GENOMICS mmadụ
okike mkpụrụ ndụ ihe nketa
Usoro ngụ ogologo na-achọpụta mgbasawanye GGC na NOTCH2NLC jikọtara ya na ọrịa nsonye intranuclear neuronal.
ONT resequencing |Illumina |Usoro exome dum |Usoro ezubere iche CRISPR-Cas9 ONT |RNA-seq |ONT 5mC methylation na-akpọ
Isi ihe
1. Site na nyocha njikọ na nnukwu ezinụlọ NIID, achọpụtara mpaghara abụọ jikọtara ọnụ.
2.ONT dabeere ogologo-agụ sequencing na Cas-9 mediated enrichment ONT sequencing chọpụtara nwere ike mkpụrụ ndụ ihe na-akpata NIID, GGC ikwugharị expansions na 5′ UTR nke NOTCH2NLC.Ọmụmụ ihe a kọrọ mgbasawanye ugboro ugboro na mkpụrụ ndụ ihe nketa nke mmadụ maka oge izizi sitere na mkpirisi akụkụ.
Usoro 3.RNA kpughere transcript antisense na-adịghị mma na mbido ma ọ bụ n'ime GGC kwugharịa mpaghara mgbasawanye na NOTCH2NLC.
ndabere
NỌrịa intranuclear intranuclear euronal (NIID) bụ ọrịa neurodegenerative na-aga n'ihu ma na-egbu egbu, nke a na-egosipụta site na ọnụnọ eosinophilic hyaline intranuclear inclusions na etiti na sistem ụjọ.Ngosipụta ụlọ ọgwụ na-agbanwe nke ukwuu na-ebute nnukwu ihe isi ike n'ịchọpụta ọrịa ruo mgbe iwebata biopsy anụ ahụ.Otú ọ dị, ụzọ ndị dabeere na histopathology ka na-ata ahụhụ site na nchọpụta na-ezighị ezi, nke na-akpọ maka nghọta mkpụrụ ndụ ihe nketa nke NIID.
Mmezu
Nyocha njikọ
Short-agụ usoro dabere na usoro genome dum (WGS) na usoro exome dum (WES) ka emere na nnukwu ezinụlọ NIID (13 metụtara na ndị otu 7 enweghị nsogbu).Nyocha njikọ na SNP ndị ewepụtara na data ndị a kpughere naanị mpaghara abụọ ejikọtara: mpaghara 3.5 Mb na 1p36.31-p36.22 (kachasị LOD = 2.32) na mpaghara 58.1 Mb na 1p22.1-q21.3 (oke LOD: 4.21). ).Otú ọ dị, ọ dịghị SNPs ma ọ bụ CNV na-akpata ọrịa a chọpụtara na mpaghara ndị a jikọtara.
GGC kwugharịa mgbasawanye na NOTCH2NLC
NA na-ahazi usoro nke dabere na anopore na 13 metụtara yana ndị òtù 4 na-emetụtaghị site na ezinụlọ 8 (onye ọzọ metụtara ya bụ usoro nke Pacbio na-agụ ogologo usoro ikpo okwu.).Data agụchara ogologo oge gosipụtara ọrịa metụtara GGC megharịa mgbasawanye na 5′ UTR nke mkpụrụ ndụ ihe nketa NOTCH2NLC gaa na mpaghara ejikọta 58.1 Mb (Njirimara 1).Achọpụtakwara mgbasawanye ndị a ugboro ugboro n'okwu NIID iri anọ na-adịtụghị oge nke RP-PCR nwalere.
Cas-9 agbazitere lekwasịrị anya sequencing na nanopore n'elu ikpo okwu ka ejiri wee nweta mkpuchi ọgụgụ dị elu na NOTCH2NLC ikwugharị (100 X-1,795 X).Usoro nkwenye ndị a kwekọrịtara nke ọma na nchoputa gara aga na mgbasawanye GGC.Ọzọkwa, {(GGA) n (GGC) n}nkwugharị ka achọpụtara dị ka ihe nwere ike ịkara mkpụrụ ndụ ihe nketa adịghị ike na-achị phenotype (Njirimara 2).
Ọgụgụ 1. Ọrịa metụtara mmebawanye ugboro ugboro achọpụtara na exon 1 nke isoforms NOTCH2NLC.
Ọgụgụ 2. Usoro nkwekọrịta nke NPTCH2NLC na-emegharị na ndị ọrịa NIID nwere (*) ma ọ bụ na-enweghị ike-na-achị phenotype
NMkpụrụ ndụ ihe nketa OTCH2NL bụ mkpụrụ ndụ ihe nketa nke mmadụ, nke ekwenyere na ọ na-ekere òkè dị mkpa na mgbanwe ụbụrụ mmadụ na ọrịa akwara ozi.Otú ọ dị, mkpụrụ ndụ ihe nketa atọ metụtara NOTCH2 (NOTCH2NLA, NOTCH2NLB na NOTCH2NLC) nwere>99.1% njirimara usoro emebeghị ruo mgbe mgbakọ genome mmadụ kacha ọhụrụ.Usoro nke enweghị njikọ na ogologo ọgụgụ na-agụ n'elu ikpo okwu nanopore egosila uru dị ịrịba ama na-edozi mpaghara nke myirịta dị elu na (GGC) n na-emegharị ya na 100% GC-ọgaranya.
GGC kwugharịa mgbasawanye na NOTCH2NLC
TA na-ahazi usoro nke ranscriptome na ndị otu 2 metụtara yana 2 na-emetụtaghị ya.A gbakọrọ omimi agụ nke ọma site n'echiche na strands antisense na elu nke exons mbụ nke paralog NOTCH2NL.Achọpụtara ihe odide mgbochi na-adịghị mma naanị na ikpe metụtara, nke na-anọdụ na mmalite ma ọ bụ n'ime mpaghara mgbasawanye ọzọ (Purple peaks na F1-14 na F1-16 na Figure 3.).Tụkwasị na nke ahụ, a chọpụtara 54 DEGs na ihe niile na-eme ka ọ baa ọgaranya na GO na MPO okwu metụtara ọrụ neuronal.
Ọnụọgụ 3. Nhazi agụ ihe omimi na elu nke exon mbụ nke NOTCH2NLC na-emetụtaghị (n'elu) na emetụta (n'okpuru).
Nkà na ụzụ
Teknụzụ Oxford Nanopore (ONT)
Nusoro nke anopore na-ekewa onwe ya na usoro nhazi usoro ndị ọzọ, n'ihi na a na-agụ nucleotides ozugbo na-enweghị usoro DNA.Dị ka otu eriri DNA na-agafe na nano-sized protein pore (nanopore), nucleotides dị iche iche na-emepụta mmiri ionic dị iche iche, nke enwere ike ijide ma bufee ya n'usoro nke ntọala.Ikpo okwu usoro usoro ONT n'onwe ya egosighi oke nka nka na ogologo ọgụgụ DNA.Ya mere, Ultra-long reads (ULRs) dị maka mgbakọ genome nke dị elu.Ọzọkwa, ọgụgụ ndị a dị ogologo nke ukwuu, nke toro ogologo iji gafee njiri mara usoro dị mgbagwoju anya ma ọ bụ mgbanwe nhazi, na-enyere aka imeri njedebe nke usoro ọgụgụ dị mkpirikpi ebe a.
Nanopore usoro
Nchọpụta mgbanwe nhazi (SV).
SUsoro na-enweghị ynthesis echekwara nke ukwuu ozi methylation DNA na ndebiri.Methylated A, T, C na G na-emepụta mmiri iyi ionic dị iche iche sitere na ndị na-enweghị methylated, nke enwere ike ịgụ ya ozugbo site na ikpo okwu.Usoro Nanopore na-enye ike ịkọwapụta genome dum nke ma 5mC na 6mA na mkpebi otu-nucleotide.
Ntụaka
Jun Sone, et.al.Usoro ngụ ogologo na-achọpụta mgbasawanye GGC na NOTCH2NLC jikọtara ya na ọrịa nsonye intranuclear neuronal.Genetics okike (2019)
Teknụzụ na isi ihe bu n'obi kesaa ngwa kacha aga nke ọma na nso nso a nke teknụzụ usoro usoro dị elu dị iche iche na mpaghara nyocha dị iche iche yana echiche mara mma na nhazi nnwale na ngwuputa data.
Oge nzipu: Jan-06-2022