Ukulandelana kwe-mRNA yobude obugcwele-Nanopore
Izici
● Ukuthwebula i-poly-A mRNA okulandelwa ukuhlanganiswa kwe-cDNA nokulungiselela umtapo wolwazi
● Ukulandelana kwemibhalo yobude obugcwele
● Ukuhlaziywa kwe-Bioinformamatic okusekelwe ekuqondaneni nofuzo lwereferensi
● Ukuhlaziywa kwe-Bioinformamatic akubandakanyi nje kuphela ukuvezwa ezingeni lofuzo kanye neleveli ye-isoform kodwa nokuhlaziywa kwe-lncRNA, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation kanye nesakhiwo sofuzo.
Izinzuzo Zesevisi
●Ukulinganisa kwenkulumo ezingeni le-isoform: ukunika amandla ukuhlaziywa kwenkulumo okunemininingwane nokunembile, ukwembula uguquko olungase lufihlwe lapho kuhlaziywa lonke uhlobo lwesisho sofuzo
●Izidingo Zedatha Eyehlisiwe:Uma kuqhathaniswa ne-Next-Generation Sequencing (NGS), ukulandelana kwe-Nanopore kubonisa izimfuneko zedatha eziphansi, okuvumela amaleveli alinganayo okugcwala kwesisho sofuzo ngedatha encane.
●Ukunemba okuphezulu kokulinganisa kwenkulumo: kokubili ezingeni lofuzo kanye ne-isoform
●Ukuhlonza ulwazi olwengeziwe lwe-transcriptomic: enye i-polyadenylation, izakhi zofuzo ezihlanganisiwe kanye ne-lcnRNA kanye nezakhi zazo ezihlosiwe
●Ubungcweti Olubanzi: Ithimba lethu liletha ingcebo yolwazi kuwo wonke amaphrojekthi, njengoba seliqede amaphrojekthi we-transcriptome we-Nanopore angaphezu kuka-850 futhi lacubungula amasampula angaphezu kuka-8,000.
●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 | 6/12 Gb | Isilinganiso sekhwalithi yesikolo: Q10 |
Izimfuneko zesampula:
Ama-Nucleotides:
| Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| ≥ 100 | ≥ 1.0 | 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≥7.0; Okwezilwane: RIN≥7.5; 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
Ama-Nucleotides:
Ukulethwa kwesampula
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
Ukugeleza Komsebenzi Wesevisi
Izicubu:
Idizayini yokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
● Ukucubungula idatha engahluziwe
● Ukuhlonza umbhalo
● Okunye ukuhlanganisa
● Ukulinganisa kokuvezwa ezingeni lofuzo kanye nezinga le-isoform
● Ukuhlaziywa kwenkulumo okuhlukile
● Isichasiselo somsebenzi nokunothisa (ama-DEG nama-DET)
Ukuhlaziya okuhlukile kokuhlanganisa
I-Alternative Polyadenylation Analysis (APA)
Ukubikezela kwe-lncRNA
Isichasiselo sofuzo lwenoveli
Ukuhlanganiswa kwama-DET
Amanethiwekhi Amaprotheni-Amaprotheni kuma-DEGs
Hlola intuthuko egqugquzelwa izinsiza zokulandelana ze-mRNA ze-BMKGene's Nanopore ngeqoqo elikhethiwe lokushicilelwe.
Gong, B. et al. (2023) 'Ukwenziwa kusebenze kwe-Epigenetic kanye nokubhala okubhaliwe kwe-secretory kinase FAM20C njenge-oncogene ku-glioma', Ijenali yeGenetics and Genomics, 50(6), pp. 422–433. doi: 10.1016/J.JGG.2023.01.008.
Yena, Z. et al. (2023) 'Ukulandelana kwe-transcriptome yobude obugcwele be-lymphocyte kusabela ku-IFN-γ kuveza ukusabela kwamasosha omzimba oku-Th1 ku-flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
UMa, Y. et al. (2023) 'Ukuhlaziywa okuqhathanisayo kwe-PacBio kanye nezindlela zokulandelana ze-ONT RNA zokuhlonza ubuthi be-Nemopilema Nomurai', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
Yu, D. et al. (2023) 'Ukuhlaziywa kwe-Nano-seq kuveza ukuthambekela okuhlukile kokusebenza phakathi kwama-exosomes nama-microvesicles atholakala ku-hUMSC', i-Stem Cell Research and Therapy, 14(1), amakhasi 1-13. doi: 10.1186/S13287-023-03491-5/AMATHEBULA/6.
