Ubushinwa De novo Uburebure bwuzuye Transcriptome ikurikirana -Uruganda rwa PacBio nababikora |BMK

De novoUburebure bwuzuye Transcriptome ikurikirana -PacBio

De novo Full-length Transcriptome sequencing -PacBio Featured Image

De novobyuzuye-byuzuye bikurikirana, bizwi kandi nkaDe novoIso-Seq ifata ibyiza bya PacBio ikurikirana muburebure bwo gusoma, ituma ikurikiranwa rya molekile ya cDNA yuzuye nta kiruhuko.Ibi birinda rwose amakosa yose yatanzwe mumyandikire yinteko kandi yubaka unigene hamwe na isoform-urwego rwo gukemura.Iyi unigene igizwe itanga amakuru akomeye nka "reference genome" kuri transcriptome-urwego.Mubyongeyeho, guhuza hamwe nibisekuru bizakurikiraho bikurikirana, iyi serivise iha imbaraga umubare wuzuye wa isoform-urwego rwimvugo.

Ihuriro ： Urutonde rwa PacBio II

Isomero: Isomero ry'inzogera ya SMRT

Twandikire

Ibisobanuro bya serivisi

Ibisubizo bya Demo

Inyigo

Inyungu za serivisi

ØGusoma mu buryo butaziguye uburebure bwa cDNA kuva 3'- kurangiza kugeza 5'- iherezo

ØIso-form urwego rwo gukemura muburyo bukurikirana

ØInyandiko-mvugo hamwe nukuri kandi ubunyangamugayo

ØBihujwe cyane nubwoko bwa vaiours

ØUbushobozi bunini bukurikirana hamwe na 4 ya PacBio Sequel II ikurikirana

ØInararibonye cyane hamwe na 700 ya Pacbio ishingiye kuri RNA ikurikirana

ØBMKCloud ishingiye kubisubizo: Gutanga amakuru yihariye kubucukuzi buboneka kurubuga.

ØSerivise nyuma yo kugurisha ifite agaciro kumezi 3 iyo umushinga urangiye

Ibisobanuro bya serivisi

Ihuriro: Urutonde rwa PacBio II

Isomero rikurikirana: Poly A- ikungahaye kuri mRNA isomero

Basabwe gutanga umusaruro: 20 Gb / icyitegererezo (Ukurikije amoko)

FLNC （%）： ≥75%

* FLNC: Uburebure bwuzuye butari chimeric transcipts

Isesengura rya bioinformatics

üGutunganya amakuru mabi

üKumenyekanisha inyandiko

üImiterere y'uruhererekane

üUmubare w'amagambo

üImikorere Ibisobanuro

Icyitegererezo gisabwa no gutanga

Icyitegererezo gisabwa:

Nucleotide:

Umubare	Igiteranyo ≥ 3 μg;Umwanzuro.≥ 300 ng / μl;Inkingi ≥ 10 μl
Ubunyangamugayo	Kubimera: RIN ≥ 7.5;Ku nyamaswa: RIN ≥ 8;28S / 18S ≥ 1.0;Ntarengwa cyangwa ntamwanya wo hejuru
Isuku	OD260 / 280 ≥ 1.8;OD260 / 230 ≥ 1.0;Impanuka nziza kuri 260 nm
Icyitonderwa	Poroteyine cyangwa ADN byanduye birashoboka cyane ko bigira ingaruka kubipimo no kurwego rwa QC.

Tissue: Uburemere (bwumye):≥1 g
* Kuri tissue ntoya ya mg 5, turasaba kohereza flash ya flash ikonje (muri azote yuzuye).

Guhagarika akagari:Kubara Akagari = 3 × 10⁶- 1 × 10⁷
* Turasaba kohereza lysate ya selile ikonje.Mugihe iyo selile ibara ntoya ya 5 × 105, birashoboka ko flash ikonjeshwa muri azote yuzuye, bikaba byiza gukuramo mikoro.

Ingero zamaraso:Umubumbe wa ml

Microorganism:Misa ≥ 1 g

Basabwe Gutanga Icyitegererezo

Ibirimwo:
2 ml centrifuge tube (Tin foil ntabwo isabwa)
Icyitegererezo cyerekana: Itsinda + kwigana urugero A1, A2, A3;B1, B2, B3 ... ...

Kohereza:

1. Urubura rwumye: Ingero zigomba gupakirwa mumifuka hanyuma zigashyingurwa mu rubura.
2.Imiyoboro ihamye: Ingero za RNA zirashobora gukama mumashanyarazi ya RNA (urugero RNAstable®) hanyuma ikoherezwa mubushyuhe bwicyumba.

Akazi ka serivisi

Igishushanyo mbonera

Icyitegererezo

Gukuramo RNA

Kubaka isomero

Urukurikirane

Isesengura ryamakuru

Serivisi nyuma yo kugurisha

Mbere: Eukaryotic mRNA ikurikirana-Illumina

Ibikurikira: Uburebure bwuzuye mRNA ikurikirana-Nanopore

1. Gukwirakwiza uburebure bwa FLNC

Uburebure bwuzuye-butari chimeric soma (FLNC) bwerekana uburebure bwa cDNA mukubaka amasomero.Ikwirakwizwa rya FLNC ni ikimenyetso cyingenzi mugusuzuma ubwiza bwububiko bwibitabo.

mRNA-FLNC-read-length-distribution

FLNC soma ikwirakwizwa ry'uburebure

2.Kuzuza uburebure bwa ORF mukarere

Dukoresha TransDecoder kugirango tumenye uturere twa kodegisi ya poroteyine hamwe na aside amine ikurikirana kugirango tubyare unigene, ikubiyemo amakuru yuzuye atagabanije mu byitegererezo byose.

mRNA-Complete-ORF-length-distribution

Kuzuza uburebure bwa ORF mukarere

3.Isesengura ryinzira ya KEGG

Inyandiko-mvugo yerekanwe mu buryo butandukanye (DETs) irashobora kumenyekana muguhuza amakuru ya NGS ashingiye kuri RNA akurikirana kumirongo miremire yuzuye yakozwe na PacBio ikurikirana.Izi DET zirashobora gutunganywa kubisesengura bitandukanye, urugero isesengura rya KEGG.

mRNA-DEG-KEGG-pathway-enrichment

DET KEGG inzira yo gutunganya -Umwanya muto

Urubanza rwa BMK

Iterambere ryiterambere rya populus stem transcriptome

Byatangajwe: Gutera Ikinyamakuru Ikoranabuhanga, 2019

Ingamba zikurikirana:
Icyegeranyo cy'icyitegererezo:uturere twibanze: apex, internode yambere (IN1), internode ya kabiri (IN2), internode ya gatatu (IN3), internode (IN4) na internode (IN5) kuva Nanlin895
Urutonde rwa NGS:RNA yabantu 15 bahujwe nkicyitegererezo kimwe cyibinyabuzima.Ibinyabuzima bitatu byigana kuri buri ngingo byatunganijwe kugirango NGS ikurikirane
Urutonde rwa TGS:Uturere twibiti twagabanijwemo uturere dutatu, ni ukuvuga apex, IN1-IN3 na IN4-IN5.Buri karere katunganijwe kugirango PacBio ikurikirane hamwe nubwoko bune bwamasomero: 0-1 kb, 1-2 kb, 2-3 kb na 3-10 kb.

Ibisubizo by'ingenzi

1.Ibisobanuro byose hamwe 87150 byuzuye byuzuye, aho, 2081 isoforms yubuvanganzo hamwe na 62058 bishya byanditseho isoforms.
2.1187 lncRNA na 356 gen fusion byagaragaye.
3.Ku gukura kwambere kugera kumikurire ya kabiri, hamenyekanye inyandiko-mvugo itandukanye kuva 995 itandukanye.Muri DEGs zose, 1216 zari ibintu byandikirwa, ibyinshi bikaba bitaratangazwa.
4.Isesengura ryitunganyirize rya GO ryagaragaje akamaro ko kugabana ingirabuzimafatizo no kugabanya okiside mu mikurire y'ibanze n'iya kabiri.

Ubundi buryo bwo gutondeka ibyabaye hamwe na isoforms zitandukanye
Isesengura rya WGCNA kubintu byandikirwa

Reba

Chao Q, Gao ZF, Zhang D, n'abandi.Iterambere ryiterambere rya populus stem transcriptome.Gutera Biotechnol J. 2019; 17 (1): 206-219.doi: 10.1111 / pbi.12958