiri, 15838 yakaratidza zvakasiyana-siyana zvinyorwa kubva ku995 zvakasiyana-siyana zvakaratidzwa majeni zvakaonekwa.Mune ese maDEGs, 1216 aive ezvinyorwa zvinyorwa, mazhinji acho asati ataurwa.
    4.GO enrichment analysis yakaratidza kukosha kwesero kupatsanurwa uye oxidation-kuderedza maitiro mukukura kwekutanga uye kwechipiri.

    • PB-yakazara-kureba-RNA-Sequencing-case-study

      Alternative splicing zviitiko uye akasiyana isoforms

    • PB-yakazara-kureba-RNA-alternative-splicing

      WGCNA kuongororwa pane zvinyorwa zvinyorwa

    Reference

    Chao Q, Gao ZF, Zhang D, nevamwe.Mamiriro ekusimudzira ePopulus stem transcriptome.Plant Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958

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