Yakazara-kureba mRNA kutevedzana -PacBio

Yakazara-kureba mRNA sequencing -PacBio Featured Image

De novofull-length transcriptome sequencing, inozivikanwawo seDe novoIso-Seq inotora zvakanakira PacBio sequencer mukureba kwekuverenga, iyo inogonesa kutevedzana kweakazara-kureba cDNA mamorekuru pasina chero mabreak.Izvi zvinodzivirira zvachose chero zvikanganiso zvinogadzirwa mumatanho egungano ezvinyorwa uye zvinovaka unigene seti ine isoform-level resolution.Iyi unigene seti inopa ruzivo rwakasimba rwemajini se "reference genome" pa transcriptome-level.Uye zvakare, kusanganisa nechizvarwa chinotevera chekuteedzera data, iyi sevhisi inopa iyo chaiyo quantification yeisoform-level kutaura.

Platform: PacBio Sequel II

Raibhurari: SMRT bhero raibhurari

Taura nesu

Service Details

Demo Results

Nyaya Yokudzidza

Basa Rinobatsira

● Kuverenga zvakananga kwemorekuru wecDNA yakareba kubva pa3'- kumagumo kusvika pa5'- kumagumo

● Iso-form level resolution mukugadzirisa maitiro

● Zvinyorwa nekururama kwepamusoro uye kutendeseka

● Inoenderana zvakanyanya nemhando dzevaiours

● Huru hwekutevedzana hune 4 PacBio Sequel II sequencing mapuratifomu ane

● Yakanyanya ruzivo nepamusoro pe700 Pacbio-based RNA sequencing mapurojekiti

● BMKCloud-based result delivery: Customized data-mining inowanikwa pachikuva.

● Mushure mekutengesa masevhisi anoshanda kwemwedzi mitatu pakupedzwa kweprojekiti

Sevhisi Zvinotsanangurwa

Platform: PacBio Sequel II

Sequencing raibhurari: Poly A- enriched mRNA raibhurari

Inokurudzirwa kuburitsa data: 20 Gb/sample (Zvichienderana nemhando)

FLNC (%): ≥75%

*FLNC: Yakazara-kureba isiri-chimeric transcipts

Bioinformatics inoongorora

● Raw data processing

● Kuzivikanwa kwezvinyorwa

● Kutevedzana kwemaitiro

● Kuenzana Kwemashoko

● Chiratidzo cheBasa

Sample Zvinodiwa uye Delivery

Sample Zvinodiwa:

Nucleotides:

Conc.(ng/μl)

Mari (μg)

Kuchena

Kuperera

≥ 120

≥ 0.6

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Yakaganhurirwa kana isina mapuroteni kana kusvibiswa kweDNA kunoratidzwa pagel.

Kumiti: RIN≥7.5;

Zvemhuka: RIN≥8.0;

5.0≥ 28S/18S≥1.0;

kukwirira kwakadzika kana kusina kwekutanga

Tissue: Huremu (yakaoma):≥1 g
*Kune tishu diki pane 5 mg, tinokurudzira kutumira flash yakaomeswa nechando (mumvura nitrogen) sampuli yetishu.

Kumiswa kwesero:Nhamba yemasero = 3 × 10⁶1 × 10⁷
* Isu tinokurudzira kutumira chando cell lysate.Kana iyo sero inoverenga idiki pane 5 × 10⁵, flash yakaomeswa mu nitrogen yemvura inokurudzirwa, iyo inosarudzika kune micro extraction.

Mienzaniso yeropa:Kuwanda≥1 mL

Microorganism:Husa ≥ 1 g

Yakakurudzirwa Sample Delivery

Container:
2 ml centrifuge chubhu (Tin foil haina kukurudzirwa)
Sample labeling: Boka+ replicate eg A1, A2, A3;B1, B2, B3...

Shipment:

1. Dry-ice: Samples dzinoda kurongedzerwa mumabhegi uye kuvigwa muoma-aizi.
2. RNAstable tubes: RNA samples inogona kuomeswa muRNA stabilization tube (eg RNAstable®) uye inotumirwa mukamuri yekupisa.

Kuyerera Kwebasa Rebasa

Kuedza kugadzira

Muenzaniso wekutumira

RNA kubviswa

Kuvakwa kweraibhurari

Sequencing

Data analysis

Mushure mekutengesa masevhisi

Zvakapfuura: Eukaryotic mRNA Sequencing-Illumina

Zvinotevera: Yakazara-Kureba mRNA Sequencing-Nanopore

1.FLNC kureba kugoverwa

Hurefu hwehurefu hwakazara isiri-chimeric kuverenga(FLNC) inoratidza kureba kwecDNA mukuvaka raibhurari.FLNC kureba kugovera chiratidzo chakakosha mukuongorora mhando yekuvaka raibhurari.

mRNA-FLNC-kuverenga-kureba-kugovera

FLNC kuverenga kureba kugoverwa

2.Pedzisa ORF nharaunda kureba kugoverwa

Isu tinoshandisa TransDecoder kufanotaura mapuroteni ekukodha matunhu uye anoenderana amino acid kutevedzana kugadzira unigene seti, iyo ine yakazara isina-yedundant zvinyorwa zvinyorwa mumasampuli ese.

mRNA-Yakazara-ORF-kureba-kugovera

Zadzisa ORF kureba kwedunhu kugovera

3.KEGG nzira yekuvandudza kuongorora

Zvinyorwa zvakasiyana-siyana (DETs) zvinogona kuonekwa nekubatanidza NGS-based RNA sequencing data pane yakazara-yakareba zvinyorwa zvinyorwa zvinogadzirwa nePacBio sequencing data.Aya maDET anogona kuenderera mberi nekugadziridzwa kune akasiyana mashandiro ekuongorora, semuenzaniso KEGG nzira yekuvandudza ongororo.

mRNA-DEG-KEGG-nzira-kupfumisa

DET KEGG nzira yekupfumisa -Dot plot

Nyaya yeBMK

Mamiriro ekusimudzira ePopulus stem transcriptome

Rakatsikiswa: Plant Biotechnology Journal, 2019

Sequencing strategy:
Sample collection:stem matunhu: apex, yekutanga internode (IN1), yechipiri internode (IN2), yechitatu internode (IN3), internode (IN4) uye internode (IN5) kubva kuNanlin895
NGS-kutevedzana:RNA yevanhu gumi nevashanu vakaiswa semumwechete biological sample.Zvitatu zvebhayoloji zvinodzokororwa pamapoinzi ega ega akagadziriswa kutevedzana kweNGS
TGS-kutevedzana:Matunhu akapatsanurwa kuita matunhu matatu, kureva apex, IN1-IN3 uye IN4-IN5.Dunhu rega rega rakagadziriswa PacBio kutevedzana nemhando ina dzemaraibhurari: 0-1 kb, 1-2 kb, 2-3 kb uye 3-10 kb.

Mibairo yakakosha

1. Huwandu hwe87150 hwakazara-hurefu hwakanyorwa hwakaonekwa, umo, 2081 novel isoforms uye 62058 novel imwe spliced isoforms yakaonekwa.
2.1187 lncRNA uye 356 fusion majini akaonekwa.
3.Kubva pakukura kwekutanga kusvika pakukura kwechipiri, 15838 yakaratidza zvakasiyana-siyana zvinyorwa kubva ku995 zvakasiyana-siyana zvakaratidzwa majeni zvakaonekwa.Mune ese maDEGs, 1216 aive ezvinyorwa zvinyorwa, mazhinji acho asati ataurwa.
4.GO enrichment analysis yakaratidza kukosha kwesero kupatsanurwa uye oxidation-kuderedza maitiro mukukura kwekutanga uye kwechipiri.