Zogulitsa

Kutalika Kwathunthu kwa mRNA Sequencing-Nanopore

Kutsatizana kwa RNA kwakhala chida chamtengo wapatali chowunikira mwatsatanetsatane zolembalemba.Mosakayikira, kutsatizana kwachikale kwa kuwerenga mwachidule kunapindula zambiri zofunika pano.Komabe, nthawi zambiri imakumana ndi zoletsa pakuzindikiritsa kwautali wa isoform, kuchuluka, kukondera kwa PCR.

Nanopore sequencing imadzisiyanitsa ndi nsanja zina zotsatizana, chifukwa ma nucleotides amawerengedwa mwachindunji popanda kaphatikizidwe ka DNA ndipo amapanga kuwerengera nthawi yayitali pa makumi a kilobases.Izi zimapereka mphamvu zowerengera molunjika ndikuwoloka zolembedwa zazitali zonse ndikuthana ndi zovuta mumaphunziro amlingo wa isoform.

nsanja:Nanopore PromethION

Laibulale:cDNA-PCR

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Nkhani Yophunzira

Ubwino wa Utumiki

● Tsankho locheperako

● Kuvumbula mamolekyu aatali a cDNA

● Deta yochepera yofunikira kuti mulembe zolemba zomwezo

● Kuzindikiritsa ma isoform angapo pa jini

● Kuchulukira kwa mawu mu mulingo wa isoform

Mafotokozedwe a Utumiki

Library

nsanja

Zomwe zaperekedwa (Gb)

Kuwongolera Kwabwino

cDNA-PCR(Poly-A enriched)

Nanopore PromethION P48

6 Gb / zitsanzo (malingana ndi mitundu)

Chiyerekezo chautali ＞70%

Chigoli chapakati: Q10

Bioinformatics kusanthula

●Yaiwisi data processing

● Kuzindikiritsa zolembedwa

● Kuphatikizika kwina

● Kuchulukitsidwa kwa mafotokozedwe a jini ndi mulingo wa isoform

● Kusanthula kwamaganizidwe osiyanasiyana

● Kafotokozedwe ka ntchito ndi kulemeretsa (DEGs ndi DETs)

Zitsanzo Zofunika ndi Kutumiza

Zitsanzo Zofunika:

Nucleotides:

Conc.(ng/μl)

Mtengo (μg)

Chiyero

Umphumphu

≥100

≥ 0.6

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≥7.0;

Kwa nyama: RIN≥7.5;

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 × 10⁶-1 × 10⁷

*Tikupangira kutumiza ma cell a frozen lysate.Ngati seloyo imawerengera yaying'ono kuposa 5 × 10⁵, kung'anima kozizira mu nayitrogeni wamadzimadzi kumalimbikitsidwa, komwe kumakhala koyenera kutulutsa pang'ono.

Zitsanzo za magazi: Voliyumu≥1 ml

Kutumiza Kwachitsanzo Kovomerezeka

Chidebe: 2 ml centrifuge chubu (zojambula za malata ndizosavomerezeka)

Zitsanzo zolembera: Gulu+ lofanana mwachitsanzo A1, A2, A3;B1, B2, B3...

Kutumiza: 2, Dry-ice: Zitsanzo ziyenera kunyamula m'matumba ndikukwiriridwa mu ayezi wouma.

RNAstable machubu: RNA zitsanzo akhoza zouma mu RNA kukhazikika chubu (mwachitsanzo RNAstable®) ndi kutumizidwa mu firiji.

Kuyenda kwa Ntchito Yantchito

Nucleotides:

Kupereka zitsanzo

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Kuyenda kwa Ntchito Yantchito

Thupi:

Kuyesera kupanga

Kupereka zitsanzo

Kusintha kwa RNA

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Zam'mbuyo: Kutsata kutalika kwa mRNA -PacBio

Ena: 10x Genomics Visium Spatial Transcriptome

1.Kusanthula kwa mawu osiyanasiyana - Chiwembu cha Volcano

Kusanthula kwamafotokozedwe osiyanasiyana kumatha kusinthidwa mumagulu onse a jini kuti azindikire ma jini ofotokozera mosiyanasiyana (DEGs) komanso mulingo wa isoform kuti azindikire mosiyana.

3(1)

zolembedwa zowonetsedwa (DETs)

2.Hierarchical clustering heatmap

3.Alternative splicing chizindikiritso ndi gulu

Mitundu isanu ya zochitika zina zophatikizana zitha kuneneratu ndi Astalavista.

4.Kuzindikiritsa zochitika za Alternative poly-adenylation (APA) ndi Motif pa 50 bp kumtunda kwa poly-A

BMK Case

Chizindikiritso china cholumikizirana komanso kuchuluka kwa mulingo wa isoform ndi nanopore kutalika kwautali wa transcriptome sequencing

Lofalitsidwa:Kulumikizana Kwachilengedwe, 2020

Njira yotsatirira:

Magulu: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E kusintha);3. Ma B-maselo achibadwa

Njira yotsatirira: kutsatizana kwa laibulale ya MinION 2D, kutsatizana kwa library ya PromethION 1D;zowerengera zazifupi kuchokera ku zitsanzo zomwezo

Sequencing nsanja: Nanopore MinION;Nanopore PromethION;

Zotsatira zazikulu

1.Isoform-level Alternative Splicing Identification

Kuwerengera kwanthawi yayitali kumathandizira kuzindikira SF3B1 yosinthika^K700E-malo osinthika a splice pa isoform-level.35 njira zina za 3′SS ndi 10 zina 5′SS zinapezeka kuti ndizosiyana kwambiri pakati pa SF3B1^K700Endi SF3B1^WT.Zosintha 33 mwa 35 zidapezedwa kumene ndikutsatiridwa kwanthawi yayitali.

2.Isoform-level Alternative Splicing quantification

Kufotokozera kwa intron retention (IR) isoforms mu SF3B1^K700Endi SF3B1^WTadawerengedwa kutengera kutsata kwa nanopore, kuwulula kutsika kwapadziko lonse kwa ma isoform a IR mu SF3B1.^K700E.

Buku

Tang AD , Soulette CM , Baren MJV , et al.Kufotokozera kwautali kwanthawi yayitali kwa SF3B1 mutation mu chronic lymphocytic leukemia kumawonetsa kutsika kwa introns zosungidwa [J].Nature Communications.