I-Non-Reference based mRNA Sequencing-NGS
Izici
● Ukuthwebula i-poly mRNA ngaphambi kokulungiswa komtapo wolwazi
● Ingazimele kunoma iyiphi i-genome yereferensi: ngokusekelwe ekuhlanganisweni kwe-de novo yemibhalo ebhaliwe, okukhiqiza uhlu lwama-unigenes anezichasiselo zolwazi oluningi (NR, Swiss-Prot, COG, KOG, eggNOG, Pfam, GO, KEGG)
● Ukuhlaziya okuphelele kwe-bioinformatic ye-gene expression kanye ne-transcript structure
Izinzuzo Zesevisi
●Ubungcweti Olubanzi: Ngomlando wokucubungula amasampula angaphezu kuka-600,000 ku-BMKGENE, ahlanganisa izinhlobo zamasampula ahlukahlukene njengamasiko amaseli, izicubu, noketshezi lomzimba, ithimba lethu liletha ingcebo yesipiliyoni kuyo yonke iphrojekthi. Sivale ngempumelelo amaphrojekthi angaphezu kuka-100,000 mRNA-Seq ezizindeni ezihlukene zocwaningo.
●Ukulawulwa Kwekhwalithi Okuqinile: sisebenzisa amaphuzu okulawula abalulekile kuzo zonke izigaba, kusukela kusampula nokulungiselela umtapo wezincwadi ukuya ekulandelaneni kanye ne-bioinformatics. Lokhu kuqapha ngokucophelela kuqinisekisa ukulethwa kwemiphumela ehlala njalo yekhwalithi ephezulu.
● Isichasiselo Esibanzi: sisebenzisa imininingo egciniwe eminingi ukuze sichasise ngokusebenza Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG) futhi senze ukuhlaziya okunothisayo okuhambisanayo, sinikeze imininingwane ngezinqubo zeselula nezamangqamuzana ezingaphansi kwempendulo ye-transcriptome.
●Ukusekelwa Kwangemva Kokuthengisa: Ukuzinikela kwethu kudlulela ngalé kokuphothulwa kwephrojekthi ngenkathi yesevisi yezinyanga ezi-3 ngemva kokuthengisa. Ngalesi sikhathi, sinikezela ngokulandelelwa kwephrojekthi, usizo lokuxazulula inkinga, namaseshini e-Q&A ukuze siphendule noma yimiphi imibuzo ehlobene nemiphumela.
Izidingo Zesampula Nokulethwa
| Umtapowolwazi | Isu lokulandelanisa | Idatha iyanconywa | Ikhwalithi yokulawula |
| I-Poly A inothisiwe | I-Ilumina PE150 I-DNBSEQ-T7 | 6-10 Gb | Q30≥85% |
Izimfuneko zesampula:
Ama-Nucleotides:
| Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Amaprotheni anomkhawulo noma awekho nhlobo noma ukungcola kwe-DNA okuboniswa kujeli. | Okwezitshalo: RIN≥4.0; Okwezilwane: RIN≥4.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselwe noma okungenakho |
● Izitshalo:
Izimpande, Isiqu noma I-Petal: 450 mg
Iqabunga noma Imbewu: 300 mg
Isithelo: 1.2 g
● Isilwane:
Inhliziyo noma Amathumbu: 300 mg
I-Viscera noma Ubuchopho: 240 mg
Imisipha: 450 mg
Amathambo, Izinwele noma Isikhumba: 1g
● Ama-arthropods:
Izinambuzane: 6g
I-crustacea: 300 mg
● Igazi lonke: 1 ithubhu
● Amaseli: 106 amaseli
Ukulethwa Kwesampula Okunconyiwe
Isitsha: 2 ml centrifuge tube (Tin foil ayinconywa)
Ukulebula okuyisampula: Ukuphindaphinda kweqembu+isb A1, A2, A3; B1, B2, B3.
Ukuthunyelwa:
1. Iqhwa elomile: Amasampula adinga ukupakishwa ezikhwameni futhi agqitshwe eqhweni elomile.
2. Amashubhu e-RNAstable: Amasampula e-RNA angomiswa ngeshubhu lokuzinzisa le-RNA(isb. i-RNAstable®) futhi athunyelwe ekamelweni lokushisa.
Ukugeleza Komsebenzi Wesevisi
Idizayini yokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
I-Bioinformatics
Ukuhlanganiswa kwe-Transcriptome nokukhetha kwe-unigene
Unigene isichasiselo
Ukuhlanganiswa kwesampula nokuhlolwa kokuphindaphindeka kwezinto eziphilayo
Izakhi zofuzo ezivezwe ngokuhlukile (DEGs)
Isichasiselo esisebenzayo sama-DEGs
Ukuthuthukisa Okusebenzayo kwama-DEGs
Hlola intuthuko egqugquzelwa izinsiza zokulandelana ze-Eukaryotic NGS mRNA ze-BMKGene ngeqoqo elikhethiwe lokushicilelwe.
Shen, F. et al. (2020) 'I-De novo transcriptome assembly kanye ne-sex-biased gene expression in the gonads of Amur catfish (Silurus asotus)', Genomics, 112(3), pp. 2603–2614. doi: 10.1016/J.YGENO.2020.01.026.
Zhang, C. et al. (2016) 'Ukuhlaziywa kwe-Transcriptome ye-sucrose metabolism ngesikhathi sokuvuvukala kwe-bulb nokuthuthukiswa ku-anyanisi (Allium cepa L.)', Frontiers in Plant Science, 7(September), p. 212763. doi: 10.3389/FPLS.2016.01425/BIBTEX.
Zhu, C. et al. (2017) 'De novo assembly, characterization and annotation for the transcriptome of Sarcocheilichthys sinensis', PLoS ONE, 12(2). doi: 10.1371/JOURNAL.PONE.0171966.
Zou, L. et al. (2021) 'Ukuhlaziywa kwe-De novo transcriptome kunikeza imininingwane yokubekezelela usawoti we-Podocarpus macrophyllus ngaphansi kokucindezeleka kosawoti', BMC Plant Biology, 21(1), pp. 1–17. doi: 10.1186/S12870-021-03274-1/AMAFIGURES/9.
