I-PacBio 2+3 Isixazululo Esigcwele Ubude be-mRNA
Izici
● Idizayini yokufunda:
Isampula elihlanganisiwe elilandelana ne-PacBio ukuze kutholakale ama-isoforms alotshiwe
Amasampula ahlukene (okuphindaphindwayo kanye nezimo okufanele zihlolwe) alandelweI-NGS ukulinganisa inkulumo elotshiweyo
● Ukulandelana kwe-PacBio kumodi ye-CCS, okukhiqiza ukufundwa kwe-HiFi
● Ukulandelana kwemibhalo yobude obugcwele
● Ukuhlaziya akudingi i-reference genome; nokho, ingase isetshenziswe
● Ukuhlaziywa kwe-Bioinformamatic akuhlanganisi nje kuphela ukubonakaliswa kufuzo kanye neleveli ye-isoform kuphela kodwa nokuhlaziywa kwe-lncRNA, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation, nesakhiwo sofuzo.
Izinzuzo
● Ukunemba Okuphezulu: I-HiFi ifundeka ngokunemba >99.9% (Q30), uma iqhathaniswa ne-NGS
● Ukuhlaziywa Kokuhlukanisa Okuhlukile: ukulandelana kwayo yonke imibhalo kunika amandla ukukhonjwa kwe-isoform kanye nokwenza izinhlamvu.
● Inhlanganisela ye-PacBio kanye ne-NGS Amandla: ukunika amandla ukulinganisa kokusho ezingeni le-isoform, ukwembula uguquko olungase lufihlwe lapho kuhlaziywa lonke uhlobo lwesisho sofuzo
● Ingcweti Ebanzi: Ngomlando wokuqedela amaphrojekthi wombhalo obhalwe ngobude obugcwele we-PacBio angaphezu kuka-1100 nokucubungula amasampula angaphezu kuka-2300, ithimba lethu liletha ingcebo yesipiliyoni kuyo yonke iphrojekthi.
● 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 |
| Ilabhulali ye-PolyA ethuthukisiwe ye-mRNA CCS | I-PacBio Sequel II I-PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| I-Poly A inothisiwe | I-Ilumina PE150 | 6-10 Gb | Q30≥85% |
Ama-Nucleotides
|
| Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| I-Illumina Library | ≥ 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 |
| Umtapo wezincwadi we-PacBio | ≥ 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. | Izitshalo: RIN≥7.5 Izilwane: RIN≥8.0 5.0≥28S/18S≥1.0; ukuphakama okulinganiselwe noma okungenakho |
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.
Kufaka ukuhlaziya okulandelayo:
Ukulawulwa kwekhwalithi yedatha eluhlaza
I-Alternative Polyadenylation Analysis (APA)
Ukuhlaziywa kokulotshiweyo kwe-Fusion
Ukuhlaziya Okuhlukile Kokuhlukanisa
Ukuhlaziywa kwe-Benchmarking Single-Copy Orthologs (BUSCO).
Ukuhlaziywa kokulotshiweyo kwenoveli: ukubikezelwa kokulandelana kwekhodi (CDS) kanye nesichasiselo esisebenzayo
Ukuhlaziywa kwe-lncRNA: ukubikezela kwe-lncRNA kanye nezinhloso
I-MicroSatelite Identification (SSR)
Ukuhlaziywa kwe-Differentially Expressed Transcripts (DETs).
Ukuhlaziywa Kwezakhi Zofuzo Ezivezwe Ngokuhlukile (DEGs).
Isichasiselo esisebenzayo sama-DEG nama-DET
Ukuhlaziywa kwe-BUSCO
Ukuhlaziya Okuhlukile Kokuhlukanisa
I-Alternative Polyadenylation Analysis (APA)
Izakhi Zofuzo Ezivezwe Ngokuhlukile (DEGs) kanye Nemibhalo Ebhaliwe (DETs9 anlaysis
Amanethiwekhi wokusebenzisana we-Protein-Protein we-DET nama-DEGs
Hlola intuthuko eqhutshwa i-BMKGene's PacBio 2+3 ye-mRNA yobude obugcwele ngokulandelana ngeqoqo elikhethiwe lokushicilelwe.
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Wang, Lijun et al. (2019) 'Inhlolovo yobunkimbinkimbi be-transcriptome kusetshenziswa ukuhlaziya kwesikhathi sangempela se-PacBio okuhlanganiswe 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/AMAFIGURE/7.
