Non-Reference based mRNA Sequencing-Illumina

mRNA sequencing inotora inotevera-chizvarwa kutevedzana maitiro (NGS) kutora mutumwa RNA(mRNA) fomu Eukaryote panguva chaiyo iyo mamwe mabasa akakosha ari kuita.Chinyorwa chakareba kwazvo chakanatswa chainzi 'Unigene' uye chakashandiswa sereferensi yekuongorora kunotevera, inova nzira inoshanda yekudzidza mamorekuru nemambure ekutonga emarudzi pasina kutaurwa.

Mushure mekubatanidza data yetranscriptome uye unigene inoshanda annotation

(1) Ongororo yeSSR, kufanotaura kweCDS uye chimiro chemajini zvichafanogadzirwa.

(2) Quantification ye unigene kutaura mumuenzaniso wega wega uchaitwa.

(3) Unigenes akasiyaniswa pakati pemasamples (kana mapoka) achawanikwa zvichibva pane unigene kutaura.

(4) Kubatanidza, chirevo chekushanda uye kuvandudza kuongororwa kweunigenes yakasiyana-siyana ichaitwa.

Taura nesu

Service Details

Bioinformatics

Demo Results

Nyaya Yokudzidza

Features

● Yakazvimirira pane chero referensi genome,

● Iyo data inogona kushandiswa kuongorora chimiro uye kutaura kwezvinyorwa

● Ziva nzvimbo dzekuchekeresa dzakasiyana

Basa Rinobatsira

● BMKCloud-yakavakirwa mhedzisiro yekuunza: Mibairo inounzwa sefaira re data uye inopindirana mushumo kuburikidza neBMKCloud papuratifomu, iyo inobvumira mushandisi-ane hushamwari kuverenga kwezvakaoma ongororo zvinobuda uye yakasarudzika migodhi yedata pachigadziko cheyakajairwa bioinformatics kuongororwa.

● Mushure mekutengesa masevhisi: Mushure mekutengesa masevhisi anoshanda kwemwedzi mitatu pakupedzwa kweprojekiti, kusanganisira kutevera mapurojekiti, kupfura-dambudziko, mhinduro Q&A, nezvimwe.

Sample Zvinodiwa uye Delivery

Sample Zvinodiwa:

Nucleotides:

Conc.(ng/μl)

Mari (μg)

Kuchena

Kuperera

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Yakaganhurirwa kana isina mapuroteni kana kusvibiswa kweDNA kunoratidzwa pagel.

Kumiti: RIN≥6.5;

Zvemhuka: RIN≥7.0;

5.0≥28S/18S≥1.0;

kukwirira kwakadzika kana kusina kwekutanga

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

Kumiswa kwesero: Kuwanda kwesero = 3 × 10⁷
* Isu tinokurudzira kutumira chando cell lysate.Kana iyo sero inoverenga idiki pane 5 × 10⁵, flash yakaomeswa munitrogen yemvura inokurudzirwa.

Mienzaniso yeropa:
PA×geneBloodRNATube;
6mLTRIzol uye 2mL ropa(TRIzol:Ropa=3:1)

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 kuiswa mumabhegi uye kuvigwa muoma-ice.
2.RNAstable tubes: RNA samples inogona kuomeswa muRNA stabilization tube (eg RNAstable®) uye inotumirwa mukupisa kwekamuri.

Kuyerera Kwebasa Rebasa

Kuedza kugadzira

Muenzaniso wekutumira

RNA kubviswa

Kuvakwa kweraibhurari

Sequencing

Data analysis

Mushure mekutengesa masevhisi

Zvakapfuura: Yakareba isiri-coding kutevedzana-Illumina

Zvinotevera: Eukaryotic mRNA Sequencing-Illumina

Bioinformatics

1.mRNA(denovo) Nheyo yeGungano

NeTiriniti, kuverenga kwakakamurwa kuita zvidimbu zvidiki, zvinozivikanwa seK-mer.Aya maK-mers anobva ashandiswa sembeu kuti dziwedzererwe mumakontig uyezve chikamu zvichibva pane contig overlappings.Pakupedzisira, De Bruijn akaiswa pano kuti aone zvinyorwa muzvikamu.

mRNA-(De-novo)-Pfungwa-ye-Utatu

mRNA (De novo) Mhedziso yeUtatu

2.mRNA (De novo) Kugoverwa kweGene Expression Level

RNA-Seq inokwanisa kuwana fungidziro inonzwisisika yejini kutaura.Kazhinji, iyo inoonekwa mhando yezvinyorwa zvekutaura FPKM kubva ku10 ^ -2 kusvika 10 ^ 6

mRNA-(De-novo)-Kugovera-kwe-FPKM-density-mune-imwe-sample

mRNA (De novo) Kugovera kweFPKM density mumuenzaniso wega wega

3.mRNA (De novo) GO Enrichment Analysis yeDEGs

GO (Gene Ontology) dhatabhesi ndeye yakarongeka biological annotation system ine yakajairwa mazwi emajini uye gene zvigadzirwa zvinoshanda.Iine mazinga akawanda, apo iyo yakaderera nhanho, iyo inonyanya kutarisisa mabasa.

mRNA-(De-novo)-GO-classification-of-DEGs-pa-second-level

mRNA (De novo) GO kupatsanurwa kweDEGs padanho rechipiri

Nyaya yeBMK

Transcriptome Ongororo yeSucrose Metabolism panguva yekuzvimba kweBhubhu uye Kubudirira muOni (Allium cepa L.)

Rakatsikiswa: miganhu musayenzi yemiti,2016

Sequencing strategy

Illumina HiSeq2500

Sample collection

Utah Yellow Sweet Spain cultivar "Y1351" yakashandiswa muchidzidzo ichi.Nhamba yemasampuli akaunganidzwa aive
Zuva regumi nemashanu mushure mekuzvimba (DAS) yegirobhu (2-cm dhayamita uye 3-4 g uremu), 30th DAS (5-cm dhayamita uye 100-110 g uremu), uye ~3 pa40th DAS (7-cm dhayamita uye 260-300 magiramu).

Mibairo yakakosha

1. mumufananidzo weVenn, huwandu hwe146 DEGs hwakaonekwa pamativi ese matatu ematanho ebudiriro.
2. "Carbohydrate transport uye metabolism" yaimiririrwa chete ne585 unigenes (kureva, 7% yeCOG inotsanangurwa).
3.Unigenes yakabudirira kutsanangurwa kune GO dhatabhesi yakaiswa muzvikamu zvitatu zvakakosha pamatanho matatu akasiyana ekugadzirisa girobhu.Vazhinji vanomiririrwa mu "biological process" chikamu chikuru vaive "metabolic process", inoteverwa ne "cellular process".Muchikamu chikuru che "molecular function" mapoka maviri ainyanya kumiririrwa aive "binding" uye "catalytic chiitiko".

PB-yakazara-kureba-RNA-Sequencing-case-study

Histogram yemasumbu e orthologous mapoka (COG) kupatsanura

Histogram yegene ontology (GO) kupatsanurwa kweunigenes inotorwa kubva kuma bulbs mumatanho matatu ekukura.

Venn dhayagiramu inoratidza majini anoratidzwa zvakasiyana mumatanho maviri ekukura kwegirobhu rehanyanisi

Reference

Zhang C, Zhang H, Zhan Z, nevamwe.Transcriptome Analysis yeSucrose Metabolism panguva yeBulb Kuzvimba uye Kubudirira muanyanisi (Allium cepa L.)[J].Frontiers in Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425