I-TRANSCRIPTOMICS
indalo
UNXIBELELWANO
Ukuchazwa kobude obupheleleyo bokuguqulwa kwe-SF3B1 kwi-lymphocytic leukemia engapheliyo ibonisa ukuthotywa kwe-introns ezigciniweyo.
Ushicilelo olupheleleyo | Ulandelelwano lweNanopore| Uhlalutyo olulolunye lwe-isoform
Imvelaphi
SUkuguqulwa kwe-omatic kwi-splicing factor SF3B1 iye yaxelwa ngokubanzi ukuba idibanise neentlobo zomhlaza, kubandakanywa i-lymphocytic leukemia engapheliyo (CLL), i-melanoma ye-uveal, umhlaza wamabele, njl. Ukongeza, izifundo ezimfutshane ezifundwayo zibonise iipateni zokuqhawula ngokungafaniyo ezibangelwa ukuguqulwa kweSF3B1. Nangona kunjalo, amaphononongo kwezi patheni zokwahlula-hlula kudala anqunyelwe kwinqanaba lomnyhadala kunye nokunqongophala kolwazi kumgangatho we-isoform ngenxa yomda wokukhutshelwa okufutshane okufundwayo. Apha, i-platform yokulandelelanisa i-nanopore yaziswa ukuvelisa imibhalo yobude obugcweleyo, exhobisa ukuguqulwa kwamandla kwi-AS isoforms.
Uyilo loMfuniselo
Iimvavanyo
Ukwahlulahlula:1. CLL-SF3B1 (WT) 2. CLL-SF3B1 (K700E ukuguqulwa); 3. Iiseli ze-B eziqhelekileyo
Iqhinga lokulandelelanisa:Ulandelelwano lwethala leencwadi eliMncinci le-2D, ulandelelwano lwethala leencwadi le-PromethION 1D; idatha yokufunda emfutshane kwiisampuli ezifanayo
Iqonga lolandelelwano:I-ONT MiniION; I-ONT PromethION;
Uhlalutyo lwe-Bioinformatic
Iziphumo
AItotali yezigidi ezingama-257 ezifundwe zaveliswa kwiisampulu ze-CLL ezi-6 kunye neeseli ze-B ezi-3. Kwi-avareji ye-30.5% yolu fundo luchongwe njengemibhalo ebhalwe ngobude obupheleleyo.
FUhlalutyo olupheleleyo lwe-isoform ye-RNA (FLAIR) yaphuhliswa ukuvelisa i-isoforms yokuzithemba okuphezulu. I-FLAIR inokushwankathelwa ngolu hlobo:
Ni-anopore ifunda ulungelelwaniso: chonga ubume bombhalo jikelele obusekelwe kwi-genome yereferensi;
Sisilungiso se-plice junction: iimpazamo ezichanekileyo zolandelelwano (ezibomvu) kunye nesiza se-splice ukusuka kwii-introns ezine-annotated, ii-introns ezivela kwidatha efundwayo emfutshane okanye zombini;
Ci-ollapse: shwankathela i-isoforms emele ngokusekwe kumatyathanga okuhlangana kwe-splice (iseti yokupasa yokuqala). Khetha i-isofrom yokuzithemba okuphezulu ngokusekelwe kwinani lokufundwa kwenkxaso (I-Threshold: 3).
Umzobo 1. Uhlalutyo lwe-FLAIR ukuchonga i-isoforms epheleleyo ehambelana nokuguqulwa kwe-SF3B1 kwi-CLL
FI-LAIR Ichonge i-326,699 ephezulu yokuzithemba i-isoforms edibeneyo, i-90% yayo i-isoforms entsha. Uninzi lwezi isoforms zingachazwanga zifunyenwe ziyindibaniselwano yenoveli ye-splice juntions (142,971), ngelixa ezinye ii-isoforms zenoveli ziqulethwe nokuba yi-intron egciniweyo (21,700) okanye inoveli exon (3594).
LUlandelelwano lokufunda ngokuqhubekayo luxhobisa ukuchongwa kwe-SF3B1-K700E eguqulweyo ye-splice sites kwinqanaba le-isoform. Ngama-35 ezinye ii-3'SS kunye ne-10 ezizezinye ezi-5'SS zifunyenwe zicandeke kakhulu ngokwahlukileyo phakathi kwe-SF3B1-K700E kunye ne-SF3B1-WT. I-33 kwezingama-35 ezitshintshiweyo zisanda kufunyanwa ngolandelelwano lokufunda ixesha elide. Kwidatha ye-Nanopore, ukuhanjiswa komgama phakathi kwe-SF3B1-K700E-eguqulelwe i-3'SS kwiindawo eziphakamileyo ze-canonical zijikeleze -20 bp, ehluke kakhulu kwi-distribution yolawulo, efana nento echazwe kwi-CLL yokulandelelana okufutshane. I-Isoforms ye-ERGIC3 gene yahlaziywa, apho i-isoform yenoveli equlethe i-proximal splice site yafunyanwa ininzi kakhulu kwi-SF3B1-K700E. Zombini ezikude kunye ne-3'SS ezikude ziye zadityaniswa neepateni ze-AS ezahlukileyo ezivelisa ii-isoform ezininzi.
Umfanekiso 2. Enye indlela ye-3′ yokuhlanganisa iipateni ezichongiweyo kunye nedatha yokulandelelana kwe-nanopore
Uhlalutyo lokusetyenziswa kwesiganeko se-IR lunexesha elide lulinganiselwe kuhlalutyo olusekelwe kufundo olufutshane ngenxa yokuzithemba ekuchongweni kwe-IR kunye nobungakanani. Ukubonakaliswa kwe-IR isoforms kwi-SF3B1-K700E kunye ne-SF3B1-WT zaye zabalwa ngokusekelwe kulandelelwano lwe-nanopore, ebonisa ukulawulwa kwehlabathi jikelele kwe-IR isoforms kwi-SF3B1-K700E.
Umfanekiso 4. Ukuqina kwezolimo kunye noqhagamshelwano lothungelwano kwiinkqubo ezintathu zokufama (A no-B); Uhlalutyo lwehlathi olungenamkhethe(C) kunye nobudlelwane phakathi kokuqina kwezolimo kunye ne-AMF colonization (D)
Umzobo 3. Iziganeko zokugcinwa kwe-Intron ziphantsi kakhulu kwi-CLL SF3B1-K700E
Iteknoloji
I-Nanopore Ulandelelwano lokuFunda ixesha elide
NUlandelelwano lwe-anopore yi-molecule ye-real-time yombane yokulandelelanisa itekhnoloji.
DI-DNA ene-ouble-stranded okanye i-RNA iya kubophelela kwiprotheyini ye-nanoporous efakwe kwi-biofilm kunye nokukhulula phantsi kwesikhokelo se-motor protein.
DIintambo ze-NA/RNA zidlula kwiprotheyini yesitishi se-nanopore ngesantya esithile phantsi kwesenzo somohluko wombane.
Miiolecules zivelisa imiqondiso eyahlukeneyo yombane ngokwesakhiwo sekhemikhali.
Rukufumanisa ixesha lexesha lolandelelwano kufezekiswa ngokufowuna okusisiseko.
Ukusebenza kolandelelwano olupheleleyo lwe-transcriptome
√ Ugcino lwedatha
Ufundo oluphindaphindwayo olu-7 olufunekayo ukufikelela kwidatha enokuthelekiseka.
√ Ukuchongwa koLwakhiwo olushicilelweyo
Ukuchongwa kweentlobo ngeentlobo ezahlukeneyo zesakhiwo kunye nemvumelwano yobude obugcweleyo bokufundwa kombhalo ngamnye
√ Uhlalutyo lweyantlukwano kwinqanaba lokubhaliweyo -Diza utshintsho olufihlwe kufundo olufutshane
Isalathiso
Tang AD , Soulette CM , Baren MJV , et al. Ubude obupheleleyo be-transcript characterization ye-SF3B1 utshintsho kwi-lymphocytic leukemia engapheliyo ibonisa ukuthotywa kwe-introns ezigciniweyo[J]. Unxibelelwano lweNdalo.
ITekhnoloji kunye neeNgcaciso ijolise ekwabelaneni ngeyona ndlela iphumeleleyo yamvanje yosetyenziso lweetekhnoloji zolandelelwano oluphezulu kwiindawo ezahlukeneyo zophando kunye neengcinga ezikrelekrele kuyilo lovavanyo kunye nokwembiwa kwedatha.
Ixesha lokuposa: Jan-08-2022