BMKCloud Log in
条形banner-03

Nā huahana

ʻO ke kaʻina mRNA lōʻihi piha -PacBio

De novoka lōʻihi transcriptome sequencing, ʻike ʻia hoʻiDe novoLawe ʻo Iso-Seq i nā pōmaikaʻi o PacBio sequencer i ka lōʻihi heluhelu, e hiki ai i ka hoʻonohonoho ʻana i nā molekala cDNA piha piha me ka ʻole o ka haki.Hōʻalo loa kēia i nā hewa i hana ʻia i nā ʻanuʻu hui transcript a kūkulu i nā pūʻulu unigene me ka hoʻonā isoform-level.Hāʻawi kēia unigene set i ka ʻike genetic ikaika e like me "genome reference" ma ka pae transcriptome.Eia kekahi, e hui pū me ka ʻikepili sequencing hanauna e hiki mai ana, hāʻawi kēia lawelawe i ka helu kūpono o ka hōʻike isoform-level.

Papahana: PacBio Sequel II
Hale Waihona Puke: SMRT bele waihona

  • :
  • Nā kikoʻī lawelawe

    Nā hopena hōʻike

    Nānā Hiʻohiʻona

    Pono lawelawe

    2

    ● Heluhelu pololei i ka mole cDNA lōʻihi piha mai ka hopena 3'- a hiki i ka hopena 5'-

    ● Iso-form pae hoʻonā ma ke kaʻina hana

    ● Nā palapala me ka pololei kiʻekiʻe a me ka pololei

    ● kūpono loa i nā ʻano waiours

    ● Hiki ke hoʻopili nui me 4 PacBio Sequel II sequencing platform i lako

    ● Loaʻa i ka ʻike me ka ʻoi aku o 700 Pacbio-based RNA sequencing projects

    ● Hāʻawi i nā hualoaʻa ma muli o BMKCloud: Loaʻa ka ʻili ʻikepili i hana ʻia ma ka paepae.

    ● Pono nā lawelawe ma hope o ke kūʻai aku no 3 mahina ma hope o ka pau ʻana o ka papahana

    Nā kikoʻī lawelawe

    Papahana: PacBio Sequel II

    Waihona waihona: Poly A- hoʻonaʻauao mRNA waihona

    Hāʻawi ʻia ka ʻikepili i manaʻo ʻia: 20 Gb/lapana (Ma muli o nā ʻano ʻano)

    FLNC(%): ≥75%

    *FLNC: Nā transcipts non-chimeric lōʻihi piha

    Nā loiloi bioinformatics

    ● Hoʻoponopono ʻikepili maka
     
    ● ʻIke kope kope
     
    ● Hoʻonohonoho kaʻina
     
    ● Ka helu ʻana
     
    ● Palapala Hana

    pacbio lōʻihi piha

    Nā Koina Laʻana a me ka Hoʻouna ʻana

    Nā Koina Laʻana:

    Nucleotides:

    Conc.(ng/μl)

    Ka nui (μg)

    Maemae

    Pono pono

    ≥ 120

    ≥ 0.6

    OD260/280=1.7-2.5

    OD260/230=0.5-2.5

    Hoʻopaʻa ʻia a ʻaʻole hoʻi i ka protein a i ʻole DNA contamination i hōʻike ʻia ma ka gel.

    No nā mea kanu: RIN≥7.5;

    No nā holoholona: RIN≥8.0;

    5.0≥ 28S/18S≥1.0;

    palena a ʻaʻole kiʻekiʻe kumu

    Tissue: Kaumaha(maloʻo):≥1 g
    *No ka ʻiʻo liʻiliʻi ma mua o 5 mg, manaʻo mākou e hoʻouna i kahi hāpana kikoo uila (i loko o ka nitrogen wai).

    Hoʻokuʻu kelepona:Helu kelepona = 3×106- 1×107
    * Manaʻo mākou e hoʻouna i ka lysate cell frozen.Inā ʻoi aku ka liʻiliʻi o kēlā cell ma mua o 5 × 105, ʻōlelo ʻia ka flash frozen i loko o ka nitrogen wai, ʻoi aku ka maikaʻi no ka micro extraction.

    Nā laʻana koko:Volume≥1 mL

    Microorganism:Ka nui ≥ 1 g

    Manaʻo ʻia ka hāʻawi laʻana

    ipu:
    2 ml centrifuge paipu (ʻaʻole ʻōlelo ʻia ʻo Tin foil)
    Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3;B1, B2, B3... ...

    Hoʻouna:

    1. Ka hau maloo: Pono e hoʻopili ʻia nā laʻana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
    2. Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i loko o ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i loko o ka lumi wela.

    Holo Hana Hana

    Laʻana QC

    Hoʻolālā hoʻokolohua

    hāʻawi laʻana

    Hāʻawi laʻana

    Hoʻokolohua hoʻokele

    RNA unuhi

    Hoʻomākaukau hale waihona puke

    Kūkulu hale waihona puke

    Ke kaʻina ʻana

    Ke kaʻina ʻana

    ʻIkepili ʻikepili

    ʻIkepili ʻikepili

    Mahope o ke kuai ana

    Nā lawelawe ma hope o ke kūʻai aku


  • Mua:
  • Aʻe:

  • 1.FLNC puunaue loihi

    Hōʻike ka lōʻihi o ka heluhelu non-chimeric piha piha (FLNC) i ka lōʻihi o ka cDNA i ka hana hale waihona.He hōʻailona koʻikoʻi ka hāʻawi ʻana i ka lōʻihi o FLNC i ka loiloi ʻana i ka maikaʻi o ke kūkulu hale waihona.

    mRNA-FLNC-heluhelu-lōʻihi-kahele

    Heluhelu ʻia ka lōʻihi o FLNC

    2. Hoʻopau i ka māhele ORF māhele lōʻihi

    Hoʻohana mākou i ka TransDecoder e wānana i nā ʻāpana coding protein a me nā kaʻina amino acid e hoʻohua ai i nā pūʻulu unigene, kahi i loaʻa i ka ʻike transcript ʻole-redundant i nā laʻana āpau.

    mRNA-Complete-ORF-lōʻihi-puʻunaue

    Hoʻopiha piha i ka māhele ʻāina ORF

    3.KEGG ala hoʻonui hoʻonaʻauao

    Hiki ke ʻike ʻia nā transcripts like ʻole (DETs) ma o ka hoʻohālikelike ʻana i ka ʻikepili sequencing RNA e pili ana i ka NGS ma nā pūʻulu transcript lōʻihi i hana ʻia e ka ʻikepili sequencing PacBio.Hiki i kēia mau DET ke hana hou no ka nānā 'ana i nā hana like 'ole, no ka la'ana, ka ho'onui 'ana i ke ala KEGG.

    mRNA-DEG-KEGG-alanui-hoonui

    ʻO ka hoʻonui ʻana i ke alahele DET KEGG -Dot plot

    Hihia BMK

    ʻO ka hoʻomohala hoʻomohala o ka Populus stem transcriptome

    Paʻi ʻia: Mea Puke Biotechnology Journal, 2019

    Hoʻolālā kaʻina hana:
    ʻOhi laʻana:ʻāpana ʻāpana: piko, internode mua (IN1), internode ʻelua (IN2), internode ʻekolu (IN3), internode (IN4) a me internode (IN5) mai Nanlin895
    NGS-sequence:Ua hui ʻia ʻo RNA o 15 mau kānaka i hoʻokahi laʻana olaola.ʻEkolu hoʻopiʻi koʻikoʻi o kēlā me kēia helu i hana ʻia no ke kaʻina NGS
    TGS-sequence:Ua māhele ʻia nā ʻāpana kumu i ʻekolu ʻāpana, ʻo ia hoʻi ka piko, IN1-IN3 a me IN4-IN5.Ua hana ʻia kēlā me kēia māhele no ka hoʻonohonoho ʻana o PacBio me nā ʻano hale waihona puke ʻehā: 0-1 kb, 1-2 kb, 2-3 kb a me 3-10 kb.

    Nā hopena koʻikoʻi

    1. Ua ʻike ʻia he 87150 mau moʻolelo holoʻokoʻa, kahi i ʻike ʻia ai 2081 novel isoforms a me 62058 novel alternative spliced ​​isoforms.
    Ua ʻike ʻia ka 2.1187 lncRNA a me 356 fusion genes.
    3. Mai ka ulu mua a hiki i ka ulu lua, 15838 i hōʻike ʻokoʻa ʻia nā transcripts mai 995 mau genes i hōʻike ʻokoʻa ʻia.Ma nā DEG a pau, he 1216 nā kumu kākau, ʻaʻole i hōʻike ʻia ka hapa nui o ia mau mea.
    Ua hōʻike ʻia ka loiloi hoʻonui 4.GO i ke koʻikoʻi o ka hoʻokaʻawale cell a me ke kaʻina hana hoʻemi oxidation i ka ulu mua a me ke kula kiʻekiʻe.

    • PB-lōʻihi-RNA-Sequencing-case-study

      Nā hanana splicing ʻokoʻa a me nā isoform like ʻole

    • PB-lōʻihi-RNA-ʻokoʻa-splicing

      ʻO ka nānā ʻana o WGCNA ma nā kumu transcription

    Kuhikuhi

    Chao Q, Gao ZF, Zhang D, et al.ʻO ka dynamics hoʻomohala o ka Populus stem transcriptome.Mea kanu Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958

    kiʻi i kahi ʻōlelo

    E kākau i kāu leka ma aneʻi a hoʻouna mai iā mākou

    E hoʻouna i kāu leka iā mākou: