Cov khoom

Tag nrho-ntev mRNA sequencing - PacBio

Full-length mRNA sequencing -PacBio Featured Duab

De novfull-length transcriptome sequencing, tseem hu uaDe novIso-Seq siv qhov zoo ntawm PacBio sequencer hauv kev nyeem ntev, uas ua rau muaj kev sib luag ntawm tag nrho-ntev cDNA molecules yam tsis muaj kev so.Qhov no ua kom tsis txhob muaj qhov tsis raug tsim los ntawm cov ntawv sau ua ke thiab tsim cov unigene teeb nrog isoform-theem daws teeb meem.Cov txheej txheem unigene no muab cov ntaub ntawv muaj txiaj ntsig zoo li "reference genome" ntawm transcriptome-theem.Tsis tas li ntawd, kev sib txuas nrog cov ntaub ntawv txuas mus ntxiv, qhov kev pabcuam no txhawb nqa qhov tseeb ntawm isoform-theem qhia.

Platform: PacBio Sequel II

Lub tsev qiv ntawv: SMRT tswb lub tsev qiv ntawv

Tiv tauj peb

Cov ntsiab lus kev pabcuam

Cov txiaj ntsig Demo

Case Study

Kev Pabcuam Zoo

● Direct nyeem-tawm ntawm tag nrho-ntev cDNA molecule ntawm 3'- kawg rau 5'- kawg

● Iso-form theem daws teeb meem nyob rau hauv cov qauv ua ntu zus

● Cov ntawv sau nrog qhov tseeb thiab ncaj ncees

● Zoo heev rau vaiours hom

● Loj sequencing muaj peev xwm nrog 4 PacBio Sequel II sequencing platforms nruab

● Muaj kev paub dhau los nrog ntau dua 700 Pacbio-raws li RNA sequencing tej yaam num

● BMKCloud-raws li kev xa tawm: Cov ntaub ntawv kho kom haum-tsim muaj nyob rau ntawm lub platform.

● Cov kev pab cuam tom qab muag siv tau rau 3 lub hlis tom qab qhov project tiav

Service Specifications

Platform: PacBio Sequel II

Sequencing library: Poly A- enriched mRNA library

Cov ntaub ntawv pom zoo: 20 Gb / qauv (nyob ntawm hom)

FLNC (%): ≥75%

* FLNC: Full-ntev non-chimeric transcipts

Bioinformatics tsom xam

● Raw cov ntaub ntawv ua

● Kev txheeb xyuas cov ntaub ntawv

● Sequence structure

● Kev nthuav qhia Quantification

● Cov lus piav qhia ua haujlwm

Sample Requirements thiab Delivery

Cov Qauv Xav Tau:

Nucleotides:

Conc. (ng / μl)

Tus nqi (μg)

Purity

Kev ncaj ncees

≥ 120

≥ 0.6

OD 260/280 = 1.7-2.5

OD 260/230 = 0.5-2.5

Txwv tsis pub lossis tsis muaj protein ntau lossis DNA kis tau pom ntawm gel.

Rau cov nroj tsuag: RIN≥7.5;

Rau tsiaj: RIN≥8.0;

5.0 ≥ 28S / 18S≥1.0;

txwv los yog tsis muaj qhov nce siab

Cov ntaub so ntswg: Nyhav (qhuav):≥1g
* Rau cov ntaub so ntswg me dua 5 mg, peb pom zoo kom xa flash khov (hauv kua nitrogen) cov ntaub so ntswg.

Cell ncua:Cell suav = 3 × 10⁶- 1 × 10⁷
* Peb pom zoo kom nkoj khov cell lysate.Yog hais tias lub xov tooj ntawm tes suav me dua 5 × 10⁵, flash khov hauv kua nitrogen yog pom zoo, uas yog qhov zoo dua rau kev rho tawm micro.

Cov ntshav kuaj:Ntim ≥ 1 mL

Microorganism:Qhov hnyav ≥ 1 g

Pom zoo coj mus kuaj

Ntim:
2 ml centrifuge raj (Tin foil tsis pom zoo)
Sample labeling: Pab pawg + replicate xws li A1, A2, A3;B1, B2, B3....

Kev xa khoom:

1. Dry-ice: Cov qauv yuav tsum tau ntim rau hauv hnab thiab faus rau hauv cov dej khov qhuav.
2. RNAstable raj: RNA cov qauv tuaj yeem qhuav hauv RNA stabilization raj (xws li RNAstable®) thiab xa mus rau hauv chav tsev kub.

Kev Pabcuam Ua Haujlwm Flow

Kev sim tsim

Qauv xa tuaj

RNA rho tawm

Kev tsim kho tsev qiv ntawv

Sequencing

Cov ntaub ntawv tsom xam

Kev pab cuam tom qab muag

Yav dhau los: Eukaryotic mRNA Sequencing-Illumina

Tom ntej: Full-Length mRNA Sequencing-Nanopore

1.FLNC qhov ntev faib

Ntev ntawm tag nrho-ntev uas tsis yog-chimeric nyeem (FLNC) qhia qhov ntev ntawm cDNA hauv kev tsim tsev qiv ntawv.FLNC qhov ntev kev faib tawm yog qhov tseem ceeb hauv kev ntsuas qhov zoo ntawm kev tsim tsev qiv ntawv.

mRNA-FLNC-read-length-distribution

FLNC nyeem ntev kev faib tawm

2.Complete ORF cheeb tsam ntev faib

Peb siv TransDecoder los kwv yees cov cheeb tsam ntawm cov protein coding thiab cov amino acid sib txuas ua ke los tsim cov unigene cov ntaub ntawv, uas muaj cov ntaub ntawv ua tiav cov ntaub ntawv tsis rov qab hauv txhua qhov qauv.

mRNA-Complete-ORF-ntev-distribution

Ua kom tiav ORF cheeb tsam ntev kev faib tawm

3.KEGG pathway enrichment analysis

Cov ntawv teev lus sib txawv (DETs) tuaj yeem txheeb xyuas tau los ntawm kev ua raws NGS-raws li RNA sequencing cov ntaub ntawv ntawm cov ntawv sau tag nrho uas tsim los ntawm PacBio sequencing cov ntaub ntawv.Cov DETs no tuaj yeem ua tiav ntxiv rau ntau yam kev ntsuas kev ua haujlwm, xws li KEGG txoj kev txhim kho kev txheeb xyuas.

mRNA-DEG-KEGG-pathway-enrichment

DET KEGG pathway enrichment -Dot plot

BMK Cas

Kev loj hlob dynamics ntawm Populus qia transcriptome

Luam tawm: Plant Biotechnology Journal, xyoo 2019

Sequence Strategy:
Qauv sau:Cov cheeb tsam qia: apex, thawj internode (IN1), thib ob internode (IN2), thib peb internode (IN3), internode (IN4) thiab internode (IN5) los ntawm Nanlin895
NGS-sequence:RNA ntawm 15 tus neeg tau sib sau ua ib qho piv txwv lom neeg.Peb qhov kev hloov pauv roj ntsha ntawm txhua lub ntsiab lus tau ua tiav rau NGS ib ntus
TGS-sequence:Cov cheeb tsam qia tau muab faib ua peb cheeb tsam, xws li apex, IN1-IN3 thiab IN4-IN5.Txhua cheeb tsam tau ua tiav rau PacBio sequencing nrog plaub hom tsev qiv ntawv: 0-1 kb, 1-2 kb, 2-3 kb thiab 3-10 kb.

Cov txiaj ntsig tseem ceeb

1.Ib tag nrho ntawm 87150 daim ntawv teev npe tag nrho tau txheeb xyuas, uas, 2081 cov isoforms tshiab thiab 62058 tshiab lwm txoj kev sib txuas isoforms tau txheeb xyuas.
2.1187 lncRNA thiab 356 fusion noob tau txheeb xyuas.
3.Los ntawm thawj qhov kev loj hlob mus rau theem nrab kev loj hlob, 15838 qhov sib txawv ntawm cov ntawv sau los ntawm 995 qhov txawv ntawm cov noob tau txheeb xyuas.Nyob rau hauv tag nrho cov DEGs, 1216 yog transcription yam, feem ntau tseem tsis tau tshaj tawm.
4.GO enrichment tsom xam qhia qhov tseem ceeb ntawm cell division thiab oxidation-txo cov txheej txheem nyob rau hauv thawj thiab theem nrab kev loj hlob.