HUMAN GENOMICS
ntuj genetics
Kev nyeem ntawv ntev ntev txheeb xyuas GGC rov nthuav dav hauv NOTCH2NLC cuam tshuam nrog cov kab mob neuronal intranuclear suav nrog
ONT resequencing |Illumina |Tag nrho exome sequencing |CRISPR-Cas9 ONT lub hom phiaj sequencing |RNA-seq |ONT 5mC methylation hu
Qhov tseem ceeb
1.Los ntawm kev txheeb xyuas Linkage ntawm tsev neeg NIID loj, ob thaj chaw txuas tau raug txheeb xyuas.
2.ONT-based long-read sequencing thiab Cas-9 mediated enrichment ONT sequencing pom muaj peev xwm ua rau NIID, GGC rov nthuav dav hauv 5′ UTR ntawm NOTCH2NLC.Txoj kev tshawb no tau tshaj tawm rov nthuav dav hauv tib neeg cov noob caj noob ces thawj zaug uas hloov zuj zus los ntawm kev sib tshooj segmental.
3.RNA sequencing qhia txawv txawv antisense transcripts thaum pib lossis hauv GGC rov nthuav dav thaj tsam hauv NOTCH2NLC.
Keeb kwm
Neuronal intranuclear inclusion disease (NIID) yog ib qho mob hnyav thiab tuag taus neurodegenerative, uas yog tus cwj pwm los ntawm qhov muaj eosinophilic hyaline intranuclear inclusions hauv nruab nrab thiab peripheral paj hlwb.Nws qhov kev hloov pauv hloov pauv tau tshwm sim ua rau muaj teeb meem loj hauv kev kuaj mob kom txog rau thaum kev taw qhia ntawm daim tawv nqaij biopsy.Txawm li cas los xij, cov txheej txheem histopathology tseem raug kev txom nyem los ntawm misdiagnosis, uas yog hu rau kev nkag siab txog caj ces ntawm NIID.
Kev ua tiav
Linkage Analysis
Short-read sequencing raws li tag nrho genome sequencing (WGS) thiab tag nrho exome sequencing (WES) tau ua nyob rau hauv tsev neeg NIID loj (13 tus neeg cuam tshuam thiab 7 tus tswvcuab tsis cuam tshuam).Kev txheeb xyuas kev sib txuas ntawm SNPs tau muab rho tawm los ntawm cov ntaub ntawv no tsuas yog qhia ob thaj chaw txuas: thaj tsam 3.5 Mb ntawm 1p36.31-p36.22 (qhov siab tshaj LOD = 2.32) thiab thaj tsam 58.1 Mb ntawm 1p22.1-q21.3 (qhov siab tshaj LOD: 4.21 ).Txawm li cas los xij, tsis muaj kab mob SNPs lossis CNVs tau txheeb xyuas hauv cov cheeb tsam txuas no.
GGC rov nthuav dav hauv NOTCH2NLC
Nanopore-based sequencing tau ua tiav ntawm 13 tus neeg raug cuam tshuam thiab 4 tus tswvcuab tsis raug cuam tshuam los ntawm 8 tsev neeg (lwm tus tswvcuab cuam tshuam tau ua raws los ntawm Pacbio ntev nyeem lub platform.).Cov ntaub ntawv ntev-nyeem tau nthuav tawm cov kab mob cuam tshuam nrog GGC rov nthuav dav hauv 5' UTR ntawm NOTCH2NLC gene mapping rau 58.1 Mb txuas thaj tsam (Daim duab 1).Cov kev nthuav dav ntxiv no kuj tau txheeb xyuas nyob rau hauv tag nrho 40 qhov xwm txheej NIID kuaj los ntawm RP-PCR.
Cas-9 kev sib kho lub hom phiaj sib txuas ntawm nanopore platform tau ua haujlwm kom ua tiav kev nyeem ntawv ntau dua ntawm NOTCH2NLC rov ua dua (100 X-1,795 X).Cov kev pom zoo ua ntu zus tau pom zoo nrog cov kev tshawb pom yav dhau los ntawm GGC rov nthuav dav.Tsis tas li ntawd, {(GGA)n (GGC)n}n rov ua dua tau txheeb xyuas tias yog qhov muaj peev xwm genetic marker rau qhov tsis muaj zog-dominant phenotype (Daim duab 2).
Daim duab 1. Kab mob ntsig txog rov nthuav dav pom ntawm exon 1 ntawm NOTCH2NLC isoforms.
Daim duab 2. Kev pom zoo ua ntu zus ntawm NPTCH2NLC rov ua dua hauv NIID cov neeg mob uas muaj (*) lossis tsis muaj zog-dominant phenotype
NOTCH2NL noob yog tib neeg cov noob caj noob ces, uas ntseeg tau ua lub luag haujlwm tseem ceeb hauv tib neeg lub hlwb hloov pauv thiab kab mob paj hlwb.Txawm li cas los xij, peb NOTCH2 cov noob muaj feem xyuam (NOTCH2NLA, NOTCH2NLB thiab NOTCH2NLC) nrog> 99.1% cov cim kab ke tsis raug daws kom txog rau thaum tib neeg genome los ua ke.Synthesis-free and long-read sequencing on nanopore platform tau qhia qhov zoo tshaj plaws hauv kev daws cov cheeb tsam zoo sib xws thiab (GGC)n rov ua dua nrog 100% GC-nplua nuj.
GGC rov nthuav dav hauv NOTCH2NLC
Transcriptome sequencing tau ua tiav ntawm 2 cuam tshuam thiab 2 tus tswv cuab tsis cuam tshuam.Normalized nyeem qhov tob yog xam ntawm kev nkag siab thiab antisense strands nyob rau hauv upstream ntawm thawj exons ntawm NOTCH2NL paralogs.Cov ntaub ntawv pov thawj tsis txaus ntseeg tau pom tsuas yog nyob rau hauv cov xwm txheej cuam tshuam, uas zaum hauv qhov pib lossis hauv cheeb tsam rov nthuav dav (Piv txwv li hauv F1-14 thiab F1-16 hauv daim duab 3.).Tsis tas li ntawd, 54 DEGs tau txheeb xyuas thiab txhua tus tau ua kom muaj txiaj ntsig hauv GO thiab MPO cov ntsiab lus ntsig txog cov haujlwm neuronal.
Daim duab 3. Normalized nyeem qhov tob nyob rau sab sauv ntawm thawj exon ntawm NOTCH2NLC nyob rau hauv unaffected (saum toj no) thiab cuam tshuam (hauv qab).
Technology
Oxford Nanopore Technologies (ONT).
Nanopore sequencing distinguishes nws tus kheej los ntawm lwm cov sequencing platforms, nyob rau hauv hais tias cov nucleotides nyeem ncaj qha tsis muaj DNA synthesis txheej txheem.Raws li ib txoj hlua DNA dhau los ntawm nano-loj protein pore (nanopore), cov nucleotides sib txawv tsim cov ionic tam sim no sib txawv, uas tuaj yeem raug ntes thiab xa mus rau hauv qib ntawm cov hauv paus.ONT sequencing platform nws tus kheej tsis qhia meej txog kev txwv ntawm qhov ntev ntawm DNA nyeem.Yog li ntawd, Ultra-ntev nyeem ntawv (ULRs) yog muaj rau genome los ua ke ntawm qhov zoo.Tsis tas li ntawd, cov ntawv nyeem ntev heev, uas ntev txaus los hla cov txheej txheem sib txawv lossis cov qauv sib txawv, pab kov yeej cov kev txwv ntawm kev nyeem luv luv ntawm no.
Nanopore sequencing
Structure variation (SV) identification
Synthesis-free sequencing loj khaws cia DNA methylation cov ntaub ntawv ntawm tus qauv.Methylated A, T, C thiab G tsim cov ionic tam sim no los ntawm cov tsis muaj methylated, uas tuaj yeem nyeem ncaj qha los ntawm lub platform.Nanopore sequencing txhawb nqa tag nrho-genome profileing ntawm ob qho tib si 5mC thiab 6mA ntawm ib leeg-nucleotide daws teeb meem.
Kev siv
Jun Sone, thiab.al.Kev nyeem ntawv ntev ntev qhia txog GGC rov nthuav dav hauv NOTCH2NLC cuam tshuam nrog cov kab mob neuronal intranuclear suav nrog.Nature Genetics (2019)
Tech thiab Highlights Lub hom phiaj ntawm kev sib koom ua tiav daim ntawv thov tam sim no ntawm kev sib txawv high-throughput sequencing technologies nyob rau hauv ntau yam reseach arena nrog rau cov tswv yim ci ntsa iab hauv kev sim tsim thiab cov ntaub ntawv mining.
Post lub sij hawm: Jan-06-2022