Zogulitsa

Kutsata kutalika kwa mRNA -PacBio

Kutsata kwamRNA kwautali -PacBio Chithunzi Chowonetsedwa

Pa novokutsatizana kwa ma transcriptome kutalika, komwe kumadziwikanso kutiPa novoIso-Seq imatenga ubwino wa PacBio sequencer mu kutalika kwa kuwerenga, zomwe zimathandiza kutsatizana kwa ma molekyulu a cDNA aatali popanda kusweka.Izi zimapewa kwathunthu zolakwika zilizonse zomwe zimapangidwa pamasitepe amisonkhano yolembera ndikupanga ma seti a unigene okhala ndi isoform-level resolution.Ma seti a unigenewa amapereka chidziwitso champhamvu cha majini monga "reference genome" pa transcriptome-level.Kuphatikiza apo, kuphatikiza ndi deta yotsatizana ya m'badwo wotsatira, ntchitoyi imapatsa mphamvu kuwerengera kolondola kwa mawu amtundu wa isoform.

Pulatifomu: PacBio Sequel II

Library: SMRT belu laibulale

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Nkhani Yophunzira

Ubwino wa Utumiki

● Kuwerenga molunjika kwa molekyu ya cDNA yaitali kuchokera pa 3'- kumapeto mpaka 5'- mapeto

● Kusintha kwa mlingo wa Iso-form mu ndondomeko yotsatizana

● Zolemba zolondola kwambiri komanso zowona mtima

● Zogwirizana kwambiri ndi mitundu ya vaours

● Kuthekera kwakukulu kotsatizana ndi nsanja za 4 PacBio Sequel II zokhala ndi zida

● Wodziwa zambiri ndi ma 700 Pacbio-based RNA sequencing mapulojekiti

● BMKCloud yochokera ku zotsatira zobweretsera: Customize data-mining kupezeka pa nsanja.

● Ntchito zogulitsa pambuyo pogulitsa ndizovomerezeka kwa miyezi itatu polojekiti ikamalizidwa

Mafotokozedwe a Utumiki

Platform: PacBio Sequel II

Laibulale yotsatizana: Library ya Poly A-enriched mRNA

Zomwe zaperekedwa: 20 Gb/chitsanzo (Malingana ndi mitundu)

FLNC (%): ≥75%

*FLNC: Zolemba zazitali zopanda chimeric

Bioinformatics kusanthula

● Kukonza deta yaiwisi

● Kuzindikiritsa zolembedwa

● Mayendedwe ake

● Kukwanira kwa Mawu

● Mawu a Ntchito

Zitsanzo Zofunika ndi Kutumiza

Zitsanzo Zofunika:

Nucleotides:

Conc.(ng/μl)

Mtengo (μg)

Chiyero

Umphumphu

≥ 120

≥ 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≥7.5;

Kwa nyama: RIN≥8.0;

5.0≥ 28S/18S≥1.0;

kukwera kochepa kapena kopanda koyambira

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

Kuyimitsidwa kwa cell:Chiwerengero cha 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:Kuchuluka ≥1 mL

Microorganism:Kulemera ≥ 1 g

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 kupakidwa m'matumba ndikukwiriridwa mu ayezi wouma.
2. RNAstable chubu: 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: Eukaryotic mRNA Sequencing-Illumina

Ena: Kutalika Kwathunthu kwa mRNA Sequencing-Nanopore

1.FLNC kutalika kugawa

Kutalika kwa utali wathunthu wopanda chimeric read(FLNC) kumawonetsa kutalika kwa cDNA pakumanga laibulale.Kugawa kwautali wa FLNC ndichizindikiro chofunikira pakuwunika mtundu wa kamangidwe ka library.

kugawa kwa mRNA-FLNC-kuwerenga-utali

FLNC kuwerenga kutalika kugawa

2.Malizani kugawa kwa dera la ORF

Timagwiritsa ntchito TransDecoder kulosera zigawo zama protein ndi ma amino acid omwe amatsatiridwa kuti apange ma seti a unigene, omwe amakhala ndi zidziwitso zonse zosafunikira pazitsanzo zonse.

mRNA-Complete-ORF-utali-kugawa

Malizitsani kugawa kwa dera la ORF

3.KEGG njira yowonjezeretsa kusanthula

Differentially expression transcripts(DETs) zitha kudziwika mwa kugwirizanitsa deta yotsatiridwa ndi NGS-based RNA pamaseti aatali athunthu opangidwa ndi data ya PacBio.Ma DET awa atha kukonzedwanso kuti afufuze ntchito zosiyanasiyana, mwachitsanzo KEGG njira yolemeretsa.

mRNA-DEG-KEGG-njira-kulemeretsa

DET KEGG njira yolemeretsa -Dot chiwembu

BMK Case

Kukula kwamphamvu kwa Populus stem transcriptome

Lofalitsidwa: Plant Biotechnology Journal, 2019

Njira yotsatirira:
Zosonkhanitsira zitsanzo:madera apakati: apex, internode yoyamba (IN1), internode yachiwiri (IN2), internode yachitatu (IN3), internode (IN4) ndi internode (IN5) kuchokera ku Nanlin895
NGS-kutsatizana:RNA ya anthu 15 adaphatikizidwa ngati chitsanzo chimodzi chachilengedwe.Zofananira zitatu zachilengedwe za mfundo iliyonse zidakonzedwa kuti zitsatidwe ndi NGS
TGS-kutsatizana:Magawo atsinde adagawidwa m'magawo atatu, mwachitsanzo, apex, IN1-IN3 ndi IN4-IN5.Chigawo chilichonse chinakonzedwa kuti PacBio atsatidwe ndi mitundu inayi ya malaibulale: 0-1 kb, 1-2 kb, 2-3 kb ndi 3-10 kb.

Zotsatira zazikulu

1. Zolemba zonse za 87150 zautali zidadziwika, momwemo, ma isoform 2081 ndi ma isoform 62058 amitundu ina adadziwika.
2.1187 lncRNA ndi 356 fusion majini adadziwika.
3.Kuchokera ku kukula kwapachiyambi mpaka kukula kwachiwiri, zolemba za 15838 zosiyana siyana kuchokera ku 995 zomwe zimafotokozedwa mosiyanasiyana zinadziwika.M'ma DEG onse, 1216 anali zinthu zolembera, zambiri zomwe sizinafotokozedwebe.
Kusanthula kwabwino kwa 4.GO kunawonetsa kufunikira kwa kugawanika kwa maselo ndi njira yochepetsera oxidation mu kukula kwa pulayimale ndi yachiwiri.