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Iiproteomics
I-Proteomics igxile kwiiproteni—abaphathi bemisebenzi yobomi edlala indima ebalulekileyo kulawulo lokubhalwa kwezinto eziphilayo. Ihlalutya ukwakheka, amanqanaba okubonakaliswa, kunye neemeko zokuguqulwa kwazo zonke iiproteni ezitshintsha ngokuguquguqukayo kwizicwili okanye kwiiseli, ijongana nempembelelo ebalulekileyo ye-proteome abubuninzi be-dynamics kwiinkqubo ezahlukeneyo zobomi. Isetyenziswa kakhulu kumayeza, kwezolimo, nakwimfuyo. I-Qualitative Proteomics isebenzisa iteknoloji yokuchonga iiproteni ze-HPLC-MS/MS ukuchonga iisampuli kuquka imicu yejeli, i-IP, kunye neesampuli ze-CO-IP/Pull down. I-Quantitative Proteomics ifezekisa ukulinganiswa okuchanekileyo kunye nokuchongwa kwazo zonke iiproteni ezivezwa yi-genome okanye kwinkqubo exutyiweyo eyinkimbinkimbi. Iitekhnoloji ze-quantitative proteomics zangoku zihlulwe ngokubanzi kwiindlela ezibhalwe ilebhile (TMT) kunye nezingabhalwanga ilebhile (ezingenalebhile, i-DIA, i-PRM). I-BMKGENE ibonelela ngezisombululo ze-proteomics ezinamaqonga amaninzi kunye netekhnoloji eninzi.
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I-Metabolomics
I-Metabolomics, isifundo esisezantsi se-genomics, ikakhulu ijolise kwizinto ezincinci ze-molecule ezinobunzima be-molecular obungaphantsi kwe-1500 Da. Ivumela ii-metabolites ukuba zibonise iimpendulo zezinto eziphilayo kwi-stimuli zangaphandle kunye notshintsho lwe-physiological/pathological ngononophelo ngakumbi. Utshintsho lwe-genetic-olubangelwa kukutshintsha kwamanqanaba e-metabolite nalo lukwindawo yalo yophando, lubonelela ngembono entsha yophando.
I-BMKGENE inikezela ngoluhlu olupheleleyo lweenkonzo ze-metabolomics, kuquka i-metabolomics engajoliswanga, i-metabolomics ejoliswa kakhulu, kunye ne-metabolomics ejoliswayo. Ukusebenzisa i-liquid chromatography-mass spectrometry (LC-MS) okanye i-gas chromatography-mass spectrometry (GC-MS), utshintsho olutshintshayo kwii-metabolites ezininzi ze-molecule ezincinci kwizinto eziphilayo ngaphambi nasemva kokukhuthazwa kwangaphandle kunokubonwa. Umongo wezi nkonzo usekwe ekuchongeni ii-metabolites ezinomahluko omkhulu phakathi kwamaqela ovavanyo kunye namaqela olawulo kunye nokuhlola ngakumbi ulwalamano lwazo notshintsho lwe-physiological/pathological kunye neendlela ezisisiseko.
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I-Exosomal mRNA/LncRNA/CirrcRNA Sequencing-Illumina
Ii-Exosomes zii-vesicles ezincinci ezikhutshwa ziiseli, zihlala zinobubanzi obuphakathi kwee-nanometers ezingama-30 ukuya kwi-100. Ezi vesicles ziqulathe ii-RNA ezahlukeneyo. Ii-Exosomes zikholelwa ukuba zidlala indima ebalulekileyo kunxibelelwano phakathi kweeseli, iimpendulo zomzimba, kunye nophuhliso lwezifo, kwaye zinokusasazwa kwezinye iindawo zomzimba ngolwelo lomzimba olufana ne-plasma, amathe, kunye nomchamo. Ziphethe ii-biomolecules ezithile ukulawula imisebenzi yeeseli ezamkelayo, zichaphazela imeko ye-physiological yeseli. Ii-Exosomes zikwathathwa njengezidlala indima ebalulekileyo ekuphuhlisweni kwesifo, kubandakanya umhlaza, izifo ze-neurodegenerative, kunye neemeko zokudumba. Uphando ngee-exosomes lubonelela ngengqiqo entsha kunye neendlela zokuxilonga, unyango, kunye nokuthintela izifo.
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Ukulandelelana kwe-RNA encinci ye-Exosomal-Illumina
Ii-Exosomes zii-vesicles ezincinci ezikhutshwa ziiseli, zihlala zinobubanzi obuphakathi kwee-nanometers ezingama-30 ukuya kwi-100. Ezi vesicles ziqulathe ii-RNA ezahlukeneyo. Phakathi kweentlobo ze-RNA kwii-exosomes, eyona ixhaphakileyo nefundwa kakhulu yi-microRNA (miRNA). I-miRNA luhlobo lwee-RNA ezincinci ezingabhalisiyo iikhowudi ezimalunga ne-18-25 nucleotides ubude. Zilawula ukuthuliswa kwe-gene emva kokubhalwa ngokubophelela kwindawo ye-3′ engaguqulelwanga (3′ UTR) yee-mRNA ezijoliswe kuzo, ngaloo ndlela zilawula ukubonakaliswa kwe-gene. Umzekelo, ii-exosomes ezikhutshwa ziiseli ezithile ze-tumor ziqulathe ii-miRNA ezithile, ezifana ne-miR-126 kunye ne-miR-92a. Ezi miRNAs zinokuchaphazela ukubonakaliswa kwe-gene kwiiseli ezamkelayo kwaye zikhuthaze i-tumor angiogenesis (Tomohiro Umezu, et al., Oncogene, 2012).
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I-BMKMANU S3000_Spatial Transcriptome
I-Spatial transcriptomics yindlela esivumela ukuba sibambe kwaye sibone ukubonakaliswa kwezakhi zofuzo ngaphakathi kwizicubu. Oku kunokuba luncedo kakhulu ukuqonda indlela iiseli ezisebenzisana ngayo.
Kukho amaqonga ahlukeneyo ale ndlela. Kulo mba, i-BMKGene iphuhlise i-BMKManu 3000 Spatial transcriptome Chip, iqonga eliphucula ukusebenza kobugcisa, lifikelela kwisisombululo esingaphantsi kweeseli kwaye livumela useto lwesisombululo esinamanqanaba amaninzi.
Le chip ifaka amabala ayi-4.2 yezigidi isebenzisa ubuchwepheshe obunelungelo lobunikazi bee-microwells ezifakwe ii-beads ezigcwele ii-probes ezine-barcode. Ngale ndlela, emva kokubanjwa nokwandiswa, sifumana ithala leencwadi le-cDNA elinongiweyo ngeesampulu ezine-barcode ezihambelana ne-Illumina.
Kwidatha, indibaniselwano yebarcode yendawo kunye nee-UMIs iqinisekisa ukuchaneka kunye nokuchaneka kwedatha evelisiweyo. Ukudibanisa konke okungentla, i-BMKManu ibonelela ngoseto lwedatha oluguquguqukayo kakhulu.
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Iilayibrari ezenziwe kwangaphambili ze-DNBSEQ
I-DNBSEQ, eyaphuhliswa yi-MGI, yitekhnoloji entsha ye-NGS eye yakwazi ukunciphisa iindleko zokulandelelana nokunyusa umthamo. Ukulungiswa kweelayibrari ze-DNBSEQ kubandakanya ukuqhekeka kwe-DNA, ukulungiswa kwe-ssDNA, kunye nokwandiswa kwesangqa esijikelezayo ukuze kufunyanwe ii-nanoballs ze-DNA (DNB). Ezi emva koko zilayishwa kumphezulu oqinileyo kwaye emva koko zilandelelaniswe yi-combinatorial Probe-Anchor Synthesis (cPAS). Itekhnoloji ye-DNBSEQ idibanisa iingenelo zokuba nesantya esiphantsi sempazamo yokukhulisa kunye nokusebenzisa iipateni zempazamo ezinoxinano oluphezulu kunye nee-nanoballs, nto leyo ekhokelela kulandelelwano olune-throughput ephezulu kunye nokuchaneka.
Inkonzo yethu yokulandelelanisa iilayibrari eyenziwe kwangaphambili ivumela abathengi ukuba balungiselele iilayibrari zokulandelelanisa ii-Illumina ezivela kwimithombo eyahlukeneyo (i-mRNA, i-genome epheleleyo, i-amplicon, iilayibrari ezili-10x, phakathi kwezinye), eziguqulwa zibe ziilayibrari ze-MGI kwiilabhoratri zethu ukuze zilandelelaniswe kwi-DNBSEQ-T7, nto leyo evumela ukuba kubekho idatha ephezulu ngeendleko eziphantsi.
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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.
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Isisombululo se-mRNA esipheleleyo se-PacBio 2+3
Nangona ukulandelelana kwe-mRNA okusekwe kwi-NGS kusisixhobo esisebenzisekayo sokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuxhomekeka kwayo kwiindlela ezimfutshane zokufunda kuthintela ukusebenza kwayo kuhlalutyo oluntsonkothileyo lwe-transcriptomic. Kwelinye icala, ukulandelelana kwe-PacBio (Iso-Seq) kusebenzisa itekhnoloji yokufunda ixesha elide, okuvumela ukulandelelana kwee-transcript ze-mRNA ezipheleleyo. Le ndlela yenza kube lula ukuphonononga ngokubanzi ukwahlulahlula okuthe tye, ukuhlanganiswa kwezakhi zofuzo, kunye ne-poly-adenylation, nangona kungelulo ukhetho oluphambili lokulinganisa ukubonakaliswa kwezakhi zofuzo. Indibaniselwano ye-2+3 ivala umsantsa phakathi kwe-Illumina kunye ne-PacBio ngokuxhomekeka kwiindlela ze-PacBio HiFi ukuchonga iseti epheleleyo ye-isoforms ze-transcript kunye nokulandelelana kwe-NGS ukulinganisa ii-isoforms ezifanayo.
Amaqonga: I-PacBio Revio kunye ne-Illumina NovaSeq
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Uhlalutyo loMbutho weGenome-wide
Injongo yeGenome-Wide Association Studies (GWAS) kukuchonga iintlobo ngeentlobo zemfuza (iigenotype) ezinxulumene neempawu ezithile (iiphenotype). Ngokuhlola iimpawu zemfuza kuyo yonke i-genome kubantu abaninzi, i-GWAS ikhupha unxulumano lwe-genotype-phenotype ngokusebenzisa uhlalutyo lwezibalo lwamanqanaba abantu. Le ndlela ifumana ukusetyenziswa okubanzi ekuphandeni izifo zabantu nasekuhloleni iijini ezisebenzayo ezinxulumene neempawu ezinzima kwizilwanyana okanye kwizityalo.
Apha kwi-BMKGENE, sinikezela ngeendlela ezimbini zokwenza i-GWAS kumaqela amakhulu: ukusebenzisa i-Whole-Genome Sequencing (WGS) okanye ukukhetha indlela yokulandelelanisa i-genome encitshisiweyo, i-Specific-Locus Amplified Fragment (SLAF) ephuhliswe ngaphakathi endlwini. Ngelixa i-WGS ifanele ii-genome ezincinci, i-SLAF ivela njengendlela engabizi kakhulu yokufunda amaqela amakhulu anee-genome ezinde, inciphisa ngokufanelekileyo iindleko zokulandelelana, ngelixa iqinisekisa ukusebenza kakuhle kokufumanisa uphawu lwe-genetic.
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Ulandelelwano lwe-RNA ye-nuclei enye
Uphuhliso lweendlela zokubamba iiseli enye kunye nokwakha ilayibrari ngokwezifiso, kunye nokulandelelana okuphezulu, kutshintshe izifundo zokubonakaliswa kweejini kwinqanaba leseli. Olu phuculo luvumela uhlalutyo olunzulu nolupheleleyo lwamaqela eeseli antsonkothileyo, ukoyisa imida enxulumene nokubonakaliswa kwejini okuqhelekileyo kuzo zonke iiseli kunye nokugcina ukungafani kokwenyani kwezi qela. Ngelixa ulandelelwano lwe-RNA yeseli enye (scRNA-seq) luneengenelo ezingenakuphikiswa, luhlangabezana nemingeni kwiithishu ezithile apho ukudalwa kokumiswa kweseli enye kubonakala kunzima kwaye kufuna iisampuli ezintsha. Kwi-BMKGene, sijongana nalo mqobo ngokunikezela ngokulandelana kwe-RNA ye-nucleus enye (snRNA-seq) sisebenzisa itekhnoloji ye-10X Genomics Chromium yanamhlanje. Le ndlela yandisa uluhlu lweesampuli ezilungele uhlalutyo lwe-transcriptome kwinqanaba leseli enye.
Ukuhlukaniswa kweenuclei kwenziwa nge-chip entsha ye-10X Genomics Chromium, enenkqubo ye-microfluidics yeendlela ezisibhozo ene-double crossings. Ngaphakathi kwale nkqubo, ii-gel beads eziquka ii-barcodes, ii-primers, ii-enzymes, kunye ne-nucleus enye zifakwe kwii-oil drops ezinobukhulu be-nanoliter, zenze i-Gel Bead-in-Emulsion (GEM). Emva kokwakhiwa kwe-GEM, i-cell lysis kunye nokukhululwa kwe-barcode kwenzeka ngaphakathi kwe-GEM nganye. Emva koko, ii-mRNA molecules ziguqulelwa kwi-reverse transcription zibe yi-cDNAs, zifaka ii-barcodes ze-10X kunye ne-Unique Molecular Identifiers (UMIs). Ezi cDNAs emva koko ziphantsi kolwakhiwo lwelayibrari oluqhelekileyo, oluququzelela uphando oluqinileyo nolupheleleyo lweeprofayili zokubonakaliswa kwezakhi zofuzo kwinqanaba leseli enye.
Iqonga: Iqonga le-10 × Genomics Chromium kunye ne-Illumina NovaSeq
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Ulandelelwano lweGenome yezityalo/yezilwanyana
Ukulandelelana kweGenome epheleleyo (WGS) yindlela esetyenziswa ukufumanisa ukulandelelana kweDNA ngokupheleleyo kwigenome yesidalwa ngexesha elinye.
Ngokwesiqhelo, le nkonzo yahlulwe yangamaqela amabini ahlukeneyo ngokuxhomekeke ekubeni kukho i-genome ebhekisayo:
- De novoukulandelelana kwe-genome epheleleyo.Kule meko, i-genome eza kulandelelaniswa ayinayo i-genome enokubhekiswa kuyo, kwaye ngenxa yeso sizathu, injongo yolu landelelwano kukuyivelisa (okanye ukuphucula esele ikho). Le ndlela kufuneka isebenzise zombini, idatha ye-Illumina kunye nolandelelwano olude lokufundwa ukuphucula i-genome assembly ngokwenza ukudibana phakathi kokufundwa.
- Ukulandelelanisa kwakhona.Ibhekisa kulandelelwano lwe-genome olupheleleyo lwabantu abahlukeneyo beentlobo ngeentlobo ezine-genome ezaziwayo. Ngokwale siseko, umahluko we-genomic wabantu okanye abemi unokuchongwa ngakumbi.
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I-10x Genomics Visium Spatial Transcriptome
I-Spatial transcriptomics yiteknoloji entsha evumela abaphandi ukuba baphande iipateni zokubonakaliswa kwezakhi zofuzo ngaphakathi kwezicubu ngelixa begcina imeko yazo yendawo. Elinye iqonga elinamandla kweli candelo yi-10x Genomics Visium edityaniswe nokulandelelana kwe-Illumina. Umgaqo we-10X Visium usekwe kwi-chip ekhethekileyo enendawo yokubamba ekhethiweyo apho kubekwa khona amacandelo ezicubu. Le ndawo yokubamba iqulethe amabala aneebhakhowudi, ngalinye lihambelana nendawo eyahlukileyo ngaphakathi kwezicubu. Iimolekyuli ze-RNA ezibanjiweyo ezivela kwizicubu emva koko ziphawulwa ngee-unique molecular identifiers (UMIs) ngexesha lenkqubo yokubhala engasemva. La mabala aneebhakhowudi kunye nee-UMI zivumela ukumapha ngokuchanekileyo kwendawo kunye nokulinganisa ukubonakaliswa kwezakhi zofuzo kwisisombululo seseli enye. Ukudibanisa iisampuli ezineebhakhowudi kunye nee-UMI kuqinisekisa ukuchaneka kunye nokuchaneka kwedatha evelisiweyo. Ngokusebenzisa obu buchwepheshe be-Spatial Transcriptomics, abaphandi banokufumana ukuqonda okunzulu malunga nokuhlelwa kwendawo kweeseli kunye nokusebenzisana okuntsonkothileyo kweemolekyuli okwenzeka ngaphakathi kwezicubu, okubonelela ngengqiqo ebalulekileyo kwiinkqubo ezisisiseko seenkqubo zebhayoloji kwiindawo ezininzi, kubandakanya i-oncology, i-neuroscience, i-biology yophuhliso, i-immunology, kunye nezifundo zezityalo.
Iqonga: 10X Genomics Visium kunye ne-Illumina NovaSeq
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I-Nanopore ye-mRNA yoBude obupheleleyo
Nangona ukulandelelana kwe-mRNA okusekwe kwi-NGS kusisixhobo esisebenzisekayo sokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuxhomekeka kwayo ekufundeni okufutshane kuyayithintela ukusebenza kwayo kuhlalutyo oluntsonkothileyo lwe-transcriptomic. Kwelinye icala, ukulandelelana kwe-nanopore kusebenzisa itekhnoloji yokufunda ixesha elide, okuvumela ukulandelelana kwe-mRNA transcripts epheleleyo. Le ndlela inceda uphando olupheleleyo lwe-splicing eyahlukileyo, i-gene fusions, i-poly-adenylation, kunye nokulinganiswa kwe-mRNA isoforms.
Ukulandelelana kwe-nanopore, indlela exhomekeke kwimiqondiso yombane ye-nanopore enye-molecule real-time, inika iziphumo ngexesha langempela. Ikhokelwa ziiproteni zemoto, i-DNA enemicu emibini ibopha kwiiproteni ze-nanopore ezifakwe kwi-biofilm, ikhululeke njengoko idlula kwitshaneli ye-nanopore phantsi komahluko we-voltage. Imiqondiso yombane eyahlukileyo eveliswa ziziseko ezahlukeneyo kwi-DNA strand iyafunyanwa kwaye ihlelwe ngexesha langempela, iququzelela ulandelelwano oluchanekileyo noluqhubekayo lwe-nucleotide. Le ndlela intsha iyoyisa imida yokufunda okufutshane kwaye ibonelela ngeqonga elinamandla lohlalutyo oluntsonkothileyo lwe-genomic, kubandakanya nezifundo ezintsonkothileyo ze-transcriptomic, kunye neziphumo ezikhawulezileyo.
Iqonga: I-Nanopore PromethION 48
