Zogulitsa

Non-Reference based mRNA Sequencing-Illumina

Kutsatizana kwa mRNA kumatengera njira yotsatirira ya m'badwo wotsatira (NGS) kuti ijambule messenger RNA(mRNA) mawonekedwe a Eukaryote panthawi inayake yomwe ntchito zina zapadera zikuyatsidwa.Zolemba zazitali kwambiri zomwe zidapangidwa zimatchedwa 'Unigene' ndipo zimagwiritsidwa ntchito ngati njira yotsatirira pakuwunikiridwa kotsatira, yomwe ndi njira yabwino yophunzirira momwe mamolekyu amayendera komanso maulamuliro amtundu wamtunduwu popanda kutchulapo.

Pambuyo pa kusonkhanitsa deta ya transcriptome ndi kumasulira kwa unigene

(1) Kusanthula kwa SSR, kuneneratu kwa CDS ndi kapangidwe ka jini zidzakonzedweratu.

(2) Kuchulukitsa kwa mawu a unigene mu chitsanzo chilichonse kudzachitidwa.

(3) Ma unigene omwe amawonetsedwa mosiyanasiyana pakati pa zitsanzo (kapena magulu) adzapezeka potengera mawu osagwirizana

(4) Kuphatikizika, kutanthauzira kogwira ntchito ndi kusanthula kopindulitsa kwa ma unigene ofotokozedwa mosiyanasiyana kudzachitika

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Bioinformatics

Zotsatira za Demo

Nkhani Yophunzira

Mawonekedwe

● Kusadalira ma genome aliwonse,

● Detayo ingagwiritsidwe ntchito kusanthula kalembedwe ndi kafotokozedwe ka zolembedwazo

● Dziwani malo odulira osiyanasiyana

Ubwino wa Utumiki

● Kupereka zotsatira zochokera ku BMKCloud: Zotsatira zimaperekedwa ngati fayilo ya data ndi lipoti lothandizira kudzera pa nsanja ya BMKCloud, yomwe imalola kuti anthu aziwerenga momasuka za zotsatira zovuta zowunikira ndi migodi ya deta yosinthidwa malinga ndi kusanthula kwa bioinformatics.

● Ntchito zogulitsa pambuyo pa malonda: Ntchito zogulitsa pambuyo pake ndizovomerezeka kwa miyezi 3 polojekiti ikamalizidwa, kuphatikizapo kutsata mapulojekiti, kuthetsa mavuto, zotsatira za Q & A, ndi zina zotero.

Zitsanzo Zofunika ndi Kutumiza

Zitsanzo Zofunika:

Nucleotides:

Conc.(ng/μl)

Mtengo (μg)

Chiyero

Umphumphu

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Zochepa kapena zopanda mapuloteni kapena DNA kuipitsidwa komwe kumawonetsedwa pa gel.

Kwa zomera: RIN≥6.5;

Kwa nyama: RIN≥7.0;

5.0≥28S/18S≥1.0;

kukwera kochepa kapena kopanda koyambira

Thupi: Kulemera kwake (kuuma): ≥1 g
*Pa minofu yaing'ono kuposa 5 mg, timalimbikitsa kutumiza zitsanzo za minofu yowuma (mu madzi a nayitrogeni).

Kuyimitsidwa kwa ma cell: kuchuluka kwa ma cell = 3 × 10⁷
*Tikupangira kutumiza ma cell a frozen lysate.Ngati seloyo imawerengera yaying'ono kuposa 5 × 10⁵, kung'anima kozizira mumadzi a nayitrogeni akulimbikitsidwa.

Zitsanzo za magazi:
PA×geneBloodRNATube;
6mLTRIzol ndi 2mL magazi(TRIzol:Blood=3:1)

Kutumiza Kwachitsanzo Kovomerezeka

Chotengera:
2 ml centrifuge chubu (Tin zojambulazo sizovomerezeka)
Zitsanzo zolembera: Gulu+ lofanana mwachitsanzo A1, A2, A3;B1, B2, B3...

Kutumiza:
1.Dry-ice: Zitsanzo ziyenera kuikidwa m'matumba ndikukwiriridwa mu ayezi wouma.
2.RNAstable machubu: RNA zitsanzo akhoza zouma mu RNA kukhazikika chubu (mwachitsanzo RNAstable®) ndi kutumizidwa mu firiji.

Kuyenda kwa Ntchito Yantchito

Kuyesera kupanga

Kupereka zitsanzo

Kusintha kwa RNA

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Zam'mbuyo: Kutsata kwanthawi yayitali kosalemba-Illumina

Ena: Eukaryotic mRNA Sequencing-Illumina

Bioinformatics

1.mRNA(denovo) Principle of Assembly

Ndi Utatu, zowerengera zimagawika m'zidutswa zing'onozing'ono, zotchedwa K-mer.Ma K-mers awa amagwiritsidwa ntchito ngati mbewu kuti ziwonjezedwe mu ma contig kenako chigawo chotengera kuphatikizika kwa contig.Pomaliza, De Bruijn adagwiritsidwa ntchito pano kuti azindikire zolembedwa m'zigawozo.

mRNA-(De-novo)-Mwachidule-za-Utatu

mRNA (De novo) Chidule cha Utatu

2.mRNA (De novo) Kugawa kwa Gene Expression Level

RNA-Seq imatha kukwaniritsa kuyerekezera kwamphamvu kwa jini.Nthawi zambiri, mawonekedwe owoneka bwino a FPKM amachokera ku 10 ^ -2 mpaka 10 ^ 6

mRNA-(De-novo)-Kugawa-kwa-FPKM-kachulukidwe-mu-chitsanzo chilichonse

mRNA (De novo) Kugawidwa kwa kachulukidwe ka FPKM mu chitsanzo chilichonse

3.mRNA (De novo) GO Enrichment Analysis of DEGs

Nawonso database ya GO (Gene Ontology) ndi kachitidwe kolongosoka kamene kali ndi mawu okhazikika a jini ndi zinthu za jini.Lili ndi magawo angapo, pomwe mlingowo umakhala wotsika, ntchito zake zimakhala zenizeni.

mRNA-(De-novo)-GO-gulu-la-DEGs-mulingo wachiwiri

mRNA (De novo) GO gulu la DEGs pamlingo wachiwiri

BMK Case

Kusanthula kwa Transcriptome kwa Sucrose Metabolism pa Kutupa kwa Bulb ndi Kukula kwa Anyezi (Allium cepa L.)

Lofalitsidwa: malire mu sayansi ya zomera,2016

Njira yotsatirira

Illumina HiSeq2500

Zosonkhanitsa zitsanzo

Mitundu ya Utah Yellow Sweet Spain "Y1351" idagwiritsidwa ntchito mu kafukufukuyu.Chiwerengero cha zitsanzo zosonkhanitsidwa chinali
Tsiku la 15 mutatha kutupa (DAS) ya babu (2-cm m'mimba mwake ndi 3-4 g kulemera), 30th DAS (5-cm m'mimba mwake ndi 100-110 g kulemera), ndi ~3 pa 40th DAS (7-cm m'mimba mwake ndi 260-300 magalamu).

Zotsatira zazikulu

1. mu chithunzi cha Venn, chiwerengero cha 146 DEGs chinapezeka pamagulu atatu onse a chitukuko.
2. "Mayendedwe a Carbohydrate ndi metabolism" adayimiridwa ndi 585 unigenes (ie, 7% ya COG yotchulidwa).
3. Ma Unigene ofotokozedwa bwino kunkhokwe ya GO adagawidwa m'magulu atatu a magawo atatu osiyanasiyana akukulitsa babu.Ambiri oimiridwa mu "biological process" gulu lalikulu linali "metabolic process", kutsatiridwa ndi "ma cell process".M'gulu lalikulu la "mamolekyu" magulu awiri omwe amaimiridwa kwambiri anali "zomangamanga" ndi "zothandizira".

PB-full-length-RNA-Sequencing-case-study

Histogram ya magulu a magulu a orthologous (COG) classification

Gulu la histogram of gene ontology (GO) la mayunijeni otengedwa ku mababu mu magawo atatu akukula

Chithunzi cha Venn chosonyeza majini owonetsedwa mosiyana mu magawo awiri aliwonse a kukula kwa babu ya anyezi

Buku

Zhang C, Zhang H, Zhan Z, et al.Kusanthula kwa Transcriptome kwa Sucrose Metabolism pa Kutupa kwa Bulb ndi Kukula kwa Anyezi (Allium cepa L.)[J].Frontiers in Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425