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I-Genome-wide Association Analysis
Inhloso ye-Genome-Wide Association Studies (GWAS) ukukhomba okuhlukile kofuzo (ama-genotype) axhunywe nezici ezithile (phenotypes). Ngokuhlolisisa izimpawu zofuzo kulo lonke i-genome ngenani elikhulu labantu, i-GWAS ikhipha izinhlangano ze-genotype-phenotype ngokuhlaziywa kwezibalo zezinga labantu. Le ndlela yokwenza ithola ukusetshenziswa okubanzi ekucwaningeni izifo zabantu kanye nokuhlola izakhi zofuzo ezisebenzayo ezihlobene nezici eziyinkimbinkimbi ezilwaneni noma ezitshalweni.
Kwa-BMKGENE, sinikeza izindlela ezimbili zokuqhuba i-GWAS kubantu abaningi: ukusebenzisa i-Whole-Genome Sequencing (WGS) noma ukukhetha indlela encishisiwe yokumelela igenome yokulandelanisa, i-Specific-Locus Amplified Fragment (SLAF) ethuthukisiwe endlini. Nakuba i-WGS ifanele ama-genome amancane, i-SLAF ivela njengendlela engabizi kakhulu yokutadisha abantu abaningi abanama-genome amade, inciphisa ngempumelelo izindleko zokulandelana, kuyilapho iqinisekisa ukutholakala komaka okuphezulu kofuzo.
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I-Single-nuclei RNA Sequencing
Ukuthuthukiswa kokuthwebula iseli elilodwa nezindlela zokwakha umtapo wolwazi wangokwezifiso, kuhlanganiswe nokulandelana komphumela ophezulu, kuguqule izifundo zesisho sofuzo ezingeni leseli. Lokhu kuphumelela kuvumela ukuhlaziywa okujulile nokubanzi kakhulu kwezibalo zamangqamuzana ayinkimbinkimbi, kweqa imikhawulo ehambisana nokuvezwa kwezakhi zofuzo okumaphakathi kuwo wonke amaseli kanye nokulondoloza ukuhlukahluka kwangempela phakathi kwalezi zibalo. Nakuba ukulandelana kwe-RNA yeseli elilodwa (scRNA-seq) kunezinzuzo ezingenakuphikwa, kuhlangabezana nezinselele kumathishu athile lapho ukwakhiwa kokumiswa kweseli elilodwa kuba nzima futhi kudinga amasampula amasha. Kwa-BMKGene, sibhekana nalesi sithiyo ngokunikeza ukulandelana kwe-nucleus eyodwa ye-RNA (snRNA-seq) sisebenzisa ubuchwepheshe besimanje be-10X Genomics Chromium. Le ndlela yenza banzi i-spectrum yamasampula akwazi ukuhlaziya i-transcriptome ezingeni leseli elilodwa.
Ukuhlukaniswa kwe-nuclei kufezwa nge-chip ye-10X Genomics Chromium, enohlelo lwe-microfluidics lweziteshi eziyisishiyagalombili oluwela kabili. Ngaphakathi kwalolu hlelo, ubuhlalu bejeli obuhlanganisa amabhakhodi, ama-primers, ama-enzyme, kanye ne-nucleus eyodwa kuhlanganiswe namaconsi kawoyela alingana ne-nanoliter, okwenza i-Gel Bead-in-Emulsion (GEM). Ukulandela ukwakheka kwe-GEM, i-cell lysis kanye nokukhululwa kwebhakhodi kwenzeka ngaphakathi kwe-GEM ngayinye. Kamuva, ama-molecule e-mRNA abhalwa ngokuhlehlayo abe ama-cDNA, afaka amabhakhodi angu-10X kanye Nezihlonzi Zemolekyuli Ehlukile (UMIs). Lawa ma-cDNA abe esengaphansi kokwakhiwa komtapo wolwazi wokulandelana okujwayelekile, okusiza ukuhlola okuqinile nokuphelele kwamaphrofayili wokuvezwa kofuzo ezingeni leseli elilodwa.
Inkundla: 10× Genomics Chromium kanye ne-Illumina NovaSeq Platform
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Ukulandelana Kohlobo Lonke Lwezitshalo/Izilwane
I-Whole Genome Sequencing (WGS), eyaziwa nangokuthi i-requencing, ibhekisela kukho konke ukulandelana kofuzo lwabantu abahlukene bezinhlobo zofuzo ezaziwayo eziyireferensi. Ngalesi sisekelo, ukuhlukana kwe-genomic kwabantu noma imiphakathi kungabuye kukhonjwe. I-WGS inika amandla ukuhlonzwa kwe-Single Nucleotide Polymorphism (SNP), Ukususwa Kokufaka (InDel), Ukuhluka Kwesakhiwo (SV), kanye Nokwehluka Kwenombolo Yekhophi (CNV). Ama-SV ahlanganisa ingxenye enkulu yesisekelo sokuhluka kunama-SNP futhi anomthelela omkhulu ku-genome, okuthinta kakhulu izinto eziphilayo. Nakuba ukuphinda kufundwe okufushane kuphumelela ekuhlonzeni ama-SNP nama-InDels, ukuphinda kufundwe isikhathi eside kuvumela ukuhlonza okunembe kakhudlwana kwezingcezu ezinkulu nokuhluka okuyinkimbinkimbi.
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I-10x Genomics Visium Spatial Transcriptome
I-Spatial transcriptomics ubuchwepheshe obusezingeni eliphezulu obuvumela abacwaningi ukuthi baphenye amaphethini wokusho izakhi zofuzo ngaphakathi kwezicubu ngenkathi belondoloza umongo wabo wendawo. Inkundla eyodwa enamandla kulesi sizinda yi-10x Genomics Visium ehambisana nokulandelana kwe-Illumina. Umgomo we-10X Visium ulele ku-chip ekhethekile enendawo ekhethiwe yokuthwebula lapho kubekwa khona izingxenye zezicubu. Le ndawo yokuthwebula iqukethe izindawo ezinebhakhodi, ngalinye lihambisana nendawo ehlukile ngaphakathi kwethishu. Ama-molecule e-RNA athunjiwe asuka kusicubu abe eselebula ngezihlonzi eziyingqayizivele zamangqamuzana (UMIs) phakathi nenqubo yokuhlehla yokubhala. Lezi zindawo ezinebhakhodi kanye nama-UMI anika amandla ukumepha indawo okunembayo kanye nomthamo wenkulumo yofuzo ekulungisweni kweseli elilodwa. Inhlanganisela yamasampula anebhakhodi yendawo kanye nama-UMI kuqinisekisa ukunemba nokucaciswa kwedatha ekhiqizwayo. Ngokusebenzisa lobu buchwepheshe be-Spatial Transcriptomics, abacwaningi bangathola ukuqonda okujulile kwenhlangano yendawo yamangqamuzana kanye nokusebenzisana okuyinkimbinkimbi kwamangqamuzana okwenzeka ngaphakathi kwezicubu, okuhlinzeka ngemininingwane ebalulekile ezindleleni eziyisisekelo zezinqubo zebhayoloji emikhakheni eminingi, okubandakanya i-oncology, neuroscience, biology yentuthuko, i-immunology. , kanye nezifundo ze-botanical.
I-Platform: 10X Genomics Visium kanye ne-Illumina NovaSeq
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Ukulandelana kwe-mRNA yobude obugcwele-Nanopore
Ngenkathi ukulandelana kwe-mRNA okususelwe ku-NGS kuyithuluzi eliguquguqukayo lokulinganisa ukusho kofuzo, ukuthembela kwayo ekufundeni okufushane kuvimbela ukusebenza kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic. Ngakolunye uhlangothi, ukulandelana kwe-nanopore kusebenzisa ubuchwepheshe obufundwe isikhathi eside, okuvumela ukulandelana kwemibhalo yobude obugcwele ye-mRNA. Le ndlela isiza ukuhlola okuphelele kokunye ukuhlanganisa, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation, kanye nokulinganisa kwe-mRNA isoforms.
Ukulandelana kwe-Nanopore, indlela ethembele kumasiginali kagesi we-nanopore single-molecule yesikhathi sangempela, inikeza imiphumela ngesikhathi sangempela. Iqondiswa amaphrotheni enjini, i-DNA enemicu ekabili ibophezela kumaprotheni e-nanopore ashumekwe ku-biofilm, ivuleke njengoba idlula kumzila we-nanopore ngaphansi komehluko we-voltage. Izimpawu zikagesi ezihlukile ezikhiqizwe izisekelo ezihlukene kuchungechunge lwe-DNA ziyatholwa futhi zihlukaniswe ngesikhathi sangempela, kube lula ukulandelana kwe-nucleotide okunembile nokuqhubekayo. Le ndlela yokusungula inqoba imikhawulo yokufunda okufushane futhi inikeza inkundla eguquguqukayo yokuhlaziywa kwe-genomic okuyinkimbinkimbi, okuhlanganisa izifundo eziyinkimbinkimbi ze-transcriptomic, ezinemiphumela esheshayo.
I-Platform: Nanopore PromethION 48
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Ukulandelana kwe-mRNA yobude obugcwele -PacBio
Ngenkathi ukulandelana kwe-mRNA okususelwe ku-NGS kuyithuluzi eliguquguqukayo lokulinganisa ukusho kofuzo, ukuthembela kwayo ekufundeni okufushane kuvimbela ukusetshenziswa kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic. Ngakolunye uhlangothi, i-PacBio sequencing (Iso-Seq) isebenzisa ubuchwepheshe obufundwe isikhathi eside, ivumela ukulandelana kwemibhalo yobude obugcwele ye-mRNA. Le ndlela isiza ukuhlola okuphelele kokunye ukuhlanganisa, ukuhlanganisa izakhi zofuzo, kanye ne-poly-adenylation. Nokho, kukhona ezinye izinketho zokulinganisa isisho sofuzo ngenxa yenani eliphezulu ledatha edingekayo. Ubuchwepheshe bokulandelana kwe-PacBio buthembele ekulandeleni kwe-molecule eyodwa, ngesikhathi sangempela (SMRT), buhlinzeka ngenzuzo ehlukile ekuthwebuleni imibhalo yobude obugcwele ye-mRNA. Le ndlela emisha ihilela ukusebenzisa i-zero-mode waveguides (ZMWs) kanye nemithombo eyenziwe nge-microfabricated eyenza ukubonwa kwesikhathi sangempela komsebenzi we-DNA polymerase ngesikhathi sokulandelana. Ngaphakathi kwala ma-ZMW, i-DNA polymerase ye-PacBio ihlanganisa umucu ohambisanayo we-DNA, ikhiqize ukufunda okude okuhlanganisa yonke imibhalo ye-mRNA. Ukusebenza kwe-PacBio kumodi ye-Circular Consensus sequencing (CCS) kuthuthukisa ukunemba ngokulandelanisa ngokuphindaphindiwe i-molecule efanayo. Ukufundwa kwe-HiFi okukhiqiziwe kunokunemba okuqhathaniswa ne-NGS, okuqhubeka kube negalelo ekuhlaziyweni okuphelele nokuthembekile kwezici eziyinkimbinkimbi ze-transcriptomic.
I-Platform: I-PacBio Sequel II; I-PacBio Revio
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I-Eukaryotic mRNA Sequencing-NGS
Ukulandelana kwe-mRNA, ubuchwepheshe obushintshashintshashintshayo, bunika amandla ukugcwaliseka okuphelele kwayo yonke imibhalo ye-mRNA ngaphakathi kwamaseli ngaphansi kwezimo ezithile. Ngokusebenza kwalo okubanzi, leli thuluzi elisezingeni eliphezulu lembula amaphrofayili wokuchazwa kwezakhi zofuzo, izakhiwo zofuzo, nezindlela zamangqamuzana ezihambisana nezinqubo ezihlukahlukene zebhayoloji. Yamukelwe kabanzi ocwaningweni oluyisisekelo, ekuxilongweni komtholampilo, nasekuthuthukisweni kwezidakamizwa, ukulandelana kwe-mRNA kunikeza imininingwane eyinkimbinkimbi yokuguquguquka kwamaselula nokulawulwa kofuzo, okuvusa ilukuluku mayelana namandla ako emikhakheni eyahlukene.
Ipulatifomu: Illumina NovaSeq X; I-DNBSEQ-T7
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I-Non-Reference based mRNA Sequencing-NGS
Ukulandelana kwe-mRNA kunika amandla ukuphrofayili okuphelele kwayo yonke imibhalo ye-mRNA ngaphakathi kwamaseli ngaphansi kwezimo ezithile. Lobu buchwepheshe obusezingeni eliphezulu busebenza njengethuluzi elinamandla, lembula amaphrofayli ayinkimbinkimbi enkulumo yofuzo, izakhiwo zofuzo, nezindlela zamangqamuzana ezihambisana nezinqubo ezihlukahlukene zebhayoloji. Yamukelwe kabanzi ocwaningweni oluyisisekelo, ekuxilongweni komtholampilo, nasekuthuthukisweni kwezidakamizwa, ukulandelana kwe-mRNA kunikeza imininingwane ebunkingeni bokuguquguquka kwamaselula kanye nokulawulwa kofuzo.
Ipulatifomu: Illumina NovaSeq X; I-DNBSEQ-T7
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I-Illumina Yokulandelanisa Okungeyona ikhodi ende
Ama-RNA amade angafaki ikhodi (lncRNAs) made kunama-nucleotide angu-200 anamandla amancane okubhala amakhodi futhi ayizici ezibalulekile ngaphakathi kwe-RNA engakhohlisi. Kutholakala ku-nucleus ne-cytoplasm, lawa ma-RNA adlala indima ebalulekile kumthethonqubo we-epigenetic, transcriptal, kanye nowangemuva kokubhala, egcizelela ukubaluleka kwawo ekubumbeni izinqubo zamangqamuzana namangqamuzana. Ukulandelana kwe-LncRNA iyithuluzi elinamandla ekuhlukaniseni amaseli, i-Ontogenesis, kanye nezifo zabantu.
Ipulatifomu: Illumina NovaSeq
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I-RNA Encane Ukulandelanisa-Illumina
Ama-molecule amancane e-RNA (sRNA), afaka ama-microRNAs (miRNAs), ama-RNA amancane aphazamisayo (siRNAs), nama-RNA asebenzisana ne-piwi (piRNAs). Phakathi kwalokhu, ama-miRNA, cishe ama-nucleotide angu-18-25 ubude, aphawuleka kakhulu ngezindima zawo zokulawula ezibalulekile ezinqubweni ezihlukahlukene zamaselula. Ngamaphethini okukhuluma aqondene nezicubu kanye nesiteji, ama-miRNA abonisa ukongiwa okuphezulu kuzo zonke izinhlobo zezilwane.
Ipulatifomu: Illumina NovaSeq
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I-CircRNA Sequencing-Illumina
Ukulandelana kwe-RNA eyindilinga (circRNA-seq) iwukuphrofayili nokuhlaziya ama-RNA ayindilinga, ikilasi lama-molecule e-RNA akha amalophu avaliwe ngenxa yezehlakalo ezihlanganisa i-canonical splicing, okuhlinzeka le RNA ngokuzinza okwengeziwe. Ngenkathi amanye ama-circRNA aboniswe esebenza njengezipontshi ze-microRNA, elandela ama-microRNA futhi awavimbe ekulawuleni ama-mRNA awo aqondiwe, amanye ama-circRNA angase asebenzisane namaprotheni, alungise ukwakheka kofuzo, noma abe nezindima ezinqubweni zamaselula. Ukuhlaziywa kwenkulumo ye-circRNA kunikeza imininingwane ngezindima zokulawula zala ma-molecule kanye nokubaluleka kwawo ezinqubweni ezihlukahlukene zamaselula, izigaba zokuthuthuka, nezimo zezifo, okunikela ekuqondeni okujulile kobunzima bokulawulwa kwe-RNA kumongo wokuvezwa kwezakhi zofuzo.
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Ukulandelana Okuphelele kwe-Transcriptome - Illumina
Ukulandelana okuphelele kwe-transcriptome kunikeza indlela ebanzi yokuphrofayilila ama-molecule e-RNA ahlukahlukene, afaka amakhodi (mRNA) nama-RNA angafaki amakhodi (lncRNA, circRNA, kanye ne-miRNA). Le nqubo ithwebula yonke i-transcriptome yamaseli athile ngesikhathi esithile, okuvumela ukuqonda okuphelele kwezinqubo zamaselula. Okwaziwa nangokuthi "ingqikithi yokulandelana kwe-RNA," ihlose ukwambula amanethiwekhi okulawula ayinkimbinkimbi ezingeni le-transcriptome, okuvumela ukuhlaziya okujulile okufana nokuncintisana kwe-Endo native RNA (ceRNA) kanye nokuhlaziywa kwe-RNA okuhlanganyelwe. Lokhu kuphawula isinyathelo sokuqala sokwenza abalingisi abasebenzayo, ikakhulukazi ekwembuleni amanethiwekhi okulawula ahlanganisa ukusebenzisana kwe-ceRNA okusekelwe ku-circRNA-miRNA-mRNA.
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I-Chromatin Immunoprecipitation Sequencing (ChIP-seq)
I-Chromatin Immunoprecipitation (CHIP) iyindlela esebenzisa amasosha omzimba ukuze inothise ngokukhetha amaprotheni abopha i-DNA kanye nemigomo yawo ehambisanayo ye-genomics. Ukuhlanganiswa kwayo ne-NGS kuvumela ukuhlonza okubanzi kwe-genome kokuhlosiwe kwe-DNA okuhlotshaniswa nokuguqulwa kwe-histone, izici zokuloba, namanye amaprotheni abopha i-DNA. Le ndlela eguquguqukayo inika amandla ukuqhathaniswa kwezindawo ezibophayo kuzo zonke izinhlobo zamaseli ahlukahlukene, izicubu, noma izimo. Izinhlelo zokusebenza ze-ChIP-Seq zisukela ekutadisheni imithetho yokubhala kanye nezindlela zokuthuthuka ziye ekucaciseni izindlela zezifo, okuyenza ibe ithuluzi elibalulekile lokuqonda ukubusa kokwakheka kwezwe kanye nokuthuthukiswa kwemininingwane yokwelapha.
Ipulatifomu: Illumina NovaSeq