Ukulandelelana kwe-mRNA ye-Eukaryotic-NGS
Izici
● Ukulungiselela umtapo wezincwadi we-mRNA oqondiswayo ongakukhetha ukuze kuvunyelwe ukuthola idatha yokulandelana kwemigqa ethile
● Ukuhlaziywa kwe-BI okuhlukile kumboza zonke izidingo zamakhasimende ethu
Izinzuzo
●Ubuchwepheshe Obubanzi: Sicubungule amasampula angaphezu kuka-600,000, ahlanganisa izinhlobo ezahlukene zamasampula njengezakhi zamaseli, izicubu, kanye noketshezi lomzimba. Siletha ulwazi oluningi kuyo yonke iphrojekthi.
●Ukulawulwa Kwekhwalithi Okuqinile: Sisebenzisa amaphuzu okulawula ayisisekelo kuzo zonke izigaba, kusukela ekulungiseleleni amasampula kuya ekulungiseleleni umtapo wolwazi, ukulandelana kanye ne-bioinformatics. Ukuqapha kwethu ngokucophelela kuqinisekisa ukulethwa kwemiphumela esezingeni eliphezulu njalo.
●Isichasiselo Esiphelele: Sisebenzisa izizindalwazi eziningi ukuze sichaze ngokusebenza i-Differentially Expressed Genes (DEGs) futhi senze ukuhlaziywa kokunotha okuhambisanayo. Le ndlela ebanzi inikeza ukuqonda ngezinqubo zamaseli kanye nama-molecule ezisekela impendulo ye-transcriptome, okuqinisekisa ukuthi uthola lonke ulwazi olungenzeka mayelana nedatha yokuhlolwa kwakho.
●Usekelo Lwangemva Kokuthengisa: Siyaqonda ukuthi kubaluleke kangakanani ukuba khona, yingakho ukuzibophezela kwethu kudlulela ngale kokuphothulwa kwephrojekthi ngesikhathi senkonzo 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 |
| I-Poly A ecebile | I-Illumina PE150 I-DNBSEQ-T7 | 6 -10 Gb | Q30≥85% |
Izidingo Zesampula:
Ama-Nucleotide:
Izidingo Zesampula Ezincane:
| I-Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho | |
| Umtapo Wolwazi Ojwayelekile | ≥ 10 | ≥ 0.2 | 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≥4.0; Ezilwaneni: I-RIN≥4.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselwe noma okungekho kwesisekelo |
| Umtapo Wolwazi Oqondisayo | ≥ 10 | ≥ 0.2 | 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≥4.0; Ezilwaneni: I-RIN≥4.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselwe noma okungekho kwesisekelo |
| Izitshalo | Isilwane | Ama-arthropod | Igazi eliphelele | Amaseli |
| Impande, Isiqu noma Ipetali: 450 mg Iqabunga noma Imbewu: 300 mg Izithelo: 1.2 g | Inhliziyo noma Amathumbu: 300 mg I-Viscera noma Ubuchopho: 240 mg Imisipha: 450 mg Amathambo, Izinwele noma Isikhumba: 1g | Izinambuzane: 6g I-Crustacea: 300 mg | Ishubhu eli-1 | Amaseli angu-106 |
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 etafula lama-RNA: Amasampula e-RNA angomiswa eshubhu lokuzinzisa i-RNA (isb. i-RNAstable®) bese ethunyelwa ekushiseni kwegumbi.
Izidingo Zesampula Nokulethwa
Umklamo wokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelana
Ukuhlaziywa kwedatha
Izinsizakalo zangemuva kokuthengisa
I-Bioinformatics
Kumasampula ane-genome yokubhekisela etholakalayo, ukuhlaziywa kwe-BI kungokulandelayo:
ØUkulawulwa kwekhwalithi yedatha eluhlaza
ØUkulungiswa kwe-genome okubhekiselwe kuyo
ØUkuhlaziywa kwesakhiwo sombhalo oqoshiwe
ØUkulinganiswa kokubonakaliswa
ØUkuhlaziywa kokubonakaliswa kokwehluka
ØIsichasiselo somsebenzi kanye nokucebisa
Kumasampula angenayo i-genome yokubhekisela, nansi inketho etholakalayo:
• Ukulawulwa kwekhwalithi yedatha eluhlaza
•Ukuhlanganiswa ngokulungiswa kwesakhiwo
•Ukuhlaziywa kokubonakaliswa kokwehluka
•Isichasiselo somsebenzi kanye nokucebisa
Ukuqondaniswa Kwe-Genome Yereferensi
Ukugcwala kwedatha
Ukuhambisana kwesampula kanye nokuhlolwa kokuphindaphindwa kwezinto eziphilayo
Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG)
Isichasiselo Esisebenzayo sama-DEG
Ukuthuthuka Okusebenzayo kwama-DEG
Hlola intuthuko eyenziwe yizinsizakalo zokulandelana kwe-BMKGene ze-eukaryotic NGS mRNA ngokusebenzisa iqoqo lezincwadi ezikhethiwe.
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