Bolelele bo Feletseng ba mRNA Sequencing-Nanopore
Melemo ea Tšebeletso
● Leeme le tlaase la tatelano
● Ho senola limolek'hule tsa cDNA tse bolelele bo feletseng
● Lintlha tse fokolang tse hlokahalang ho koahela palo e tšoanang ea litlaleho
● Ho khetholla li-isoform tse ngata ka jini
● Tlhaloso ea polelo boemong ba isoform
Litlhaloso tsa Tšebeletso
| Laebrari | Sethala | Tlhahiso ea data e khothalelitsoeng (Gb) | Taolo ea Boleng |
| cDNA-PCR(Poly-A enriched) | Nanopore PromethION P48 | 6 Gb / sampole (Ho ipapisitse le mefuta) | Karolelano ea bolelele bo felletseng ~70% Karolelano ea lintlha tsa boleng: Q10
|
Litlhahlobo tsa Bioinformatics
●Ts'ebetso ea data e tala
● Boitsebiso ba sengoloa
● Mokhoa o mong oa ho kopanya
● Tlhaloso ea polelo boemong ba liphatsa tsa lefutso le boemo ba isoform
● Tlhahlobo e fapaneng ea polelo
● Tlhaloso ea tšebetso le ntlafatso (DEGs le DETs)
Litlhoko tsa Mohlala le Phano
Litlhoko tsa Mohlala:
Li-nucleotides:
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| ≥ 100 | ≥ 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≥7.0; Bakeng sa liphoofolo: RIN≥7.5; 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
Sesepa: 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
Tlhahiso ea Mehlala e khothalelitsoeng
Setshelo: 2 ml centrifuge tube (Tin foil ha e khothalletsoe)
Mohlala oa ho ngola: Sehlopha+katiso mohlala A1, A2, A3;B1, B2, B3....
Thomello: 2, Dry-ice: lisampole li hloka ho kenngoa ka mekotleng ebe li patoa ka leqhoa le omileng.
- Li-tubes tsa RNAstable: lisampole tsa RNA li ka omisoa ka har'a li-tube tsa RNA stabilization (mohlala, RNAstable®) ebe li romelloa ka mocheso oa kamore.
Phallo ea Mosebetsi oa Ts'ebeletso
Li-nucleotides:
Mohlala oa thomello
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
Phallo ea Mosebetsi oa Ts'ebeletso
Tishu:
Moqapi oa liteko
Mohlala oa thomello
Tlhahiso ea RNA
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
1.Tlhaloso e fapaneng ea polelo - morero oa Volcano
Tlhahlobo e fapaneng ea polelo e ka sebetsoa maemong ka bobeli a liphatsa tsa lefutso ho khetholla liphatsa tsa lefutso tse hlahisitsoeng ka mokhoa o fapaneng (DEGs) le maemong a isoform ho khetholla ka mokhoa o fapaneng.
lingoliloeng tse hlahisitsoeng (DETs)
2.'Mapa oa mocheso oa sehlopha sa maemo a holimo
3.Alternative splicing identification and classification
Mefuta e mehlano ea liketsahalo tse ling tsa splicing e ka boleloa esale pele ke Astalavista.
4.Khethollo ea liketsahalo tsa Alternative poly-adenylation (APA) le Motif ho 50 bp holimo ho poly-A
Taba ea BMK
Tlhahiso e 'ngoe ea splicing le quantification ea boemo ba isoform ka tatellano ea transcriptome ea bolelele bo felletseng ba nanopore.
E hatisitsoe:Lipuisano tsa Tlhaho,2020
Leano la tatellano:
Lihlopha: 1. CLL-SF3B1(WT);2. CLL-SF3B1(K700E phetoho);3. Lisele tsa B tse tloaelehileng
Leano la tatellano: tatellano ea laeborari ea MinION 2D, tatellano ea laeborari ea PromethION 1D;lintlha tse khutšoane tse baloang ho tsoa lisampoleng tse tšoanang
Sethala sa tatellano: Nanopore MinION;Nanopore PromethION;
Liphetho tsa bohlokoa
1.Isoform-level Alternative Splicing Identification
Tatelano e balwang nako e telele e matlafatsa boitsebahatso ba SF3B1 e fetotswengK700E-altered splice sites at isoform-level.35'SSs tse ling tse 35 le tse 10 tse ling tsa 5'SS li fumanoe li arotsoe ka mokhoa o fapaneng haholo lipakeng tsa SF3B1.K700Ele SF3B1WT.Liphetoho tse 33 ho tse 35 li sa tsoa sibolloa ka tatellano e baloang nako e telele.
2.Isoform-level Alternative Splicing quantification
Polelo ea li-isoform tsa intron retention(IR) ho SF3B1K700Ele SF3B1WTli ne li lekantsoe ho ipapisitsoe le tatellano ea nanopore, e senolang taolo e tlase ea lefats'e ea li-isoform tsa IR ho SF3B1.K700E.
Referense
Tang AD , Soulette CM , Baren MJV , et al.Tlhaloso ea bolelele bo felletseng ea phetoho ea SF3B1 ho chronic lymphocytic leukemia e senola ho theoha ha li-introns tse bolokiloeng[J].Tlhaho Puisano.
