Isixazululo se-mRNA esigcwele ubude be-PacBio 2+3
Izici
● Umklamo wokutadisha:
Isampula ehlanganisiwe ihlelwe ngokulandelana ne-PacBio ukuze kutholakale ama-isoforms okulotshiwe
Amasampula ahlukene (okukopishwayo nezimo okufanele zihlolwe) ahlelwe ngokulandelanaI-NGS yokulinganisa ukubonakaliswa kombhalo
● Ukulandelana kwe-PacBio kumodi ye-CCS, okudala ukufundwa kwe-HiFi
● Ukulandelana kwemibhalo ephelele
● Ukuhlaziywa akudingi i-genome yokubhekisela; noma kunjalo, kungasetshenziswa
● Ukuhlaziywa kwe-Bioinformatic akubandakanyi nje kuphela ukuvezwa ezingeni lezakhi zofuzo kanye ne-isoform kodwa futhi nokuhlaziywa kwe-lncRNA, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation, kanye nesakhiwo sezakhi zofuzo.
Izinzuzo
● Ukunemba Okuphezulu: I-HiFi ifundeka ngokunemba >99.9% (Q30), uma kuqhathaniswa ne-NGS
● Ukuhlaziywa Kokuhlukaniswa Okuhlukile: Ukulandelanisa yonke imibhalo kuvumela ukuhlonza kanye nokuhlukanisa ama-isoform.
● Inhlanganisela yamandla e-PacBio kanye ne-NGS: Ivumela ukulinganiswa kokubonakaliswa ezingeni le-isoform, iveze ushintsho olungafihlwa lapho kuhlaziywa ukubonakaliswa kwezakhi zofuzo konke
● Ubuchwepheshe Obubanzi: Sicubungule 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% |
| I-Poly A ecebile | I-Illumina PE150 | 6-10 Gb | Q30≥85% |
Ama-Nucleotide
|
| I-Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| Umtapo Wezincwadi we-Illumina | ≥ 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 we-PacBio | ≥ 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. | Izitshalo: I-RIN≥7.5 Izilwane: 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.
Kufaka phakathi ukuhlaziywa okulandelayo:
- Idatha eluhlaza
- Ikhwalithi yokulawula
- Ukuhlaziywa Kwe-Alternative Polyadenylation (APA)
- Ukuhlaziywa kombhalo we-Fusion
- Ukuhlaziywa Kokuhlukaniswa Okuhlukile
- Ukuhlaziywa kwe-Benchmarking Universal Single-Copy Orthologs (BUSCO)
- 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-Differentially Expressed Transcripts (DETs)
- Ukuhlaziywa kwe-Differentially Expressed Genes (DEGs)
- Isichasiselo esisebenzayo sama-DEG nama-DET
Ukuhlaziywa kwe-BUSCO
Ukuhlaziywa Kokuhlukaniswa Okuhlukile
Ukuhlaziywa Kwe-Alternative Polyadenylation (APA)
Izakhi Zofuzo Ezivezwe Ngokuhlukile (ama-DEG) kanye Nemibhalo Eqoshiwe (i-DETs9 anlaysis)
Amanethiwekhi okuxhumana kwamaphrotheni namaphrotheni ama-DET nama-DEG
Hlola intuthuko eyenziwe yi-BMKGene's PacBio 2+3 full-length mRNA sequencing ngokusebenzisa iqoqo lezincwadi ezikhethiwe.
UChao, Q. et al. (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 Ezihlobene Zama-Molecular',Ijenali Yomhlaba Wonke Yesayensi Yamamolekyuli, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
UHua, X. et al. (2022) 'Ukubikezela okusebenzayo kwezakhi zofuzo zendlela yokwenziwa kwezinto eziphilayo ezihilelekile kuma-polyphyllin asebenzayo e-Paris polyphylla',Ibhayoloji Yokuxhumana2022 5:1, 5(1), amakhasi 1–10. doi: 10.1038/s42003-022-03000-z.
ULiu, M. et al. (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/INSETS14040363/S1.
UWang, uLijun nabanye. (2019) 'Ucwaningo lobunzima be-transcriptome olusebenzisa ukuhlaziywa kwe-PacBio nge-molecule eyodwa ngesikhathi sangempela kuhlanganiswe nokulandelana kwe-Illumina RNA ukuze kuqondwe kangcono i-ricinoleic acid biosynthesis ku-Ricinus communis',I-BMC Genomics, 20(1), amakhasi 1–17. doi: 10.1186/S12864-019-5832-9/FIGURES/7.
