MAGENOMI WA ANTHU
chibadwa chibadwa
Kutsata kwanthawi yayitali kumazindikiritsa kubwereza kwa GGC mu NOTCH2NLC yolumikizidwa ndi matenda a neuronal intranuclear inclusion
ONT kubwereza |Ilumina |Kutsata kwa exome |CRISPR-Cas9 ONT yotsata kutsata |RNA-seq |ONT 5mC methylation kuitana
Mfundo zazikuluzikulu
1.Mwa kusanthula kwa Linkage pa banja lalikulu la NIID, zigawo ziwiri zogwirizana zinadziwika.
2.ONT-yotengera nthawi yayitali yowerengera ndi Cas-9 yokhazikika yolemeretsa ONT idapeza chomwe chingayambitse NIID, GGC kubwereza kukulitsa mu 5' UTR ya NOTCH2NLC.Kafukufukuyu adanenanso za kuwonjezereka kobwerezabwereza kwa majini okhudzana ndi anthu kwa nthawi yoyamba komwe kudachitika chifukwa cha kubwereza kwa magawo.
Kutsatizana kwa 3.RNA kunavumbulutsa zolembedwa zachilendo za antisense koyambirira kapena mkati mwa GGC kubwereza zigawo zakukulitsa mu NOTCH2NLC.
Mbiri
NMatenda a euronal intranuclear inclusion disease (NIID) ndi matenda opita patsogolo komanso oopsa a neurodegenerative, omwe amadziwika ndi kukhalapo kwa eosinophilic hyaline intranuclear inclusions pakati ndi zotumphukira zamanjenje.Mawonetseredwe ake osinthika kwambiri azachipatala amadzetsa zovuta pakuzindikira mpaka kuyambitsa khungu la biopsy.Komabe, njira zochokera ku histopathology zidakali ndi vuto losazindikira, zomwe zimafuna kumvetsetsa kwa majini a NIID.
Zopambana
Linkage Analysis
Skuwerengera motsatana kotsatira ma genome onse (WGS) ndi kutsata kwamtundu wonse (WES) kunachitika pabanja lalikulu la NIID (13 okhudzidwa ndi mamembala 7 osakhudzidwa).Kusanthula kwamalumikizidwe pa SNPs otengedwa ku datayi kudavumbulutsa zigawo ziwiri zokha zolumikizidwa: dera la 3.5 Mb pa 1p36.31-p36.22 (pazipita LOD = 2.32) ndi 58.1 Mb dera pa 1p22.1-q21.3 (pazipita LOD: 4.21) ).Komabe, palibe ma SNP kapena ma CNV omwe adadziwika m'madera olumikizana awa.
GGC ibwereza kukulitsa mu NOTCH2NLC
Nkutsatizana kwa anopore kunakonzedwa pa 13 yomwe inakhudzidwa ndi mamembala a 4 osakhudzidwa ochokera m'mabanja a 8 (membala wina wokhudzidwayo adatsatiridwa ndi Pacbio yowerengera nthawi yayitali.).Deta yowerengedwa kwanthawi yayitali idavumbulutsa matenda okhudzana ndi GGC akubwereza kubwereza mu 5' UTR ya NOTCH2NLC kupanga ma gene ku 58.1 Mb dera lolumikizidwa (Chithunzi 1).Kuwonjezeka kobwerezabwerezaku kunadziwikanso muzochitika zonse za 40 zaposachedwa za NIID zoyesedwa ndi RP-PCR.
CKutsata kwa chandamale cha as-9 pa nsanja ya nanopore kudagwiritsidwa ntchito kuti akwaniritse zowerengera zapamwamba pa NOTCH2NLC kubwereza (100 X-1,795 X).Kugwirizana kumeneku kumagwirizana bwino ndi zomwe zapezedwa m'mbuyomu pakukula kobwereza kwa GGC.Komanso, {(GGA)n (GGC)n} n kubwerezabwereza kunazindikiritsidwa ngati chizindikiritso cha chibadwa cha phenotype yofooka kwambiri (Chithunzi 2).
Chithunzi 1. Matenda okhudzana ndi kubwereza kuwonjezereka komwe kumadziwika pa exon 1 ya NOTCH2NLC isoforms.
Chithunzi 2. Zotsatira za Consensus za NPTCH2NLC kubwereza kwa odwala NIID omwe ali ndi (*) kapena opanda phenotype yofooka kwambiri.
NMitundu ya OTCH2NL ndi majini okhudzana ndi anthu, omwe amakhulupirira kuti amagwira ntchito yofunika kwambiri pakusintha kwaubongo wamunthu komanso matenda amitsempha.Komabe, majini atatu okhudzana ndi NOTCH2 (NOTCH2NLA, NOTCH2NLB ndi NOTCH2NLC) okhala ndi>99.1% yodziwika motsatizana sanathe kuthetsedwa mpaka msonkhano waposachedwa wa ma genome a anthu.Kutsatizana kopanda kaphatikizidwe komanso kowerengera nthawi yayitali papulatifomu ya nanopore kwawonetsa zopindulitsa pakuthana ndi zigawo zofanana kwambiri ndi (GGC)n kubwereza ndi 100% GC-rich.
GGC ibwereza kukulitsa mu NOTCH2NLC
Transcriptome sequencing idakonzedwa pa 2 yomwe idakhudzidwa ndi mamembala a 2 osakhudzidwa.Kuzama kowerengera kokhazikika kunawerengeredwa pamalingaliro ndi zingwe zotsutsana ndi ma exons oyambira a NOTCH2NL paralogs.Zolemba zachilendo zotsutsana ndi maganizo zinapezeka pokhapokha pazochitika zomwe zakhudzidwa, zomwe zimakhala pachiyambi kapena mkati mwa chigawo chowonjezereka chobwerezabwereza (Nthawi za Purple mu F1-14 ndi F1-16 mu Chithunzi 3.).Kuphatikiza apo, 54 DEGs adadziwika ndipo onse adalemeretsedwa mu GO ndi mawu a MPO okhudzana ndi ntchito za neuronal.
Chithunzi 3. Kuwerenga mozama mozama kumtunda kwa exon yoyamba ya NOTCH2NLC muzochitika zosakhudzidwa (pamwambapa) ndi zokhudzidwa (pansipa).
Zamakono
Oxford Nanopore Teghnologies (ONT)
Nkutsatizana kwa anopore kumadzisiyanitsa ndi nsanja zina zotsatizana, chifukwa ma nucleotides amawerengedwa mwachindunji popanda DNA synthesis process.DNA ya chingwe chimodzi imadutsa pore ya nano-size protein (nanopore), ma nucleotide osiyanasiyana amapanga ma ionic mafunde osiyanasiyana, omwe amatha kugwidwa ndikusamutsidwa motsatizana.ONT yotsatizana nsanja yokha sikuwonetsa malire aukadaulo pautali wa kuwerenga kwa DNA.Chifukwa chake, ma Ultra-long reads (ULRs) amapezeka pakuphatikiza ma genome apamwamba kwambiri.Kuphatikiza apo, mawerengedwe aatali kwambiri awa, omwe ndiatali wokwanira kuwoloka mawonekedwe ovuta kwambiri kapena kusintha kwamapangidwe, amathandizira kuthana ndi malire a masanjidwe apafupiafupi apa.
Nanopore sequencing
Chizindikiritso cha Structure Variation (SV).
Skutsatizana kopanda ma synthesis kumasunga zambiri za DNA methylation pa template.Methylated A, T, C ndi G amapanga mafunde a ionic osiyana kuchokera ku un-methylated, omwe amatha kuwerengedwa mwachindunji ndi nsanja.Kutsatizana kwa Nanopore kumapereka mphamvu ku mbiri ya genome yonse ya 5mC ndi 6mA pakupanga kwa single-nucleotide.
Buku
Jun Sone, et.al.Kutsatizana kwa nthawi yayitali kumazindikiritsa kuwonjezereka kwa GGC mu NOTCH2NLC yokhudzana ndi matenda a neuronal intranuclear inclusion.Nature Genetics (2019)
Tech ndi Zowunikira Cholinga chake ndi kugawana njira zamakono zotsatirira bwino kwambiri m'mabwalo osiyanasiyana ofufuza komanso malingaliro anzeru pamapangidwe oyesera ndi migodi ya data.
Nthawi yotumiza: Jan-06-2022