I-GENOMIES YABANTU
izakhi zofuzo
Ukulandelana okufundwe isikhathi eside kukhomba ukunwetshwa okuphindayo kwe-GGC ku-NOTCH2NLC okuhlobene nesifo sokufakwa kwe-neuronal intranuclear
ONT requencing |Ilumina |Ukulandelana okuphelele kwe-exome |Ukulandelana okuhlosiwe kwe-CRISPR-Cas9 ONT |I-RNA-seq |Ukushaya kwe-ONT 5mC methylation
Amaphuzu avelele
1.Ngokuhlaziywa kokuXhumanisa emndenini omkhulu we-NIID, kuhlonzwe izifunda ezimbili ezixhumene.
Ukulandelana okusekelwe ku-2.ONT okusekelwe isikhathi eside okufundwe kanye nokulandelanisa kwe-Cas-9 kokuchumanisa kwe-ONT kuthole imbangela yofuzo engaba khona ye-NIID, i-GGC iphinda ukunwetshwa ku-5′ UTR ye-NOTCH2NLC.Lolu cwaningo lubike ukunwetshwa okuphindayo ezakhini zofuzo eziqondene nomuntu okokuqala ngqa ezivele ngokuphindaphinda kwezigaba.
Ukulandelana kwe-3.RNA kwembule okulotshiwe kwe-antisense okungajwayelekile ekuqaleni noma ngaphakathi kwe-GGC phinda izifunda zokunweba ku-NOTCH2NLC.
Ingemuva
NI-euronal intranuclear inclusion disease (NIID) yisifo se-neurodegenerative esiqhubekayo futhi esibulalayo, esibonakala ngokuba khona kwe-eosinophilic hyaline intranuclear inclusions ezimisweni zezinzwa eziphakathi nendawo.Ukubonakaliswa kwayo okuguquguqukayo kwemitholampilo kuphakamisa ubunzima obukhulu ekuxilongweni kuze kube yilapho kwethulwa i-biopsy yesikhumba.Kodwa-ke, izindlela ezisekelwe ku-histopathology zisahlushwa ukungaxilongwa kahle, okudinga ukuqonda kofuzo lwe-NIID.
Izimpumelelo
Ukuhlaziya Ukuxhumana
Si-hort-read sequencing based whole genome sequencing (WGS) kanye ne-whole exome sequencing (WES) yenziwa kumndeni omkhulu we-NIID (amalungu ayi-13 athintekile namalungu ayi-7 angathintekile).Ukuhlaziywa kokuxhumanisa kuma-SNP akhishwe kule datha kwembule izifunda ezimbili kuphela ezixhunyiwe: isifunda esingu-3.5 Mb ku-1p36.31-p36.22 (isilinganiso esiphezulu se-LOD=2.32) kanye nesifunda esingu-58.1 Mb ku-1p22.1-q21.3 (ubukhulu be-LOD: 4.21 ).Kodwa-ke, awekho ama-SNP noma ama-CNV e-pathogenic ahlonziwe kulezi zifunda ezixhumene.
I-GGC iphinda izandiso ku-NOTCH2NLC
NUkulandelana kwe-anopore-based kucutshungulwe ku-13 ethintekile kanye namalungu angu-4 angathintekile avela emindenini ye-8 (elinye ilungu elithintekile lalandelwa yi-Pacbio yesikhulumi sokulandelana esifundwe isikhathi eside.).Idatha efundwe isikhathi eside yembule izifo ezihambisana nokuphinda kwe-GGC ukunwetshwa ku-5′ UTR ye-NOTCH2NLC yokwenza imephu yofuzo ukuya endaweni exhunywe ku-58.1 Mb (Umfanekiso 1).Lokhu kwandiswa okuphindayo kuphinde kwahlonzwa kuwo wonke amacala angu-40 e-NIID ahlolwe yi-RP-PCR.
CUkulandelana kwethagethi okulamule njenge-9 kuplathifomu ye-nanopore kwaqashwa ukuze kufinyelelwe ukufundwa okuphakeme kokufunda ku-NOTCH2NLC impinda (100 X-1,795 X).Lokhu kulandelana kokuvumelana kuvumelana kahle nokutholwe kwangaphambilini ekwandiseni okuphindayo kwe-GGC.Ngaphezu kwalokho, izimpinda ze-{(GGA)n (GGC)n}n zihlonzwe njengento engaba khona umaka wofuzo we-phenotype ebuthaka kakhulu (Umfanekiso 2).
Umfanekiso 1. Ukunwetshwa okuphindayo okuhlobene nezifo okuhlonzwe ku-exon 1 ye-NOCH2NLC isoforms.
Umfanekiso 2. Ukulandelana kwe-Consensus kwe-NPTCH2NLC kuphinda ezigulini ze-NIID ezine(*) noma ezingenazo i-phenotype ebusa ubuthakathaka
NIzakhi zofuzo ze-OTCH2NL ziyizakhi zofuzo eziqondene nomuntu, okukholakala ukuthi zidlala indima ebalulekile ekuguqukeni kobuchopho bomuntu kanye nezifo zemizwa.Kodwa-ke, izinhlobo ezintathu zofuzo ezihlobene ne-NOTCH2 (NOTCH2NLA, NOTCH2NLB kanye ne-NOTCH2NLC) ezine->99.1% yobunikazi bokulandelana azizange zixazululwe kwaze kwaba sekuhlanganisweni kwakamuva kofuzo lomuntu.Ukulandelana okungahlanganisi nokufundwa isikhathi eside kuplathifomu ye-nanopore kubonise izinzuzo eziphawulekayo ekuxazululeni izifunda zokufana okuphezulu kanye (GGC)n iziphinda ngo-100% GC-rich.
I-GGC iphinda izandiso ku-NOTCH2NLC
Tukulandelana kwe-ranscriptome kwacutshungulwa ku-2 abathintekayo kanye namalungu angu-2 angathintekile.Ukujula kokufunda okujwayelekile kubalwe emqondweni nemicu ye-antisense enhla komfula wama-exons okuqala amapharalogi e-NOTCH2NL.Okulotshiweyo okungajwayelekile okulwa nomuzwa kutholwe kuphela ezimweni ezithintekile, ezihlala ekuqaleni noma ngaphakathi kwesifunda sokunwetshwa esiphindayo (Iziqongo eziPurple ku-F1-14 kanye no-F1-16 kuMfanekiso 3.).Ngaphezu kwalokho, kwahlonzwa ama-DEG angu-54 futhi wonke acetshiswa emigomeni ye-GO kanye ne-MPO ehlobene nemisebenzi ye-neuronal.
Umfanekiso 3. Ukujula kokufunda okujwayelekile enhla komfula we-exon yokuqala ye-NOTCH2NLC ezimeni ezingathintekile (ngenhla) nezithintekile (ngezansi).
Ubuchwepheshe
I-Oxford Nanopore Teghnologies (ONT)
Nukulandelana kwe-anopore kuzihlukanisa kwezinye izinkundla zokulandelana, ngokuthi ama-nucleotide afundwa ngokuqondile ngaphandle kwenqubo yokuhlanganiswa kwe-DNA.Njengoba umucu owodwa we-DNA udlula ku-nano-size protein pore (nanopore), ama-nucleotide ahlukene akhiqiza imisinga ye-ionic ehlukene, engabanjwa futhi idluliselwe ngokulandelana kwezisekelo.Inkundla yokulandelana ye-ONT ngokwayo ayibonisi umkhawulo wobuchwepheshe obonakalayo kubude bokufundwa kwe-DNA.Ngakho-ke, i-Ultra-long reads (ULRs) iyatholakala ekuhlanganiseni i-genome yekhwalithi ephezulu.Ngaphezu kwalokho, lokhu kufunda okude kakhulu, okude ngokwanele ukweqa izici eziyinkimbinkimbi zokulandelana noma ukuhlukahluka kwesakhiwo, kusiza ukunqoba imikhawulo yokulandelana kokufunda okufushane lapha.
Ukulandelana kwe-Nanopore
Ukuhlonza ukuhluka kwesakhiwo (SV).
SUkulandelana okungenayo i-ynthesis kulondoloza kakhulu ulwazi lwe-DNA methylation kusifanekiso.I-Methylated A, T, C kanye ne-G ikhiqiza imisinga ye-ionic ehlukile evela kwa-un-methylated, engafundwa ngokuqondile yipulatifomu.Ukulandelana kwe-Nanopore kunika amandla iphrofayili yegenome ephelele yakho kokubili okungu-5mC no-6mA ekulungisweni kwe-nucleotide eyodwa.
Ireferensi
Jun Sone, et.al.Ukulandelana okufunde isikhathi eside kukhomba ukunwetshwa okuphindayo kwe-GGC ku-NOTCH2NLC okuhlobene nesifo sokufakwa kwe-neuronal intranuclear.I-Nature Genetics (2019)
Ubuchwepheshe Nokugqama ihlose ukwabelana ngohlelo lokusebenza oluyimpumelelo lwakamuva kakhulu lobuchwepheshe bokulandelana komphumela ophezulu ezinkundleni ezihlukahlukene zokucwaninga kanye nemibono ekhaliphile ekwakhiweni kokuhlola kanye nokumbiwa kwedatha.
Isikhathi sokuthumela: Jan-06-2022