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I-Epigenetics Ngesikhathi Sangempela: Ukuvula Ukuguqulwa kwe-RNA nge-Nanopore Direct Sequencing

Ukuze siqonde ngokugcwele ukusebenza kwamaseli, intuthuko, kanye nokuqhubeka kwesifo, kumele sibheke ngale kochungechunge lwe-DNA uqobo futhi sihlole izendlalelo ezengeziwe zolwazi lwezinto eziphilayo ezilawula umsebenzi wezakhi zofuzo: i-epigenome kanye ne-epitranscriptome.

Ngokwesiko, ukuthola ukuguqulwa kwe-epigenetic kanye ne-epitranscriptomic, njenge-5-methylcytosine (5mC), i-5-hydroxymethylcytosine (5hmC), noma i-N6-methyladenosine (m6A), kudinge ukwelashwa ngamakhemikhali, njengokuguqulwa kwe-bisulfite, noma izindlela zokucebisa ezisekelwe kuma-antibody. Lezi zindlela zinganciphisa amasampula, zingenise ukuthambekela, zingabi nesisombululo esiqondile sama-molecule owodwa, noma zilahlekelwe umongo obalulekile wokulandelana kwebanga elide.

Ngokufunda ngqo ama-molecule e-DNA ne-RNA angakhuliswanga, i-Oxford Nanopore Technologies (ONT) ishintshe indlela abacwaningi abathola futhi bahlaziye ngayo izinguquko ze-nucleotide ngesikhathi sangempela.

Umfanekiso 1.Ukulandelana kwe-Nanopore okuqondile kokuthola ukuguqulwa kwe-epigenetic kanye ne-epitranscriptomic.

Izicelo Eziyinhloko Zokulandelana Kwe-Nanopore Epigenetic

Ikhono lokulandelanisa ama-molecule amade e-nucleic acid angokwemvelo livule amathuba amasha ocwaningo lwezinto eziphilayo.

1. Ukutholwa Kwezigaba Ezingu-5mC kanye No-5hmC Ku-Cancer Genomics

Ama-genome omdlavuza avame ukubonakala nge-hypomethylation yomhlaba wonke kanye ne-hypermethylation yama-gene athile okucindezela isimila. Ngenxa yokuthi ukulandelana kwe-Nanopore kungakhiqiza ukufundwa kwe-DNA okude kakhulu okusukela kumashumi kuya kumakhulu ama-kilobase, kungenzeka ukumaka ukuguqulwa kwe-5mC kanye ne-5hmC kuzo zonke izinto eziphindaphindayo, ama-promoter, kanye nama-enhancer.

Ngaphezu kwalokho, ukufunda okude kuvumela ukuhlukaniswa kwe-haplotype, okusiza ekunqumeni ukuthi iyiphi i-allele yomzali ephethe izinguquko ezithile ze-epigenetic.

2. Ukulandelana kwe-RNA okuqondile (i-dRNA-seq) kanye ne-Epitranscriptome

I-mRNA iqukethe izinguquko ezingaphezu kwekhulu ezahlukene ngemuva kokubhalwa, kanti i-m6A iphakathi kwezinhlobo ezivame kakhulu. Ukulandelana kwe-RNA yendabuko kuguqula i-RNA ibe yi-cDNA, inqubo esusa izinguquko ze-RNA zomdabu.

I-Nanopore Direct RNA Sequencing idlula ngokuphelele ukwakheka kwe-cDNA, okuvumela ukuhlaziywa kokuguqulwa kwe-RNA, ubude bomsila we-poly(A), kanye nemicimbi yokuhlanganisa ehlukile ku-molecule efanayo ye-RNA.

Ipayipi le-Bioinformatics: Isofthiwe Yokuhlaziywa kwe-Epigenetic

Ukuhumusha izimpawu zamanje ze-ionic ezingavuthiwe zibe izingcingo eziyisisekelo kanye nezimo zokuguqulwa kudinga amathuluzi ekhompyutha ayinkimbinkimbi.

Ukubiza Ngesisekelo Nokucubungula Kokuqala
I-Dorado / Guppy: Yakhiwe yi-Oxford Nanopore, lawa ngamathuluzi ajwayelekile okubiza ama-basecalling. Izinguqulo zamanje zisebenzisa amanethiwekhi ajulile e-neural, kufaka phakathi ama-RNN namamodeli asekelwe ku-Transformer, aqeqeshwe kumasethi edatha ama-control nucleic acid aguquliwe nangakaguquki ukuze enze ukubiza ama-basecalling futhi athole izinguquko ezivamile, njenge-5mC, 5hmC, kanye ne-m6A.

Abafonayo be-Methylation Abazinikele

Ukuze kutholakale okunembile kakhulu ngale kwemiphumela ejwayelekile yokubiza ama-basecalling, kusetshenziswa amathuluzi amaningana omthombo ovulekile kabanzi:

I-Megapodal / Nanopolish: Lawa mathuluzi aqhathanisa izimpawu zamanje ze-ionic ezingavuthiwe ezibonwe ngokumelene ne-genome yokubhekisela ukuze kutholakale izimpawu ze-methylation ye-DNA, okuhlanganisa nokuguqulwa kwe-5mC.
I-DeepSignal / i-DeepMod: Lawa mathuluzi asebenzisa amamodeli okufunda okujulile, njengamanethiwekhi e-Bi-LSTM noma amanethiwekhi e-convolutional neural, ukukhipha izici ezivela kumasignali angavuthiwe nokubikezela izimo zokuguqulwa ekuxazululweni kwe-nucleotide eyodwa.

Isofthiwe Yokuguqulwa Kwe-RNA Eqondile
m6Anet: Ithuluzi lokufunda okujulile elikhethekile elenzelwe ukuthola izinguquko ze-m6A kusuka kudatha ye-Nanopore Direct RNA Sequencing.
I-EpiTools / i-Nanocompore: Lawa maphakheji asekela ukuhlaziywa okuqhathanisayo kwezimpawu zamanje ze-RNA eziqondile phakathi kwezimo ezahlukene zebhayoloji, njengezibonelo ze-wild-type vs knockdown, ukuhlonza izindawo zokuguqulwa kwe-RNA ezihlukile.

Isiphetho

Ukulandelana okuqondile kwe-nanopore kushintshe kakhulu ucwaningo lwe-epigenetics kanye ne-epitranscriptomics. Ngokunciphisa ukubandlulula kanye nomonakalo wesampula ohlotshaniswa nokuguqulwa kwamakhemikhali, ukubhalwa kabusha, kanye nokwandiswa kwe-PCR, kunikeza umbono oqondile kakhulu wama-molecule e-DNA kanye ne-RNA endabuko.

Ingabe uhlela iphrojekthi ye-epigenomics noma ye-transcriptomics ezayo? Xhumana nethimba lethu kwa-BMKGENE ukuze ufunde ukuthi izinsizakalo zethu zokulandelana ngqo ze-ONT zingasheshisa kanjani ukutholakala kwakho kwezinto eziphilayo.

Izinkomba

Leger, A., Amaral, PP, Pandolfini, L., Capitanchik, C., Capraro, F., Miano, V., Migliori, V., Toolan-Kerr, P., Sideri, T., Enright, AJ, Tzelepis, K., van Werven, FJ, Luscombe, NM., Fitra, Fitra, Barbi I-Birney, E., Leonardi, T., & Kouzarides, T. RNA ilungisa ukutholwa ngokuqhathanisa ngokulandelana kwe-RNA eqondile ye-Nanopore.Ukuxhumana Kwemvelo 12, 7198 (2021). doi:10.1038/s41467-021-27393-3

UWu, Z., uLi, J., uXia, R., uDai, J., uSu, J., uMeng, J., kanye noZhang, Y. Ukulandelana kwe-RNA okuqondile kwe-Nanopore kokuhlaziywa kokuguqulwa kwe-RNA: ukuhlolwa komsebenzi kanye nokulinganisa ithuluzi lokubala.I-Biotechnology Ethuthukisiwe 4, 9 (2026). doi:10.1007/s44307-025-00093-5