Ulandelelwano olupheleleyo lwe-mRNA -PacBio
Iimbonakalo
● ukwenziwa kwe-cDNA kwi-poly-A mRNA kulandele ukulungiswa kwethala leencwadi
● Ukulandelelanisa kwimo ye-CCS, kuvelisa ukufundwa kwe-HiFi
● Ukulandelelana kweengxelo ezipheleleyo
● Uhlalutyo aludingi i-genome ebhekisayo; nangona kunjalo, lungasetyenziswa
● Uhlalutyo lwe-Bioinformatic lwenza ukuba kuhlalutywe ii-transcripts ze-isoform lncRNA, i-gene fusions, i-poly-adenylation, kunye nesakhiwo se-gene
Iingenelo zeNkonzo
●Ukuchaneka OkuphezuluI-HiFi ifundeka ngokuchanekileyo >99.9% (Q30), xa ithelekiswa ne-NGS
● Uhlalutyo olulolunye lokudibanisaUkulandelelana kwee-transcripts zonke kwenza ukuba kuchongwe kwaye kuchazwe iimpawu ze-isoform
●Ubuchule Obubanzi: ngerekhodi lokugqiba iiprojekthi ze-transcriptome zePacBio ezingaphezulu kwe-1100 kunye nokucubungula iisampuli ezingaphezu kwama-2300, iqela lethu lizisa amava amaninzi kwiprojekthi nganye.
●Inkxaso Emva koThengiso: 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% |
Iimfuno zeSampuli:
Iinukleotide:
● Izityalo:
Ingcambu, Isiqu okanye iPetal: 450 mg
Igqabi okanye Imbewu: 300 mg
Isiqhamo: 1.2 g
● Isilwanyana:
Intliziyo okanye Amathumbu: 300 mg
I-Viscera okanye ubuchopho: 240 mg
Izihlunu: 450 mg
Amathambo, Iinwele okanye Ulusu: 1g
● Ii-arthropod:
Izinambuzane: 6g
I-Crustacea: 300 mg
● Igazi elipheleleyo: Ityhubhu e-1
● Iiseli: 106 iiseli
| I-Conc.(ng/μl) | Ubungakanani (μg) | Ubunyulu | Ingqibelelo |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ungcoliseko lweproteni okanye lwe-DNA oluncinci okanye alubonakali kwijeli. | Kwizityalo: I-RIN≥7.5; Kwizilwanyana: 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 (ayikhuthazwa 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 kumkhenkce owomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwityhubhu yokuzinzisa i-RNA (umz. i-RNAstable®) kwaye zithunyelwe kubushushu begumbi.
Ukuhamba komsebenzi wenkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampulu
Ukukhutshwa kwe-RNA
Ukwakhiwa kweThala leeNcwadi
Ukulandelelana
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengisa
Kubandakanya uhlalutyo olulandelayo:
● Ulawulo lomgangatho wedatha eluhlaza
● Uhlalutyo lwe-Alternative Polyadenylation (APA)
● Uhlalutyo lwe-Fusion transcript
● Uhlalutyo olulolunye lokudibanisa
● Uhlalutyo lwe-Universal Single-Copy Orthologs (BUSCO) olubonisa ukulinganisela
● Uhlalutyo lwemibhalo ebhaliweyo entsha: ukuqikelela ulandelelwano lwekhowudi (ii-CDS) kunye nesichazi-magama esisebenzayo
● Uhlalutyo lwe-lncRNA: ukuqikelela i-lncRNA kunye neethagethi
● Ukuchonga iMicroSatelite (SSR)
Uhlalutyo lweBUSCO
Uhlalutyo oluHlanganisiweyo oluKhethekileyo
Uhlalutyo lwe-Alternative Polyadenylation (APA)
Isichazi-magama esisebenzayo seengxelo zenoveli
Hlola uphuhliso oluququzelelwe ziinkonzo ze-BMKGene's Nanopore full-length mRNA sequencing kule ncwadi iphambili.
UMa, Y. et al. (2023) 'Uhlalutyo oluthelekisayo lweendlela zokulandelelana kwePacBio kunye ne-ONT RNA zokuchonga ubuthi beNemopilema Nomurai', iGenomics, 115(6), iphe. 110709. doi: 10.1016/J.YGENO.2023.110709.
UChao, Q. et al. (2019) 'Iindlela zophuhliso ze-Populus stem transcriptome', i-Plant Biotechnology Journal, 17(1), iphe. 206–219. doi: 10.1111/PBI.12958.
UDeng, H. et al. (2022) 'Utshintsho Olunamandla Kwi-Ascorbic Acid Content Ngexesha Lokuphuhliswa Kweziqhamo Nokuvuthwa Kwe-Actinidia latifolia (Isityalo Seziqhamo Esityebileyo Se-Ascorbate) kunye Neendlela Ezidibeneyo ZeMolecular', Ijenali Yezizwe Ngezizwe YeeSayensi ZeMolecular, 23(10), iphe. 5808. doi: 10.3390/IJMS23105808/S1.
UHua, X. et al. (2022) 'Ukuqikelela okusebenzayo kwezakhi zofuzo zendlela yokwenza izinto eziphilayo ezibandakanyeka kwi-polyphyllins esebenzayo kwi-polyphylla yaseParis', i-Communications Biology 2022 5:1, 5(1), iphe. 1–10. doi: 10.1038/s42003-022-03000-z.
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/INSECTS14040363/S1.
UWang, uLijun et al. (2019) 'Uphando lobunzima be-transcriptome kusetyenziswa uhlalutyo lwexesha langempela lwe-PacBio single-molecule oludityaniswe ne-Illumina RNA sequencing ukuze kuqondwe ngcono i-ricinoleic acid biosynthesis kwiRicinus communis', BMC Genomics, 20(1), iphe. 1–17. doi: 10.1186/S12864-019-5832-9.
