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I-Single-nucleus RNA Sequencing
Ukuthuthuka ekuthwebuleni iseli elilodwa kanye nezindlela zokwakha umtapo wolwazi ngamunye okuhlanganiswe nokulandelana komphumela ophezulu kuvumela izifundo zesisho sofuzo ngesisekelo seseli-by-cell.Inika amandla ukuhlaziya okujulile nokuphelele kwesistimu kumaseli ayinkimbinkimbi, lapho igwema khona kakhulu ukufihla ukuhlukahluka kwawo ngokuthatha isilinganiso sawo wonke amaseli.
Kodwa-ke, amanye amangqamuzana awafanele ukwenziwa abe yi-single-cell ukumiswa, ngakho-ke ezinye izindlela zokulungiselela isampula - ukukhishwa kwe-nucleus ezicutshini ziyadingeka, okungukuthi, i-nucleus ikhishwa ngokuqondile kumathishu noma ingqamuzana futhi ilungiselwe ukumiswa kwe-nucleus eyodwa ukuze kusetshenziswe eyodwa- ukulandelana kweseli.
I-BMK ihlinzeka ngesevisi yokulandelana kwe-RNA eyi-10× Genomics ChromiumTM.Le nsizakalo isetshenziswe kakhulu ezifundweni eziphathelene nezifo, njengokuhlukaniswa kwamaseli omzimba, i-tumor heterogeneity, ukuthuthukiswa kwezicubu, njll.
I-Spatial transcriptome chip: 10× Genomics
Ipulatifomu: I-Illumina NovaSeq Platform
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I-BMKMANU S1000 Spatial Transcriptome
I-Spatial transcriptomics imi phambili ekusungulweni kwesayensi, inika amandla abacwaningi ukuthi bahlole amaphethini enkulumo eyinkimbinkimbi yezakhi zofuzo phakathi kwezicubu kuyilapho belondoloza isimo sabo sendawo.Phakathi kwamapulatifomu ahlukahlukene, i-BMKGene ithuthukise i-BMKManu S1000 Spatial Transcriptome Chip, iziqhayisaukulungiswa okuthuthukisiweyokungu-5µM, ifinyelela kububanzi obuncane beselula, futhi inika amandlaizilungiselelo zokulungiswa kwamazinga amaningi.I-chip ye-S1000, equkethe cishe izindawo eziyizigidi ezingu-2, isebenzisa ama-microwell agqitshwe ngobuhlalu obulayishwe ama-probe wokuthwebula anebhakhodi yendawo.Umtapo wezincwadi we-cDNA, ohlanganiswe namabhakhodi wendawo, ulungiswa kusukela ku-chip ye-S1000 bese ulandelana ngokulandelana endaweni ye-Illumina NovaSeq.Inhlanganisela yamasampula anebhakhodi yendawo kanye nama-UMI kuqinisekisa ukunemba nokucaciswa kwedatha ekhiqizwayo.Isibaluli esiyingqayizivele se-chip ye-BMKManu S1000 silele ekusebenziseni kwayo izinto ezihlukahlukene, enikeza izilungiselelo zokuxazulula amaleveli amaningi ezingashunwa kahle kumathishu ahlukene namazinga emininingwane.Lokhu kuzivumelanisa nezimo kubeka i-chip njengenketho evelele yezifundo ezihlukahlukene ze-spatial transcriptomics, iqinisekisa ukuhlangana okunembayo kwendawo okunomsindo omncane.
Besebenzisa i-chip ye-BMKManu S1000 nobunye ubuchwepheshe be-transcriptomics yendawo, abacwaningi bangathola ukuqonda okungcono kwenhlangano yendawo yamangqamuzana kanye nokusebenzisana okuyinkimbinkimbi kwamangqamuzana okwenzeka ngaphakathi kwezicubu, kunikeze ukuqonda okubalulekile kwezinqubo eziyisisekelo zezinqubo zebhayoloji emikhakheni eminingi ehlukahlukene, kufaka phakathi. i-oncology, i-neuroscience, i-development biology, i-immunology kanye nezifundo ze-botanical.
I-Platform: BMKManu S1000 chip kanye ne-Illumina NovaSeq
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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
Nakuba ukulandelana kwe-mRNA okususelwe ku-NGS kusebenza njengethuluzi eliguquguqukayo lokulinganisa isisho sofuzo, ukuthembela kwayo ekufundeni okufushane kuvimbela ukusebenza kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic.Ukulandelana kwe-Nanopore, ngakolunye uhlangothi, kusebenzisa ubuchwepheshe okufundwa 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 kuncike kumasiginali kagesi wesikhathi sangempela se-nanopore.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 yokuhlaziya i-genomic eyinkimbinkimbi, ehlanganisa izifundo eziyinkimbinkimbi ze-transcriptomic.
Ipulatifomu: Nanopore Promethion P48
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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.I-PacBio sequencing (Iso-Seq), ngakolunye uhlangothi, 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 nakuba kungeyona inketho eyinhloko yokulinganisa 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 ukusetshenziswa kwamagagasi e-zero-mode waveguides (ZMWs), imithombo 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
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I-Eukaryotic mRNA Sequencing-Illumina
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
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I-Non-Reference based mRNA Sequencing-Illumina
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
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Ukulandelana okungafaki amakhodi okude-Illumina
Ama-RNA amade angewona amakhodi (lncRNA) angama-RNA amade kunama-nucleotide angama-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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Ukulandelana kwe-RNA encane-Illumina
Ama-molecule amancane e-RNA (sRNA), ngokuvamile angaphansi kwama-nucleotide angu-200 ubude, ahlanganisa ama-microRNA (miRNAs), ama-RNA amancane aphazamisayo (siRNAs), kanye nama-RNA (piRNAs) asebenzisana ne-piwi.Kulokhu, ama-miRNA, acishe abe ngama-nucleotide angama-20-24 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 ngokusimama okwengeziwe.Ngenkathi amanye ama-circRNA eboniswe esebenza njengezipontshi ze-microRNA, elandela ama-microRNA futhi awavimbe ekulawuleni ama-mRNA awo aqondiwe, amanye ama-circRNA angase asebenzisane namaprotheni, alungise ukubonakaliswa kofuzo, noma abe nezindima ezinqubweni zamaselula.Ukuhlaziywa kwenkulumo ye-circRNA kunikeza imininingwane ngezindima zokulawula zala ma-molecule kanye nokubaluleka kwawo ezinqubweni ezihlukahlukene zamaselula, izigaba zokukhula, 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, ahlanganisa kokubili amakhodi (mRNA) nama-RNA angafaki amakhodi (lncRNA, circRNA, kanye ne-miRNA).Le nqubo ithwebula i-transcriptome ephelele 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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Ukulandelana kwe-Prokaryotic mRNA
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.Ukucubungula kwethu kwesampula ye-prokaryotic mRNA kwenzelwe imibhalo ye-prokaryotic, ebandakanya ukuncipha kwe-rRNA kanye nokulungiselela umtapo wolwazi.
Ipulatifomu: Illumina NovaSeq X
