-
Ukusebenzisana kweChromatin okusekelwe kwiHi-C
I-Hi-C yindlela eyilelwe ukubamba ulwakhiwo lwe-genomic ngokudibanisa unxibelelwano olusekwe ekujongeni ukusondela kunye nokulandelelana okuphezulu. Le ndlela isekelwe kwi-chromatin crosslinking kunye ne-formaldehyde, ilandelwe kukugaya kunye nokudibanisa kwakhona ngendlela yokuba kuphela iziqwenga ezidityaniswe ngokudibeneyo eziya kwenza iimveliso zokudibanisa. Ngokulandelanisa ezi mveliso zokudibanisa, kunokwenzeka ukufunda ulungelelwaniso lwe-3D lwe-genome. I-Hi-C ivumela ukufunda ukusasazwa kweendawo ze-genome ezipakishwe kancinci (ii-compartments ze-A, i-euchromatin) kwaye kunokwenzeka ukuba zisebenze ngokubhala, kunye neendawo ezipakishwe kakhulu (ii-compartments ze-B, i-Heterochromatin). I-Hi-C ingasetyenziselwa ukukhomba ii-Topologically Associated Domains (ii-TAD), iindawo ze-genome ezinezakhiwo ezigobileyo kwaye kusenokwenzeka ukuba zineendlela ezifanayo zokubonakalisa, kunye nokuchonga ii-chromatin loops, iindawo ze-DNA ezinamathele kunye ziiproteni kwaye zihlala zityebile kwizinto ezilawulayo. Inkonzo yokulandelelana kwe-BMKGene ye-Hi-C inika abaphandi amandla okuphonononga ubungakanani bendawo ye-genomics, ivula iindlela ezintsha zokuqonda ukulawulwa kwe-genome kunye nefuthe layo kwimpilo nakwizifo.
-
Ulandelelwano lweChromatin Immunoprecipitation (ChIP-seq)
I-Chromatin Immunoprecipitation (CHIP) yindlela esebenzisa ii-antibodies ukuze ityebise ngokukhethekileyo iiproteni ezibopha i-DNA kunye neethagethi zazo ze-genomics ezihambelanayo. Ukudibana kwayo ne-NGS kwenza ukuba iprofayili ye-genome ngokubanzi yeethagethi ze-DNA ezinxulumene nokuguqulwa kwe-histone, izinto ezibangela ukubhalwa kwe-transcription, kunye nezinye iiproteni ezibopha i-DNA. Le ndlela inamandla ivumela ukuthelekiswa kweendawo zokubopha kwiintlobo ezahlukeneyo zeeseli, izicubu, okanye iimeko. Ukusetyenziswa kwe-ChIP-Seq kuqala ekufundeni ummiselo wokubhalwa kwe-transcription kunye neendlela zophuhliso ukuya ekucaciseni iindlela zesifo, okwenza kube sisixhobo esibalulekileyo sokuqonda iindawo zommiselo we-genomic kunye nokuqhubela phambili ukuqonda ngonyango.
Iqonga: Illumina NovaSeq
-
Ulandelelwano lwe-bisulfite ye-genome epheleleyo (WGBS)
Le ndlela, esekelwe kunyango lwe-bisulfite lwe-DNA ukuze kuguqulwe i-cytosines ezingenamethylated zibe yi-uracil (C ukuya kwi-U), ngelixa ishiya i-cytosines ezinemethylated zingatshintshanga, imi njengendlela esemgangathweni ophezulu yokuhlola ngokunzulu i-methylation ye-DNA, ingakumbi indawo yesihlanu kwi-cytosine (5-mC), umlawuli obalulekileyo wokubonakaliswa kwezakhi zofuzo kunye nomsebenzi weseli.
Le ndlela inika isisombululo esisisiseko esinye, ivumela abaphandi ukuba baphande ngokupheleleyo i-methylome baze bafumanise iipatheni ze-methylation ezingaqhelekanga kwiisampuli. Ngokusebenzisa i-WGBS, izazinzulu zinokufumana ulwazi olungenakuthelekiswa nanto malunga neendawo ze-methylation ezijikelezileyo ze-genome, zibonelela ngokuqonda okucacileyo kweendlela ze-epigenetic ezisisiseko seenkqubo ezahlukeneyo zebhayoloji kunye nezifo.
-
Uvavanyo lweChromatin efikelelekayo kwiTransposase ene-High Throughput Sequencing (ATAC-seq)
I-ATAC-seq yindlela yokulandelelanisa ephezulu esetyenziselwa uhlalutyo lokufikelela kwi-chromatin kwi-genome. Ukusetyenziswa kwayo kunika ukuqonda okunzulu ngeendlela ezintsonkothileyo zolawulo lwe-epigenetic lwehlabathi phezu kokubonakaliswa kwezakhi zofuzo. Le ndlela isebenzisa i-transposase ye-Tn5 esebenzayo kakhulu ukuze iqhekeze ngaxeshanye kwaye ivule imimandla ye-chromatin ngokufaka ii-adaptha zokulandelelanisa. Ukwandiswa kwe-PCR okulandelayo kuphumela ekudalweni kwethala leencwadi lokulandelelanisa, elivumela ukuchongwa okubanzi kwemimandla evulekileyo ye-chromatin phantsi kweemeko ezithile zesithuba-sexesha. I-ATAC-seq ibonelela ngombono opheleleyo weendawo ze-chromatin ezifikelelekayo, ngokungafaniyo neendlela ezigxila kuphela kwiindawo zokubopha izinto ze-transcription okanye kwiindawo ezithile eziguqulwe yi-histone. Ngokulandelanisa ezi ndawo zivulekileyo ze-chromatin, i-ATAC-seq ityhila iindawo ezinokuba namathuba amaninzi okulandelana okusebenzayo kunye neendawo ezinokubakho zokubopha izinto ze-transcription, zibonelela ngengqiqo ebalulekileyo kwi-dynamic modulation ye-gene expression kuyo yonke i-genome.
-
Ulandelelwano lweBisulfite oluNciphisiweyo (RRBS)
Ukulandelelana kwe-bisulfite okuncitshisiweyo (RRBS) kuxhomekeke ekutyebiseni iindawo ezityebileyo kwiziqithi zeCpG nge-MspI cleavage elandelwa kukukhethwa kobungakanani beengceba ze-200-500/600 bps. Ngenxa yoko, ziindawo ezikufutshane neziqithi zeCpG kuphela ezilandelelaniswayo, ngelixa ezo zineziqithi zeCpG ezikude zingabandakanywa kuhlalutyo. Le nkqubo, idibene ne-bisulfite sequencing, ivumela ukufunyanwa kwe-DNA methylation enesisombululo esiphezulu, kwaye indlela yokulandelelana, i-PE150, igxile ngokukodwa kwiziphelo ze-inserts endaweni ye-middle, nto leyo enyusa ukusebenza kakuhle kwe-methylation profiling.
Le ndlela ivele njengendlela engabizi kakhulu nefanelekileyo yokutshintsha i-Whole Genome Bisulfite Sequencing (WGBS) kuphando lwe-DNA methylation. Ngelixa i-WGBS inika ulwazi olupheleleyo ngokuhlola i-genome yonke kwisisombululo sesiseko esinye, ixabiso layo eliphezulu linokuba yinto ethintelayo, ethi i-RRBS inciphise lo mngeni ngokucwangcisa ngokukhetha inxalenye emele i-genome. Le ndlela sisixhobo esixabisekileyo esenza uphando lwe-DNA methylation lube nexabiso eliphantsi kwaye iqhubele phambili ulwazi ngeendlela ze-epigenetic.
