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I-Hi-C esekelwe kwi-Chromatin Interaction
I-Hi-C yindlela eyilelwe ukubamba ukucwangciswa kwe-genomic ngokudibanisa ukunxibelelana okusekwe ngokusondeleyo kunye nokulandelelana okuphezulu. Indlela isekelwe kwi-chromatin crosslinking kunye ne-formaldehyde, ilandelwa yi-digestion kunye ne-re-ligation ngendlela yokuba kuphela amaqhekeza adityaniswe ngokubambisana aya kwenza iimveliso ze-ligation. Ngokulandelelana kwezi mveliso ze-ligation, kunokwenzeka ukuba ufunde umbutho we-3D we-genome. I-Hi-C yenza ukuba kufundwe ukuhanjiswa kwezahlulo zegenome ezipakishwe kancinci (i-A compartments, euchromatin) kwaye kunokwenzeka ukuba zisebenze ngokubhaliweyo, kunye nemimandla epakishwe ngokuqinileyo (i-B compartments, Heterochromatin). I-Hi-C ingasetyenziselwa ukukhomba i-Topologically Associated Domains (TADs), imimandla ye-genome enezakhiwo ezisongiweyo kwaye kusenokwenzeka ukuba ibe neepatheni zokubonisa ezifanayo, kunye nokuchonga i-chromatin loops, imimandla ye-DNA edityaniswe kunye neeprotheni kwaye ehlala etyetyiswe kwizinto zolawulo. Inkonzo yolandelelwano ye-Hi-C ye-BMKGene ye-Hi-C ixhobisa abaphandi ukuba baphonononge ubungakanani bendawo ye-genomics, bavule iindlela ezintsha zokuqonda ukulawulwa kwegenome kunye neziphumo zayo kwimpilo nakwizifo.
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I-Chromatin Immunoprecipitation Sequencing (ChIP-seq)
I-Chromatin Immunoprecipitation (CHIP) bubuchule obusebenzisa izilwa-buhlungu ukuba zityebise ngokukhetha iiproteni ezibophelela i-DNA kunye neethagethi zazo ezihambelana ne-genomics. Ukuhlanganiswa kwayo kunye ne-NGS kwenza ukuba i-genome-wide profiling yeethagethi ze-DNA ezinxulumene nokuguqulwa kwe-histone, izinto ezibhaliweyo, kunye nezinye iiprotheni ezibophelelayo ze-DNA. Le ndlela iguquguqukayo yenza uthelekiso lweendawo ezibophelelayo kuzo zonke iintlobo zeeseli ezahlukeneyo, izicubu, okanye iimeko. Izicelo ze-ChIP-Seq zisusela ekufundeni umgaqo woshicilelo kunye neendlela zophuhliso ukuya ekucaciseni iindlela zesifo, oko kuyenza ibe sisixhobo esibalulekileyo sokuqonda ubume bolawulo lwe-genomic kunye nokuqhubela phambili kweembono zonyango.
Iqonga: Illumina NovaSeq
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I-Whole genome bisulfite sequencing(WGBS)
I-Whole Genome Bisulfite Sequencing (WGBS) ime njengendlela yomgangatho wegolide wokuhlola nzulu kwe-DNA methylation, ngokukodwa indawo yesihlanu kwi-cytosine (5-mC), umlawuli obalulekileyo we-gene expression kunye nomsebenzi weselula. Umgaqo ophantsi kwe-WGBS ubandakanya unyango lwe-bisulfite, ukuguqula ukuguqulwa kwe-cytosines engaxutywanga kwi-uracil (C ukuya ku-U), ngelixa ishiya i-cytosine ye-methylated ingatshintshi. Obu buchule bubonelela ngesisombululo esisisiseko esinye, sivumela abaphandi ukuba baphande ngokubanzi i-methylome kwaye bafumanise iipateni ze-methylation ezingaqhelekanga ezinxulumene neemeko ezahlukeneyo, ngakumbi umhlaza. Ngokusebenzisa i-WGBS, izazinzulu zinokufumana ulwazi olungenakuthelekiswa nanto kwi-genome-wide methylation landscapes, ukunika ukuqonda okucacileyo kweendlela ze-epigenetic eziphantsi kweenkqubo ezahlukeneyo zebhayoloji kunye nezifo.
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I-Assay ye-Chromatin eFumanekayo ye-Transposase ene-High throughput Sequencing (ATAC-seq)
I-ATAC-seq yindlela yolandelelwano oluphezulu olusetyenziselwa uhlalutyo lokufikeleleka kwechromatin yegenome ngokubanzi. Ukusetyenziswa kunika ukuqonda okunzulu kwiindlela ezintsonkothileyo zolawulo lwe-epigenetic lwehlabathi malunga nokuchazwa kofuzo. Indlela isebenzisa i-hyperactive Tn5 transposase ukuqhekeza ngaxeshanye kwaye ithegi imimandla yechromatin evulekileyo ngokufaka iiadaptha zolandelelwano. Ukwandiswa okulandelayo kwePCR iziphumo ekudalweni kwethala leencwadi elilandelelanayo, elivumela ukuchongwa ngokubanzi kwemimandla yechromatin evulekileyo phantsi kweemeko ezithile zexesha lesithuba. I-ATAC-seq ibonelela ngombono obanzi wokufikeleleka kwe-chromatin landscapes, ngokungafaniyo neendlela ezigxile kuphela kwiindawo ezibophelelayo ze-transcription factor okanye imimandla ethile ye-histone-modified. Ngokulandelelanisa le mimandla yechromatin ivulekileyo, i-ATAC-seq ityhila imimandla ekunokwenzeka ukuba ibe nolandelelwano lolawulo olusebenzayo kunye neendawo ezinokubakho zokukhuphela izinto ezibophelelayo, inikezela ngezimvo ezixabisekileyo kumodareyithwa oguqukayo wokubonakaliswa kofuzo kuyo yonke igenome.
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Umekelo oluNcitshisiweyo lweBisulfite Sequencing (RRBS)
I-Reduced Representation Bisulfite Sequencing (RRBS) iye yavela njengendlela engabizi kwaye esebenzayo kwi-Whole Genome Bisulfite Sequencing (WGBS) kuphando lwe-DNA methylation. Ngelixa i-WGBS ibonelela ngemibono ebanzi ngokuphonononga yonke i-genome kwisisombululo sesiseko esinye, iindleko zayo eziphezulu zinokuba ngumba osisithintelo. I-RRBS ngobuchule iwunciphisa lo mngeni ngokuhlalutya ngokukhetha inxalenye emele yegenome. Le ndlela yokusebenza ixhomekeke ekutyebiseni imimandla etyebileyo yeziqithi ze-CpG ngokucolwa kwe-MspI kulandele ukukhethwa kobungakanani bamaqhekeza angama-200-500/600 e-bps. Ngenxa yoko, kuphela imimandla esondele kwiziqithi ze-CpG ezilandelelanayo, ngelixa ezo zineziqithi ze-CpG ezikude zingabandakanywa kuhlalutyo. Le nkqubo, idibene ne-bisulfite sequencing, ivumela ukufunyanwa kwe-high-resolution ye-DNA methylation, kunye nendlela yokulandelelana, i-PE150, igxininise ngokukodwa kwiiphelo zokufaka esikhundleni se-middle, ukwandisa ukusebenza kakuhle kwe-methylation profiling. I-RRBS sisixhobo esixabisekileyo esenza ukuba uphando lwe-DNA methylation lungabizi kakhulu kwaye luqhubele phambili ulwazi lweendlela ze-epigenetic.
