Tatelano ya bolelele bo felletseng ba mRNA -PacBio
Melemo ea Tšebeletso
● Ho bala ka ho toba molek'hule ea cDNA ea bolelele bo feletseng ho tloha 3'- pheletsong ea 5'-
● Qeto ea boemo ba Iso-form ka sebopeho sa tatellano
● Lingoliloeng tse nepahetseng le tse tšepahalang
● E lumellana haholo le mefuta ea vaours
● Bokhoni bo boholo ba tatellano bo nang le sethala sa 4 PacBio Sequel II se nang le lisebelisoa.
● O na le boiphihlelo bo phahameng ka merero e fetang 700 ea tatellano ea RNA e thehiloeng ho Pacbio
● Ho fana ka sephetho se thehiloeng ho BMKCloud: Morafo o hlophisitsoeng oa data o fumanehang sethaleng.
● Litšebeletso tsa ka mor'a thekiso li sebetsa likhoeli tse 3 ha morero o phethoa
Litlhaloso tsa Tšebeletso
Sethala: PacBio Sequel II
Laeborari ea tatellano: Laeborari ea Poly A-enriched mRNA
Tlhahiso ea data e khothalelitsoeng: 20 Gb/sample (Ho ea ka mefuta)
FLNC (%): ≥75%
*FLNC: Lits'oants'o tse se nang chimeric tse bolelele bo felletseng
Litlhahlobo tsa Bioinformatics
● Ts'ebetso ea data e tala
● Boitsebiso ba sengoloa
● Sebopeho sa tatellano
● Tlhaloso ea Polelo
● Tlhaloso ea Mosebetsi
Litlhoko tsa Mohlala le Phano
Litlhoko tsa Mohlala:
Li-nucleotides:
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| ≥ 120 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Tšoaetso e fokolang kapa ha e na protheine kapa DNA e bontšitsoeng ho gel. | Bakeng sa limela: RIN≥7.5; Bakeng sa liphoofolo: RIN≥8.0; 5.0≥ 28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
Tila: Boima ba 'mele (o omileng):≥1 g
*Bakeng sa linama tse nyane ho feta 5 mg, re khothaletsa ho romela sampole ea lithane tse hatselitsoeng (ka naetrojene e metsi).
Ho emisoa ha lisele:Palo ea lisele = 3×106-1 × 107
* Re khothaletsa ho romella li-lysate tse leqhoa.Haeba sele eo e le nyane ho feta 5×105, ho khothaletsoa ho hatsetsa ka har'a naetrojene e metsi, e leng khetho e ntle bakeng sa ho ntšoa ha nyane.
Mehlala ea mali:Bophahamo ≥1 mL
Microorganism:Boima ≥ 1 g
Tlhahiso ea Mehlala e khothalelitsoeng
Setshelo:
2 ml ea centrifuge tube (ha e khothalletsoe ho etsa foil)
Mohlala oa ho ngola: Sehlopha+katiso mohlala A1, A2, A3;B1, B2, B3....
Thomello:
1. Leqhoa le omileng: lisampole li lokela ho kenngoa ka mekotleng ebe li patoa ka leqhoa le omileng.
2. Li-tubes tsa RNAstable: lisampole tsa RNA li ka omisoa ka har'a tube ea ho tsitsisa ea RNA (mohlala, RNAstable®) le ho romelloa ka mocheso oa kamore.
Phallo ea Mosebetsi oa Ts'ebeletso
Moqapi oa liteko
Mohlala oa thomello
Tlhahiso ea RNA
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
Kabo ea bolelele ba 1.FLNC
Bolelele ba bolelele bo felletseng ba non-chimeric read(FLNC) bo bonts'a bolelele ba cDNA kahong ea laeborari.Kabo ea bolelele ba FLNC ke sesupo sa bohlokoa ho lekola boleng ba kaho ea laeborari.
FLNC bala bolelele kabo
2.Ho tlatsa kabo ea bolelele ba sebaka sa ORF
Re sebelisa TransDecoder ho bolela esale pele libaka tsa likhoutu tsa protheine le tatelano ea amino acid ho hlahisa li-unigene, tse nang le tlhaiso-leseling e felletseng e sa hlokeng letho lisampoleng tsohle.
Tlatsa kabo ea bolelele ba sebaka sa ORF
3.KEGG tlhahlobo ea ntlafatso ea tsela
Lingoliloeng tse hlahisitsoeng ka mokhoa o fapaneng (DETs) li ka khetholloa ka ho hokahanya data ea tatellano ea RNA e thehiloeng ho NGS ho li-sete tsa bolelele bo felletseng tse hlahisoang ke data ea tatellano ea PacBio.Li-DET tsena li ka sebetsoa hape bakeng sa tlhahlobo e fapaneng ea ts'ebetso, mohlala, tlhahlobo ea ntlafatso ea tsela ea KEGG.
Mokhoa oa ho ntlafatsa tsela ea DET KEGG -Dot plot
Taba ea BMK
Matla a nts'etsopele ea Populus stem transcriptome
E hatisitsoe: Plant Biotechnology Journal, 2019
Leano la tatellano:
Mohlala oa pokello:libaka tsa stem: apex, internode ea pele(IN1), internode ea bobeli(IN2), internode ea boraro(IN3), internode(IN4) le internode(IN5) from Nanlin895
NGS-tatellano:RNA ea batho ba 15 e ile ea kopanngoa e le sampole e le 'ngoe ea baeloji.Liphetoho tse tharo tsa baeloji tsa lintlha ka 'ngoe li ile tsa sebetsoa bakeng sa tatelano ea NGS
TGS-tatellano:Libaka tsa stem li arotsoe ka libaka tse tharo, ke hore, apex, IN1-IN3 le IN4-IN5.Sebaka se seng le se seng se ile sa sebetsoa bakeng sa tatellano ea PacBio ka mefuta e mene ea lilaebrari: 0-1 kb, 1-2 kb, 2-3 kb le 3-10 kb.
Liphetho tsa bohlokoa
1. Kakaretso ea lingoloa tse bolelele bo felletseng tse 87150 li ile tsa hloauoa, moo ho ileng ha tsejoa li-isoform tse 2081 le li-isoform tse ling tse 62058 tsa lipale tse fapaneng.
2.1187 lncRNA le liphatsa tsa lefutso tse 356 tsa fusion li ile tsa khetholloa.
3. Ho tloha khōlong ea mantlha ho ea ho kholo ea bobeli, ho ile ha fumanoa litlaleho tse 15838 tse hlalositsoeng ka mokhoa o fapaneng ho tloha ho liphatsa tsa lefutso tsa 995 tse fapaneng.Ho li-DEG tsohle, 1216 e ne e le lintlha tse ngotsoeng, tseo boholo ba tsona li e-so tlalehoe.
Tlhahlobo ea ntlafatso ea 4.GO e senotse bohlokoa ba karohano ea lisele le ts'ebetso ea ho fokotsa oxidation kholong ea mantlha le ea bobeli.
Liketsahalo tse ling tsa splicing le li-isoform tse fapaneng
Tlhahlobo ea WGCNA mabapi le lintlha tsa mongolo
Referense
Chao Q, Gao ZF, Zhang D, et al.Matla a nts'etsopele ea Populus stem transcriptome.Semela sa Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958
