MAGENOMI EVANHU
nature genetics
Kutevedzana kwekuverenga kwenguva refu kunozivisa GGC inodzokorora kuwedzera muNOCH2NLC yakabatana neuronal intranuclear inclusion chirwere.
ONT resequencing |Ilumina |Yese exome sequencing |CRISPR-Cas9 ONT yakanangwa kutevedzana |RNA-seq |ONT 5mC methylation kufona
Pfungwa huru
1.By Linkage analysis pamhuri huru yeNIID, nzvimbo mbiri dzakabatana dzakaonekwa.
2.ONT-based long-read sequencing uye Cas-9 mediated enrichment ONT sequencing yakawana chingangove chikonzero chemajini cheNIID, GGC inodzokorora kuwedzera mu5'UTR yeNOTCH2NLC.Ichi chidzidzo chakashuma kudzokorora kuwedzera mumajini akanangana nevanhu kekutanga akashanduka kuburikidza nezvikamu zvakadzokororwa.
3.RNA kutevedzana kwakaratidza zvinyorwa zvisingaite zveantisense pakutanga kana mukati meGGC inodzokorora nzvimbo dzekuwedzera muNOCH2NLC.
Background
Neuronal intranuclear inclusion disease (NIID) chirwere chinopfuurira uye chinouraya neurodegenerative, iyo inoratidzwa nekuvapo kweeosinophilic hyaline intranuclear inclusions mukati uye necheperipheral nervous systems.Kuratidzira kwayo kwakasiyana-siyana kwekiriniki kunomutsa matambudziko makuru mukuongororwa kusvika paunzwa ganda biopsy.Nekudaro, histopathology-yakavakirwa nzira dzichiri kutambura nekusaziva, izvo zviri kudaidzira kuti genetic kunzwisisa kweNIID.
Zvakabudirira
Linkage Analysis
Short-read sequencing based whole genome sequencing (WGS) uye yese exome sequencing (WES) yakaitwa pamhuri hombe yeNIID (13 yakakanganisika uye 7 nhengo dzisina kukanganiswa).Kuongororwa kwekubatanidza paSNPs zvakatorwa kubva kune idzi data kwakaratidza chete matunhu maviri akabatana: a 3.5 Mb dunhu pa1p36.31-p36.22 (yakanyanya LOD = 2.32) uye 58.1 Mb dunhu pa1p22.1-q21.3 (yakanyanya LOD: 4.21 )Nekudaro, hapana pathogenic SNPs kana CNVs dzakaonekwa munzvimbo idzi dzakabatana.
GGC inodzokorora kuwedzera muNOCH2NLC
Nanopore-based sequencing yakagadziriswa pane 13 yakakanganiswa uye 4 nhengo dzisina kukanganiswa kubva kumhuri dze8 (imwe nhengo yakakanganiswa yakatevedzana nePacbio yakareba kuverenga kutevedzana chikuva.).Yakareba-kuverenga data yakaratidza chirwere chakabatana neGGC inodzokorora kuwedzera mu5' UTR yeNOCH2NLC gene mapping ku58.1 Mb yakabatana dunhu (Mufananidzo 1).Aya ekuwedzera ekuwedzera akaonekwawo mune ese makumi mana esporadic NIID makesi akaedzwa neRP-PCR.
Cse-9 yakapindirana chinangwa chekutevedzana papuratifomu ye nanopore yakashandiswa kuti iwane kuverenga kwepamusoro paNOCH2NLC kudzokorora (100 X-1,795 X).Uku kutevedzana kwekubvumirana kwakabvumirana zvakanaka nezvakawanikwa kare paGGC kudzokorora kuwedzera.Uyezve, {(GGA)n (GGC)n} n kudzokorora kwakaonekwa sechingangove genetic marker yeutera-inotonga phenotype (Mufananidzo 2).
Mufananidzo 1. Chirwere chakabatana kudzokorora kuwedzera kwakaonekwa pane exon 1 yeNOCH2NLC isoforms.
Mufananidzo 2. Consensus sequences yeNPTCH2NLC inodzokorora muNIID varwere vane (*) kana vasina utera-hunotonga phenotype
NOTCH2NL majini magene akatarisana nevanhu, ayo anofungidzirwa kuti anoita basa rakakosha mukushanduka kwehuropi hwevanhu uye zvirwere zvetsinga.Zvisineyi, majini matatu ane hukama neNOTCH2 (NOTCH2NLA, NOTCH2NLB uye NOTCH2NLC) ane> 99.1% sequence identity haina kugadziriswa kusvika pagungano revanhu razvino.Synthesis-isina uye yakareba-yekuverenga kutevedzana papuratifomu ye nanopore yakaratidza zvakanakira mukugadzirisa matunhu ekufanana kwepamusoro uye (GGC) n inodzokorora ne100% GC-yakapfuma.
GGC inodzokorora kuwedzera muNOCH2NLC
Transcriptome sequencing yakagadziriswa pa2 yakakanganiswa uye 2 nhengo dzisina kubatwa.Kudzika kwekuverenga kwakajairwa kwakaverengerwa pane pfungwa uye antisense tambo kumusoro kwekutanga maexons eNOCH2NL paralogs.Abnormal anti-sense zvinyorwa zvakawanikwa chete mumatambudziko akabatwa, ayo anogara pakutanga kana mukati mekudzokorora nharaunda yekuwedzera (Purple peaks muF1-14 uye F1-16 muFigure 3.).Mukuwedzera, 54 DEGs yakaonekwa uye yose yakafumiswa muGO uye MPO mazwi ane chokuita neuronal mabasa.
Mufananidzo 3. Yakajairwa kuverenga yakadzama kumusoro kweiyo exon yekutanga yeNOCH2NLC mune isina kubatwa (pamusoro) uye yakakanganiswa (pazasi) kesi.
Vadivelu Comedy Technology
Oxford Nanopore Teghnologies (ONT)
Nanopore sequencing inozvisiyanisa kubva kune mamwe mapuratifomu ekutevedzana, mukuti nucleotides inoverengwa zvakananga pasina DNA synthesis process.Sezvo tambo imwe chete DNA inopfuura nenano-sized protein pore (nanopore), nucleotides yakasiyana inogadzira akasiyana ionic currents, inogona kutorwa uye kuendeswa munhevedzano yezvigadziko.ONT yekuteedzera chikuva pachayo hairatidze pachena tekinoroji muganho pakureba kwekuverenga kweDNA.Naizvozvo, Ultra-refu kuverenga (ULRs) inowanikwa kune genome kusangana kwemhando yepamusoro.Uyezve, aya maverengero akareba kwazvo, akareba zvakakwana kuyambuka akaomesesa kutevedzana maficha kana kusiana kwechimiro, anobatsira kukunda zvipimo zvekupfupika-kuverenga kutevedzana pano.
Nanopore sequencing
Structure variation (SV) chiziviso
Synthesis-yemahara sequencing yakachengetedza zvakanyanya DNA methylation ruzivo pane template.Methylated A, T, C uye G inogadzira akasiyana ionic currents kubva kune un-methylated, iyo inogona kuverengwa zvakananga nepuratifomu.Nanopore sequencing inopa simba rose-genome profiling yeese ari maviri 5mC uye 6mA pa single-nucleotide resolution.
Reference
Jun Sone, nevamwe.al.Kutevedzana kwekuverenga kwenguva refu kunozivisa GGC kudzokorora kuwedzera muNOTCH2NLC yakabatana neuronal intranuclear inclusion chirwere.Nature Genetics (2019)
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Nguva yekutumira: Jan-06-2022