IIGENOMIK ABANTU
imfuzo yendalo
Ulandelelwano olude lokufunda luchonga i-GGC yokuphinda ukwandiswa kwi-NOTCH2NLC ehambelana nesifo se-neuronal intranuclear inclusion
ONT ilandelelana |Ilumina |Ulandelelwano olupheleleyo lwe-exome |CRISPR-Cas9 ONT ulandelelwano ekujoliswe kulo |I-RNA-seq |ONT 5mC methylation ukufowuna
Iimbalasane
1.Ngohlalutyo lonxibelelwano kusapho olukhulu lwe-NIID, imimandla emibini edibeneyo ichongiwe.
I-2.ONT-based based based sequencing and Cas-9 mediated enrichment ONT sequencing ifumene isizathu esinokubangela i-NIID, i-GGC iphinda iphinde ikhulise kwi-5' UTR ye-NOTCH2NLC.Olu phononongo lunike ingxelo yokwandiswa okuphindayo kwimizila yemfuza ethe ngqo emntwini okokuqala eyathi yavela ngokuphindaphindana kwamacandelo.
Ulandelelwano lwe-3.RNA luveze imibhalo ye-antisense engaqhelekanga ekuqaleni okanye ngaphakathi kwe-GGC iphinda imimandla yokwandisa kwi-NOTCH2NLC.
Imvelaphi
NI-euronal intranuclear inclusion disease (NIID) sisifo esiqhubekayo kunye nesibulalayo se-neurodeergenerative, esibonakaliswa kubukho be-eosinophilic hyaline intranuclear inclusions kwiinkqubo ze-nervous central kunye ne-peripheral.Ukubonakaliswa kwayo okuguquguqukayo kakhulu kweklinikhi kuphakamisa ubunzima obukhulu ekuxilongeni de kuqaliswe i-biopsy yolusu.Nangona kunjalo, iindlela ezisekwe kwi-histopathology zisaphathwa kakubi, nto leyo efuna ukuqonda ngemfuza ye-NIID.
Impumelelo
Uhlalutyo lonxulumano
Si-hort-read sequencing based whole genome sequencing (WGS) kunye nolandelelwano olupheleleyo lwe-exome (WES) lwenziwa kwintsapho enkulu ye-NIID (i-13 echaphazelekayo kunye ne-7 amalungu angachaphazelekiyo).Uhlalutyo lonxibelelwano kwii-SNP ezikhutshwe kule datha zibonakalise kuphela imimandla emibini edibeneyo: ummandla we-3.5 Mb kwi-1p36.31-p36.22 (ubuninzi be-LOD = 2.32) kunye nommandla we-58.1 Mb kwi-1p22.1-q21.3 (ubuninzi be-LOD: 4.21) ).Nangona kunjalo, akukho zi-SNP ze-pathogenic okanye ii-CNV ezichongiweyo kule mimandla edibeneyo.
I-GGC iphinda ukwandiswa kwi-NOTCH2NLC
Nukulandelelana kwe-anopore-based based sequencing kwi-13 echaphazelekayo kunye ne-4 amalungu angathintekiyo avela kwiintsapho ze-8 (elinye ilungu elichaphazelekayo lilandelelwa yi-Pacbio ende yokufunda iqonga lokulandelelana.).Idatha efundwe ixesha elide ibonakalise isifo esihambelana ne-GGC yokuphinda ukwandiswa kwi-5' UTR ye-NOTCH2NLC ye-gene mapping ukuya kwi-58.1 Mb kummandla odibeneyo (Umfanekiso 1).Olu lwandiso luphindayo lwachongwa kuzo zonke iimeko ze-NIID ezingama-40 ezivavanywe yi-RP-PCR.
Cnjengo-9 ukulandelelana kwethagethi elamliweyo kwiqonga le-nanopore laqeshwa ukuphumeza ugutyulo oluphezulu lokufunda kwi-NOTCH2NLC impinda (100 X-1,795 X).Olu landelelwano lwemvumelwano luvumelane kakuhle neziphumo zangaphambili kwi-GGC ephinda ukwandiswa.Ngaphezu koko, {(GGA) n (GGC) n} ukuphindaphinda kwachongwa njengento enokubakho i-genetic marker ye-phenotype ebuthathaka kakhulu (Umfanekiso 2).
Umfanekiso 1. Ukwandiswa okuphindayo okunxulumene nesifo kuchongwe kwi-exon 1 ye-NOCH2NLC isoforms.
Umzobo 2. Ulandelelwano lwe-Consensus ye-NPTCH2NLC iphinda kwizigulane ze-NIID kunye (*) okanye ngaphandle kwephenotype ebuthathaka.
NIijini ze-OTCH2NL luhlobo oluthile lwabantu, ekukholeleka ukuba ludlala indima ebalulekileyo kwinguquko yobuchopho bomntu kunye nezifo zemithambo-luvo.Nangona kunjalo, iijini ezintathu ezinxulumene ne-NOTCH2 (NOTCH2NLA, NOTCH2NLB kunye ne-NOTCH2NLC) ene>99.1% yesazisi solandelelwano azizange zisonjululwe de kube yindibano yamva nje yegenome yomntu.Ukulandelelana kwe-synthesis-free kunye nexesha elide lokufunda kwi-platform ye-nanopore ibonise inzuzo ephawulekayo ekuxazululeni imimandla yokufana okuphezulu kunye (i-GGC) n iphinda iphinda i-100% ye-GC-rich.
I-GGC iphinda ukwandiswa kwi-NOTCH2NLC
Tukulandelelana kwe-ranscriptome kwaqhutyelwa kwi-2 echaphazelekayo kunye namalungu angama-2 angachaphazelekanga.Ubunzulu bokufunda obuqhelekileyo bubalwe kwingqiqo kunye nemicu ye-antisense phezulu kwii-exons zokuqala ze-NOTCH2NL paralogs.Imibhalo echasene ne-anti-sense engaqhelekanga ifunyenwe kuphela kwiimeko ezichaphazelekayo, ezihlala ekuqaleni okanye ngaphakathi kwendawo yokwandisa ukuphindaphinda (i-Purple peaks kwi-F1-14 kunye ne-F1-16 kwi-Figure 3.).Ukongeza, ii-DEGs ezingama-54 zachongwa kwaye zonke zatyetyiswa kwimigaqo ye-GO kunye ne-MPO enxulumene nemisebenzi yemithambo-luvo.
Umzobo 3. Ubunzulu bokufunda obuqhelekileyo kumntla we-exon yokuqala ye-NOTCH2NLC kwiimeko ezingachaphazelekiyo (ngasentla) kunye nezichaphazelekayo (ngezantsi).
Iteknoloji
Oxford Nanopore Teghnologies (ONT)
Nulandelelwano lwe-anopore luzahlula kwezinye iiplatifomu zokulandelelana, kuba i-nucleotides ifundwa ngokuthe ngqo ngaphandle kwenkqubo ye-DNA synthesis.Njengoko i-DNA ye-strand enye idlula kwi-nano-size protein pore (nanopore), i-nucleotides eyahlukileyo ivelisa i-ionic currents ezahlukeneyo, ezinokubanjwa kwaye zidluliselwe ngokulandelelana kweziseko.Iqonga lokulandelelana kwe-ONT ngokwalo alibonisi umda ocacileyo wobugcisa kubude bokufunda kweDNA.Ke ngoko, ii-Ultra-long reads (ULRs) ziyafumaneka kwindibano ye-genome yomgangatho ophezulu.Ngaphaya koko, olu fundo lude kakhulu, lude ngokwaneleyo ukuwela iimpawu zolandelelwano oluntsonkothileyo okanye ukwahluka kolwakhiwo, lunceda ukoyisa imida yolandelelwano lokufunda okufutshane apha.
Ulandelelwano lweNanopore
Ukwahluka kolwakhiwo (SV) ukuchongwa
SUlandelelwano lwasimahla lwe-synthesis lugcine kakhulu ulwazi lweDNA methylation kwitemplate.I-Methylated A, T, C kunye ne-G ivelisa imisinga ye-ionic eyahlukileyo ukusuka kwi-un-methylated, enokufundwa ngokuthe ngqo kwiqonga.Ulandelelwano lweNanopore luxhobisa iprofayili yegenome epheleleyo yazo zombini i-5mC kunye ne-6mA kwisisombululo senucleotide enye.
Isalathiso
Jun Sone, kunye.al.Ulandelelwano olude lokufunda luchonga i-GGC yokuphinda ukwandiswa kwi-NOTCH2NLC ehambelana nesifo se-neuronal intranuclear inclusion.Imfuzo yendalo (2019)
ITekhnoloji kunye neeNgcaciso ijolise ekwabelaneni ngeyona mpumelelo yamva nje yosetyenziso lweetekhnoloji zolandelelwano oluphezulu kwiindawo ezahlukeneyo zophando kunye neengcinga ezikrelekrele kuyilo lovavanyo kunye nokwembiwa kwedatha.
Ixesha lokuposa: Jan-06-2022