PacBio 2+3 Tharollo ea Bolelele bo Feletseng ba mRNA
Likaroloana
● Moralo oa ho ithuta:
Mohlala o kopantsoeng o hlophisitsoeng le PacBio ho khetholla li-isoform tsa sengoloa
Arohaneng disampole (replicates le maemo a lokela ho lekoa) tatellano leNGS ho lekanya polelo ea sengoloa
● Sequencing ea PacBio ka mokhoa oa CCS, e hlahisang HiFi e baloa
● Tatellano ea likopi tse bolelele bo felletseng
● Tlhahlobo ha e hloke genome ea boitsebiso; leha ho le joalo, e ka sebelisoa
● Tlhahlobo ea bioinformatiki ha e akarelletse feela polelo ho liphatsa tsa lefutso le boemo ba isoform empa hape le tlhahlobo ea lncRNA, ho kopanngoa ha liphatsa tsa lefutso, poly-adenylation, le gene structure.
Melemo
● Ho Nepaha Ha Haholo: HiFi e bala ka nepo>99.9% (Q30), ha e bapisoa le NGS
● Tlhahlobo e 'ngoe ea ho Splicing: tatellano ea lingoliloeng tsohle e thusa ho tsebahatsa le ho khetholla li-isoform.
● Motsoako oa Matla a PacBio le NGS: e nolofalletsang palo ea polelo boemong ba isoform, ho senola phetoho e ka 'nang ea patoa ha ho hlahlojoa polelo eohle ea lefutso.
● Tsebo e Pharalletseng: ka rekoto ea ho phethela merero e fetang 1100 ea PacBio ea bolelele bo felletseng le ho sebetsana le lisampole tse fetang 2300, sehlopha sa rona se tlisa boiphihlelo bo bongata morerong o mong le o mong.
● Tšehetso ea Post-Sales: Boitlamo ba rona bo fetela ka nģ'ane ho ho phethoa ha morero ka nako ea tšebeletso ea likhoeli tse 3 ka mor'a thekiso. Nakong ena, re fana ka tatellano ea projeke, thuso ea ho rarolla mathata, le linako tsa Q&A ho araba lipotso life kapa life tse amanang le sephetho.
Litlhoko tsa Mohlala le Phano
| Laebrari | Leano la tatellano | Lintlha li khothalelitsoe | Taolo ea Boleng |
| PolyA e matlafalitse laeborari ea mRNA CCS | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| Poly A e atlehile | Illumina PE150 | 6-10 Gb | Q30≥85% |
Li-nucleotides
|
| Conc.(ng/μl) | Chelete (μg) | Bohloeki | Botšepehi |
| Laebrari ea Illumina | ≥10 | ≥ 0.2 | 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≥4.0; Bakeng sa liphoofolo: RIN≥4.5; 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
| Laebrari ea PacBio | ≥ 100 | ≥ 1.0 | 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. | Limela: RIN≥7.5 Liphoofolo: RIN≥8.0 5.0≥28S/18S≥1.0; bophahamo bo fokolang kapa bo se nang motheo |
Tlhahiso ea Mohlala 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:
1. Leqhoa le omileng:Mehlala e lokela ho kenngoa ka mekotleng ebe e 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.
E kenyelletsa tlhahlobo e latelang:
Taolo ea boleng ba data e tala
Alternative Polyadenylation Analysis (APA)
Tlhahlobo ea sengoloa sa Fusion
Alternative Splicing Analysis
Tlhahlobo ea Benchmarking Universal Single-Copy Orthologs (BUSCO).
Tlhahlobo ea sengoloa sa Novel: ponelopele ea tatellano ea likhoutu (CDS) le tlhaloso e sebetsang
Tlhahlobo ea lncRNA: ho bolela esale pele ka lncRNA le lipheo
MicroSatelite Identification (SSR)
Tlhahlobo ea Lingoloa tse Hlalositsoeng ka Phapang (DETs).
Tlhahlobo ea liphatsa tsa lefutso tse fapaneng (DEGs).
Tlhaloso e sebetsang ea li-DEG le li-DET
Tlhahlobo ea BUSCO
Alternative Splicing Analysis
Alternative Polyadenylation Analysis (APA)
Liphatsa tsa lefutso tse hlahisitsoeng ka mokhoa o fapaneng (DEGs) le lingoloa (DETs9 anlaysis
Marang-rang a tšebelisano ea protheine ea li-DET le li-DEG
Lekola tsoelo-pele e tsamaisoang ke BMKGene's PacBio 2+3 mRNA ea bolelele bo felletseng e tsamaisanang le pokello e hlophisitsoeng ea lingoliloeng.
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