Isisombululo se-mRNA esipheleleyo se-PacBio 2+3
Iimbonakalo
● Uyilo lophando:
Isampuli edityanisiweyo ilandelelaniswe nePacBio ukuze kuchongwe ii-isoforms ze-transcript
Iisampulu ezahlulwe (iikopi kunye neemeko eziza kuvavanywa) ezilandelelaniswaI-NGS yokulinganisa ukubonakaliswa kwe-transcript
● Ulandelelwano lwePacBio kwimo yeCCS, luvelisa ukufundwa kweHiFi
● Ukulandelelana kweengxelo ezipheleleyo
● Uhlalutyo aludingi i-genome ebhekisayo; nangona kunjalo, lungasetyenziswa
● Uhlalutyo lwe-Bioinformatic alubandakanyi kuphela ukubonakaliswa kwinqanaba le-gene kunye ne-isoform kodwa lukwabandakanya nohlalutyo lwe-lncRNA, ukuhlanganiswa kwe-gene, i-poly-adenylation, kunye nesakhiwo se-gene
Iingenelo
● Ukuchaneka OkuphezuluI-HiFi ifundeka ngokuchanekileyo >99.9% (Q30), xa ithelekiswa ne-NGS
● Uhlalutyo olulolunye lokudibanisaUkulandelelanisa zonke iitranscript kwenza ukuba kuchongwe kwaye kuchazwe iimpawu ze-isoform.
● Ukudibanisa amandla ePacBio kunye ne-NGS: Ivumela ukulinganiswa kokubonakaliswa kwinqanaba le-isoform, ityhila utshintsho olunokuthi lufihlwe xa kuhlalutywa yonke imbonakalo yejini.
● Ubuchule Obubanzi: Sicubungule iisampulu ezingaphezu kwama-2300, iqela lethu lizisa amava amaninzi kwiprojekthi nganye.
● Inkxaso Emva kokuthengisa: ukuzibophelela kwethu kudlulela ngaphaya kokugqitywa kweprojekthi kunye nexesha lenkonzo emva kokuthengiswa kweenyanga ezi-3. Ngeli xesha, sinikezela ngokulandela iprojekthi, uncedo lokusombulula iingxaki, kunye neeseshoni ze-Q&A ukuphendula nayiphi na imibuzo enxulumene neziphumo.
Iimfuno zeSampuli kunye nokuHanjiswa
| Ithala leeNcwadi | Icebo lokulandelelanisa izinto | Idatha iyacetyiswa | Ulawulo lwemeko |
| Ithala leencwadi le-mRNA CCS elinobutyebi bePolyA | I-PacBio Sequel II Uhlaziyo lwePacBio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| I-Poly A etyebileyo | I-Illumina PE150 | 6-10 Gb | Q30≥85% |
Iinucleotides
|
| I-Conc.(ng/μl) | Ubungakanani (μg) | Ubunyulu | Ingqibelelo |
| Ithala leeNcwadi le-Illumina | ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ungcoliseko lweproteni okanye lwe-DNA oluncinci okanye alubonakali kwijeli. | Kwizityalo: I-RIN≥4.0; Kwizilwanyana: I-RIN≥4.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselweyo okanye okungekho siseko |
| Ithala leencwadi lePacBio | ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ungcoliseko lweproteni okanye lwe-DNA oluncinci okanye alubonakali kwijeli. | Izityalo: I-RIN≥7.5 Izilwanyana: I-RIN≥8.0 5.0≥28S/18S≥1.0; ukuphakama okulinganiselweyo okanye okungekho siseko |
Ukuhanjiswa kweSampuli okuCetyisiweyo
Isikhongozeli: Ityhubhu ye-centrifuge ye-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
Iilebheli zesampulu: Iqela + ikopi umz. A1, A2, A3; B1, B2, B3.
Ukuthunyelwa:
1. Umkhenkce owomileyo:Iisampulu kufuneka zipakishwe kwiingxowa zize zingcwatywe emkhenkceni owomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwityhubhu yokuzinzisa i-RNA (umz. i-RNAstable®) kwaye zithunyelwe kubushushu begumbi.
Kubandakanya uhlalutyo olulandelayo:
- Idatha ekrwada
- Ulawulo lwemeko
- Uhlalutyo lwe-Alternative Polyadenylation (APA)
- Uhlalutyo lwe-Fusion transcript
- Uhlalutyo oluHlanganisiweyo oluKhethekileyo
- Uhlalutyo lwe-BEnchmarking Universal Single-Copy Orthologs (BUSCO)
- Uhlalutyo lwe-transcript entsha: ukuqikelela ulandelelwano lwekhowudi (CDS) kunye nesichazi-magama esisebenzayo
- Uhlalutyo lwe-lncRNA: ukuqikelela i-lncRNA kunye neethagethi
- Ukuchonga iMicroSatelite (SSR)
- Uhlalutyo lweeTranscripts ezichazwe ngokwahlukileyo (ii-DET)
- Uhlalutyo lweeGene ezichazwe ngokwahlukileyo (ii-DEG)
- Isichazi-magama esisebenzayo se-DEGs kunye ne-DETs
Uhlalutyo lweBUSCO
Uhlalutyo oluHlanganisiweyo oluKhethekileyo
Uhlalutyo lwe-Alternative Polyadenylation (APA)
IiGene ezichazwe ngokwahlukileyo (ii-DEG) kunye neeTranscripts (ii-DETs9 anlaysis)
Iinethiwekhi zonxibelelwano phakathi kweeproteni neeproteni ze-DET kunye nee-DEG
Hlola uphuhliso oluququzelelwe yi-BMKGene's PacBio 2+3 full-length mRNA sequencing ngokusebenzisa ingqokelela yeempapasho ezikhethiweyo.
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ULiu, M. et al. (2023) 'Uhlalutyo lwe-PacBio Iso-Seq kunye ne-Illumina RNA-Seq oludibeneyo lwe-Tuta absoluta (Meyrick) Transcriptome kunye ne-Cytochrome P450 Genes',Izinambuzane, 14(4), iphe. 363. doi: 10.3390/INSETS14040363/S1.
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