Ukulandelana kwe-mRNA yobude obugcwele -PacBio
Izinzuzo Zesevisi
● Ukufundwa okuqondile kwe-molecule ye-cDNA yobude obugcwele ukusuka ku-3'- ekupheleni kuya ku-5'- ekupheleni
● Ukulungiswa kwezinga le-iso-form kusakhiwo sokulandelana
● Okulotshiweyo ngokunemba okuphezulu nobuqotho
● Ihambisana kakhulu nezinhlobo ze-vaiours
● Umthamo omkhulu wokulandelana onezinkundla zokulandelana ezingu-4 PacBio Sequel II ezihlomelisiwe
● Nginolwazi olunzulu ngamaphrojekthi wokulandelanisa we-RNA asuselwa ku-Pacbio angaphezu kuka-700
● Ukulethwa kwemiphumela okususelwe ku-BMKCloud: Ukumbiwa kwedatha ngokwezifiso kutholakala endaweni yesikhulumi.
● Izinsizakalo zangemuva kokuthengisa zisebenza izinyanga ezi-3 lapho umsebenzi usuphothuliwe
Imininingwane Yesevisi
I-Platform: I-PacBio Sequel II
Umtapo wolwazi wokulandelanisa: Umtapo wezincwadi we-Poly A-enriched mRNA
Isivuno sedatha esinconyiwe: 20 Gb/isampula (Kuye ngezinhlobo)
I-FLNC (%): ≥75%
*I-FLNC: Imibhalo yobude obugcwele engeyona i-chimeric
I-Bioinformatics ihlaziya
● Ukucubungula idatha engahluziwe
● Ukuhlonza umbhalo
● Isakhiwo sokulandelana
● Ukulinganisa Inkulumo
● Isichasiselo Somsebenzi
Izidingo Zesampula Nokulethwa
Izimfuneko zesampula:
Ama-Nucleotides:
| Conc.(ng/μl) | Inani (μg) | Ubumsulwa | Ubuqotho |
| ≥ 120 | ≥ 0.6 | 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≥7.5; Okwezilwane: RIN≥8.0; 5.0≥ 28S/18S≥1.0; ukuphakama okulinganiselwe noma okungenakho |
Izicubu: Isisindo(somile):≥1 g
*Ngezicubu ezincane kuno-5 mg, sincoma ukuthi uthumele isampula yezicubu eziqandisiwe (ku-nitrogen ewuketshezi).
Ukumiswa kweseli:Inani lamaseli = 3×106- 1 × 107
*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×105, i-flash efriziwe ku-nitrogen eyi-liquid iyanconywa, okungcono kakhulu ekukhishweni okuncane.
Amasampula egazi:Umthamo≥1 mL
I-Microorganism:Isisindo ≥ 1 g
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
Idizayini yokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-RNA
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
1.FLNC ubude bokusabalalisa
Ubude bobude obugcwele be-non-chimeric read(FLNC) bubonisa ubude be-cDNA ekwakhiweni komtapo wolwazi.Ukusatshalaliswa kobude be-FLNC kuyinkomba ebalulekile ekuhloleni ikhwalithi yokwakhiwa komtapo wolwazi.
Ukusabalalisa ubude bokufundwa kwe-FLNC
2.Qedela ukusatshalaliswa kobude besifunda se-ORF
Sisebenzisa i-TransDecoder ukuze sibikezele izifunda zamakhodi amaprotheni kanye nokulandelana kwe-amino acid ehambisanayo ukuze sikhiqize amasethi e-unigene, aqukethe ulwazi oluphelele lokuloba olungadideki kuwo wonke amasampuli.
Qedela ukusatshalaliswa kobude besifunda se-ORF
3.KEGG ukuhlaziya indlela yokucebisa
Okulotshiweyo okuvezwe ngendlela ehlukile(DETs) kungabonakala ngokuvumelanisa idatha yokulandelanisa ye-NGS-based RNA kumasethi ombhalo wobude obugcwele akhiqizwe idatha yokulandelana kwe-PacBio.Lawa ma-DET angaphinde acutshungulwe ukuze kuhlaziywe okuhlukahlukene kokusebenza, isb. ukuhlaziywa kwendlela ye-KEGG yokunothisa.
I-DET KEGG indlela yokucebisa -Isakhiwo se-Dot
Icala le-BMK
Amandla okuthuthuka kwe-Populus stem transcriptome
Kushicilelwe: Plant Biotechnology Journal, 2019
Isu lokulandelanisa:
Iqoqo lesampula:izifunda ze-stem: i-apex, i-internode yokuqala(IN1), i-internode yesibili(IN2), i-internode yesithathu(IN3), i-internode(IN4) kanye ne-internode(IN5) evela e-Nanlin895
I-NGS-ukulandelana:I-RNA yabantu abangu-15 yahlanganiswa njengesampula yebhayoloji eyodwa.Izifaniso ezintathu zebhayoloji zephoyinti ngalinye zicutshungulwe ngokulandelana kwe-NGS
TGS-ukulandelana:Izifunda ze-stem zahlukaniswa zaba izifunda ezintathu, okungukuthi i-apex, IN1-IN3 ne-IN4-IN5.Isifunda ngasinye sacutshungulwa ukulandelana kwe-PacBio ngezinhlobo ezine zemitapo yolwazi: 0-1 kb, 1-2 kb, 2-3 kb kanye no-3-10 kb.
Imiphumela esemqoka
1.Isamba semibhalo eqoshiwe engu-87150 yobude obugcwele yahlonzwa, lapho kwahlonzwa khona amanoveli angama-2081 kanye nama-isoform angamanoveli angama-62058 angama-spliced ahlukene.
I-2.1187 lncRNA kanye nezakhi zofuzo ezihlanganisiwe ezingama-356 zihlonziwe.
3.Kusuka ekukhuleni okuyisisekelo kuya ekukhuleni kwesibili, imibhalo eyi-15838 evezwe ngokwehlukana isuka kuzakhi zofuzo ezivezwe ngokwehlukana eziyi-995 ikhonjwe.Kuwo wonke ama-DEG, angu-1216 bekuyizici zokuloba, eziningi zazo ezingakabikwa.
Ukuhlaziywa kokunothisa kwe-4.GO kwembule ukubaluleka kokuhlukaniswa kwamaseli kanye nenqubo yokunciphisa i-oxidation ekukhuleni okuyisisekelo nokwesibili.
Imicimbi ye-splicing ehlukile kanye nama-isoform ahlukene
Ukuhlaziywa kwe-WGCNA kuzici zokuloba
Ireferensi
Chao Q, Gao ZF, Zhang D, et al.Amandla okuthuthuka kwe-Populus stem transcriptome.Isitshalo se-Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958
