I-Nanopore ye-mRNA yoBude obupheleleyo
Iimbonakalo
● Ukubanjwa kwe-poly-A mRNA kulandele ukwenziwa kwe-cDNA kunye nokulungiswa kwethala leencwadi
● Ukulandelelana kweengxelo ezipheleleyo
● Uhlalutyo lwe-Bioinformatic olusekelwe ekuhambelaneni ne-genome yesalathiso
● 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 zeNkonzo
●Ubungakanani bokubonakaliswa kwinqanaba le-isoform: ukuvumela uhlalutyo oluneenkcukacha noluchanekileyo lokubonakaliswa, ukutyhila utshintsho olunokuthi lufihlwe xa kuhlalutywa yonke imbonakalo yejini
●Iimfuno zeDatha ezincitshisiweyo:Xa kuthelekiswa ne-Next-Generation Sequencing (NGS), i-Nanopore sequencing ibonisa iimfuno zedatha eziphantsi, okuvumela amanqanaba alinganayo e-gene expression quantification saturation ngedatha encinci.
●Ukuchaneka okuphezulu kokulinganiswa kokubonakaliswa: zombini kwinqanaba le-gene kunye nele-isoform
●Ukuchongwa kolwazi olongezelelweyo lwe-transcriptomic: i-polyadenylation eyahlukileyo, ii-fusion genes kunye ne-lcnRNA kunye nee-genes zazo ekujoliswe kuzo
●Ubuchule ObubanziIqela lethu lizisa amava amaninzi kwiprojekthi nganye, emva kokuba ligqibe iiprojekthi ze-transcriptome ezingaphezulu kwama-850 zeNanopore kwaye licubungule iisampulu ezingaphezu kwama-8,000.
●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 |
| I-Poly A etyebileyo | I-Nanopore PromethION 48 | 6/12 Gb | Amanqaku omgangatho ophakathi: Q10 |
Iimfuno zeSampuli:
Iinukleotide:
| 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≥7.0; Kwizilwanyana: I-RIN≥7.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselweyo okanye okungekho siseko |
● 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
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 kumkhenkce owomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwityhubhu yokuzinzisa i-RNA (umz. i-RNAstable®) kwaye zithunyelwe kubushushu begumbi.
Ukuhamba komsebenzi wenkonzo
Iinukleotide:
Ukuhanjiswa kwesampulu
Ukwakhiwa kweThala leeNcwadi
Ukulandelelana
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengisa
Ukuhamba komsebenzi wenkonzo
Izicubu:
Uyilo lovavanyo
Ukuhanjiswa kwesampulu
Ukukhutshwa kwe-RNA
Ukwakhiwa kweThala leeNcwadi
Ukulandelelana
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengisa
● Ukucubungula idatha engaphekwanga
● Ukuchongwa kwengxelo ebhaliweyo
● Ukudibanisa okuchaseneyo
● Ukulinganiswa kokubonakaliswa kwinqanaba le-gene kunye nenqanaba le-isoform
● Uhlalutyo lokwahluka kwembonakalo
● Inkcazo yomsebenzi kunye nokuphucula (ii-DEG kunye nee-DET)
Uhlalutyo olunolunye lokudibanisa
Uhlalutyo lwe-Alternative Polyadenylation (APA)
Uqikelelo lwe-lncRNA
Inkcazelo yezakhi zofuzo ezintsha
Ukuhlanganiswa kwee-DET
IiNethiwekhi zeProteni-Protein kwi-DEGs
Hlola uphuhliso oluqhutywa ziinkonzo zokulandelelana kwe-mRNA ze-BMKGene zeNanopore ezipheleleyo ngokusebenzisa ingqokelela yeempapasho ezikhethiweyo.
UGong, B. et al. (2023) 'Ukusebenza kwe-epigenetic kunye ne-transcriptional ye-secretory kinase FAM20C njenge-oncogene kwi-glioma', i-Journal of Genetics and Genomics, 50(6), iphe. 422–433. doi: 10.1016/J.JGG.2023.01.008.
He, Z. et al. (2023) 'Ulandelelwano olupheleleyo lwe-transcriptome lwee-lymphocytes ezisabela kwi-IFN-γ lubonisa impendulo yomzimba egobekileyo ye-Th1 kwi-flounder (Paralichthys olivaceus)', Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.
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.
Yu, D. et al. (2023) 'Uhlalutyo lwe-nano-seq lubonisa ukuthambekela okuhlukeneyo kokusebenza phakathi kwee-exosomes kunye nee-microvesicles ezivela kwi-hUMSC', iStem Cell Research and Therapy, 14(1), iphe. 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.
