Ukulandelana kwe-mRNA okugcwele -PacBio
Izici
● ukwenziwa kwe-cDNA kusuka ku-poly-A mRNA kulandelwe ukulungiswa komtapo wolwazi
● Ukulandelana kwemodi ye-CCS, okudala ukufundwa kwe-HiFi
● Ukulandelana kwemibhalo ephelele
● Ukuhlaziywa akudingi i-genome yokubhekisela; noma kunjalo, kungasetshenziswa
● Ukuhlaziywa kwe-Bioinformatic kwenza ukuhlaziywa kwemibhalo ye-isoform lncRNA, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation, kanye nesakhiwo sezakhi zofuzo
Izinzuzo Zesevisi
●Ukunemba Okuphezulu: I-HiFi ifundeka ngokunemba >99.9% (Q30), uma kuqhathaniswa ne-NGS
● Ukuhlaziywa Kokuhlukaniswa Okuhlukile: ukulandelana kwemibhalo yonke kwenza kube lula ukuhlonza kanye nokuhlukanisa ama-isoform
●Ubuchwepheshe Obubanzi: ngomlando wokuqeda amaphrojekthi e-transcriptome angaphezu kuka-1100 e-PacBio kanye nokucubungula amasampula angaphezu kuka-2300, ithimba lethu liletha ulwazi oluningi kuyo yonke iphrojekthi.
●Usekelo Lwangemva Kokuthengisa: ukuzibophezela kwethu kudlulela ngale kokuqedwa kwephrojekthi ngesikhathi sesevisi yezinyanga ezintathu ngemuva kokuthengisa. Phakathi nalesi sikhathi, sinikeza ukulandelwa kwephrojekthi, usizo lokuxazulula izinkinga, kanye nezikhathi ze-Q&A ukuze kuxazululwe noma yimiphi imibuzo ehlobene nemiphumela.
Izidingo Zesampula Nokulethwa
| Umtapo Wolwazi | Isu lokulandelana kwezigaba | Kunconywa idatha | Ikhwalithi yokulawula |
| Umtapo wolwazi we-mRNA CCS ocebile nge-PolyA | I-PacBio Sequel II I-PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
Izidingo Zesampula:
Ama-Nucleotide:
● Izitshalo:
Impande, Isiqu noma Ipetali: 450 mg
Iqabunga noma Imbewu: 300 mg
Izithelo: 1.2 g
● Isilwane:
Inhliziyo noma Amathumbu: 300 mg
I-Viscera noma Ubuchopho: 240 mg
Imisipha: 450 mg
Amathambo, Izinwele noma Isikhumba: 1g
● Ama-arthropod:
Izinambuzane: 6g
I-Crustacea: 300 mg
● Igazi eliphelele: Ishubhu eli-1
● Amaseli: 106 amaseli
| I-Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ukungcoliswa kwephrotheni noma i-DNA okulinganiselwe noma okungekho nhlobo kukhonjisiwe kujeli. | Kwezitshalo: I-RIN≥7.5; Ezilwaneni: I-RIN≥8.0; 5.0≥ 28S/18S≥1.0; ukuphakama okulinganiselwe noma okungekho kwesisekelo |
Ukulethwa Kwesampula Okunconyiwe
Isitsha: Ishubhu le-centrifuge elingu-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
Ukulebula kwesampula: Iqembu + ukukopisha isib. A1, A2, A3; B1, B2, B3.
Ukuthunyelwa:
1. Iqhwa elomile: Amasampula kudingeka apakishwe ezikhwameni bese egqitshwa eqhweni elomile.
2. Amashubhu e-RNAstable: Amasampula e-RNA angomiswa eshubhu lokuzinzisa i-RNA (isb. i-RNAstable®) bese ethunyelwa ekushiseni kwegumbi.
Ukugeleza Komsebenzi Wesevisi
Umklamo wokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelana
Ukuhlaziywa kwedatha
Izinsizakalo zangemuva kokuthengisa
Kufaka phakathi ukuhlaziywa okulandelayo:
● Ukulawulwa kwekhwalithi yedatha eluhlaza
● Ukuhlaziywa Kwe-Alternative Polyadenylation (APA)
● Ukuhlaziywa kombhalo we-Fusion
● Ukuhlaziywa Kokuhlukaniswa Okuhlukile
● Ukuhlaziywa kwe-Universal Single-Copy Orthologs (BUSCO) Benchmarking
● Ukuhlaziywa kwemibhalo emisha: ukubikezela ukulandelana kokubhala amakhodi (i-CDS) kanye nesichasiselo esisebenzayo
● Ukuhlaziywa kwe-lncRNA: ukubikezela kwe-lncRNA kanye nezinhloso
● Ukuhlonza i-MicroSatelite (SSR)
Ukuhlaziywa kwe-BUSCO
Ukuhlaziywa Kokuhlukaniswa Okuhlukile
Ukuhlaziywa Kwe-Alternative Polyadenylation (APA)
Isichasiselo esisebenzayo semibhalo emisha
Hlola intuthuko eyenziwe yizinsizakalo zokulandelana kwe-mRNA ze-BMKGene ze-Nanopore kulo magazini ofakiwe.
UMa, Y. nabanye. (2023) 'Ukuhlaziywa kokuqhathanisa kwezindlela zokulandelana kwe-PacBio kanye ne-ONT RNA zokuhlonza ubuthi be-Nemopilema Nomurai', i-Genomics, 115(6), ikhasi 110709. doi: 10.1016/J.YGENO.2023.110709.
UChao, Q. nabanye. (2019) 'Izindlela zokuthuthukisa ze-Populus stem transcriptome', Ijenali ye-Plant Biotechnology, 17(1), amakhasi 206–219. doi: 10.1111/PBI.12958.
UDeng, H. et al. (2022) 'Izinguquko Ezinamandla Kokuqukethwe kwe-Ascorbic Acid Ngesikhathi Sokuthuthukiswa Kwezithelo Nokuvuthwa kwe-Actinidia latifolia (Isitshalo Sezithelo Esicebile Nge-Ascorbate) kanye Nezindlela Ezihlanganisiwe Ze-Molecular', I-International Journal of Molecular Sciences, 23(10), k. 5808. doi: 10.3390/IJMS23105808/S1.
UHua, X. nabanye. (2022) 'Ukubikezela okuphumelelayo kwezakhi zofuzo zendlela yokwenziwa kwezinto eziphilayo ezihilelekile kuma-polyphyllin asebenzayo ku-polyphylla yaseParis', i-Communications Biology 2022 5:1, 5(1), amakhasi 1–10. doi: 10.1038/s42003-022-03000-z.
ULiu, M. nabanye. (2023) 'Ukuhlaziywa kwe-PacBio Iso-Seq kanye ne-Illumina RNA-Seq okuhlanganisiwe kwe-Tuta absoluta (Meyrick) Transcriptome kanye ne-Cytochrome P450 Genes', Izinambuzane, 14(4), ikhasi 363. doi: 10.3390/INSECTS14040363/S1.
UWang, uLijun nabanye. (2019) 'Ucwaningo lobunzima be-transcriptome olusebenzisa ukuhlaziywa kwesikhathi sangempela kwe-PacBio single-molecule kuhlanganiswe nokulandelana kwe-Illumina RNA ukuze kuqondwe kangcono i-ricinoleic acid biosynthesis ku-Ricinus communis', BMC Genomics, 20(1), pp. 1–17. doi: 10.1186/S12864-019-5832-9.
