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Ukuhlanganiswa Kwe-Genome Ye-T2T | Ukulandelana Okude Kakhulu
I-T2T (i-Telomere-kuya-ku-Telomere) I-Genome iyindinganiso yegolide yokuhlanganiswa kwe-genome esezingeni eliphezulu, ebhekisela ekwakhiweni kabusha kwe-genome okungenazikhala noma okungenazikhala, okusuka ku-telomere eyodwa kuya kwenye, kanye nokuphula imingcele yokuqhekeka kwe-genome evamile.
Inikwe amandla yi-core ONT ultra-long read sequencing futhi ihlanganiswe ne-multi-platform deep sequencing kanye ne-bioinformatics pipelines elungiselelwe, isixazululo se-BMKGENE T2T Genome sihlose "izifunda ezimnyama" ze-genomic ezingalawuleki kakhulu - ama-telomere (ama-nucleoprotein complexes akhethekile emaphethelweni e-chromosome eukaryotic), ama-centromere aphezulu e-organism (ama-tandem repeat array amakhulu), kanye nezinye izifunda ze-haplotype eziphindaphindayo neziyinkimbinkimbi, ebezilokhu zingaxazululeki isikhathi eside kwi-standard long-read sequencing. Ngokungafani ne-long reads evamile ehluleka ukuwela lezi zifunda futhi ibangele ukuwa kwe-sequence noma ama-chimeric contigs, i-ONT ultra-long read reads ingahlanganisa izikhala ezingahlangani kanye nezifunda eziyinkimbinkimbi. I-BMKGene izibophezele ekuletheni ama-genome e-T2T angenazikhala noma angenazikhala, asezingeni eliphezulu ezinhlobo ezahlukahlukene.
Ukwakhiwa kwe-genome ye-T2T kuvula izindawo ze-genomic eziyinkimbinkimbi ezazingafinyeleleki ngaphambili, kugcwalisa izikhala ezibalulekile zocwaningo, futhi kuhlinzeke ngedatha eyisisekelo eqinile nenembile kakhulu yezifundo ezijulile ezifaka phakathi ukuvela kwezinhlobo, ukumbiwa kwezakhi zofuzo ezisebenzayo, ukuzalanisa kwama-molecule, umuthi wokunemba kanye nolunye ucwaningo lwesayensi olusezingeni eliphezulu.
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Ama-Proteomics
I-Proteomics igxile kumaprotheni—abaphathi bemisebenzi yokuphila edlala indima ebalulekile ekulawulweni kokubhalwa kwezinto eziphilayo. Ihlaziya ukwakheka, amazinga okuvezwa, kanye nezimo zokuguqulwa kwawo wonke amaprotheni ashintsha ngokuguquguqukayo ezicutshini noma emaseli, ibhekana nomthelela obalulekile we-proteome voluminous dynamics ezinqubweni ezahlukene zokuphila. Isetshenziswa kabanzi kwezokwelapha, kwezolimo, kanye nasekufuyweni kwezilwane. I-Qualitative Proteomics isebenzisa ubuchwepheshe bokuhlonza amaprotheni e-HPLC-MS/MS ukuhlonza amasampula afaka phakathi ama-gel strips, i-IP, kanye namasampula e-CO-IP/Pull down. I-Quantitative Proteomics ifinyelela ukulinganiswa okunembile kanye nokuhlonza wonke amaprotheni avezwa yi-genome noma ohlelweni oluxubile oluyinkimbinkimbi. Ubuchwepheshe bamanje be-quantitative proteomics buhlukaniswe kakhulu ngezindlela ezibhalwe ngamalebula (TMT) kanye nezingabhalwe ngamalebula (Azinawo Amalebula, i-DIA, i-PRM). I-BMKGENE inikeza izixazululo ze-proteomics ezinamapulatifomu amaningi kanye nobuchwepheshe obuningi.
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I-Metabolomics
I-Metabolomics, isifunda esingezansi se-genomics, ihlose kakhulu izinto ezincane zama-molecule ezinesisindo sama-molecule esingaphansi kuka-1500 Da. Ivumela ama-metabolites ukuthi abonise izimpendulo zezinto eziphilayo ezintweni zangaphandle kanye nezinguquko zomzimba/ze-pathological ngendlela ebucayi kakhulu. Ukushintsha kwezakhi zofuzo-ushintsho lwezinga le-metabolite olubangelwa ukushintsha kwezakhi zofuzo nakho kungaphakathi kobubanzi bayo bocwaningo, okunikeza umbono omusha wocwaningo.
I-BMKGENE inikeza uhla oluphelele lwezinsizakalo ze-metabolomics, kufaka phakathi i-metabolomics engahlosiwe, i-metabolomics ehlosiwe kakhulu, kanye ne-metabolomics ehlosiwe. Ukusebenzisa i-liquid chromatography-mass spectrometry (LC-MS) noma i-gas chromatography-mass spectrometry (GC-MS), izinguquko eziguquguqukayo kuma-metabolites amaningi amancane ezintweni eziphilayo ngaphambi nangemva kokukhuthazwa kwangaphandle kungatholakala. Ingqikithi yalezi zinsizakalo itholakala ekuboneni ama-metabolites anomehluko omkhulu phakathi kwamaqembu okuhlola nawokulawula kanye nokuhlola kabanzi ukuhlobana kwawo nezinguquko zomzimba/ze-pathological kanye nezindlela eziyisisekelo.
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I-Exosomal mRNA/LncRNA/CirrcRNA Sequencing-Illumina
Ama-Exosome angama-vesicle amancane akhishwa amaseli, ngokuvamile asukela kuma-nanometer angu-30 kuya kwangu-100 ububanzi. Lawa ma-vesicle aqukethe i-RNA ehlukahlukene. Ama-Exosome kukholakala ukuthi adlala indima ebalulekile ekuxhumaneni phakathi kwamangqamuzana, izimpendulo zomzimba, kanye nokuthuthukiswa kwezifo, futhi angasakazwa kwezinye izingxenye zomzimba ngoketshezi lomzimba njenge-plasma, amathe, kanye nomchamo. Aphethe ama-biomolecule athile ukulawula imisebenzi yamaseli amukelayo, ethonya izimo zomzimba zamaseli. Ama-Exosome nawo abhekwa njengadlala indima ebalulekile ekuthuthukisweni kwezifo, okuhlanganisa umdlavuza, izifo eziwohlokayo zemizwa, kanye nezimo zokuvuvukala. Ucwaningo ngama-exosome lunikeza ukuqonda okusha kanye nezindlela zokuxilonga, ukwelashwa, kanye nokuvimbela izifo.
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Ukulandelana kwe-RNA Encane ye-Exosomal-Illumina
Ama-Exosomes angama-vesicles amancane akhishwa amaseli, ngokuvamile asukela kuma-nanometers angu-30 kuya kwangu-100. Lawa ma-vesicles aqukethe i-RNA ehlukahlukene. Phakathi kwezinhlobo ze-RNA kuma-exosomes, evame kakhulu futhi efundwa kabanzi yi-microRNA (i-miRNA). I-miRNA iyiklasi yama-RNA amancane angabhalisi amakhodi angaba ngu-18-25 nucleotides ubude. Aqondisa ukuthuliswa kwezakhi zofuzo ngemva kokubhalwa ngokubopha esifundeni esingahunyushwanga esingu-3′ (3′ UTR) sama-mRNA aqondiwe, ngaleyo ndlela elawula ukuvezwa kwezakhi zofuzo. Isibonelo, ama-exosomes akhishwa amaseli athile esimila aqukethe ama-miRNA athile, njenge-miR-126 kanye ne-miR-92a. Lawa ma-miRNA angathonya ukuvezwa kwezakhi zofuzo kumaseli amukelayo futhi akhuthaze i-angiogenesis yesisu (uTomohiro Umezu, et al., Oncogene, 2012).
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I-BMKMANU S3000_Spatial Transcriptome
I-Spatial transcriptomics iyindlela esivumela ukuthi sibambe futhi sibone ngeso lengqondo ukubonakaliswa kwezakhi zofuzo ngaphakathi kwezicubu. Lokhu kungabaluleka kakhulu ukuqonda ukuthi amaseli asebenzisana kanjani.
Kunezinkundla ezahlukene zale ndlela. Kulokhu, i-BMKGene ithuthukise i-BMKManu 3000 Spatial transcriptome Chip, inkundla ethuthukisa ukusebenza kobuchwepheshe, efinyelela isixazululo esingaphansi kwamangqamuzana futhi evumela ukulungiselelwa kwesisombululo esinamazinga amaningi.
Le chip ifaka izindawo eziyizigidi ezingu-4.2 isebenzisa ubuchwepheshe obunelungelo lobunikazi bama-microwell ahlanganiswe nobuhlalu obugcwele ama-probe anebhakhodi yendawo. Ngale ndlela, ngemva kokubanjwa nokwandiswa, sithola umtapo wezincwadi we-cDNA ocebile ngamasampula anebhakhodi ahambisana ne-Illumina.
Kudatha, inhlanganisela yebhakhodi yendawo kanye nama-UMI iqinisekisa ukunemba nokucaciswa kwedatha ekhiqizwe. Uma ihlanganisa konke okungenhla, i-BMKManu inikeza isethingi yedatha eguquguqukayo kakhulu.
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Amalabhulali enziwe kusengaphambili e-DNBSEQ
I-DNBSEQ, eyathuthukiswa yi-MGI, ubuchwepheshe obusha be-NGS obukwazile ukunciphisa izindleko zokulandelana nokwandisa umthamo. Ukulungiswa kwemitapo yolwazi ye-DNBSEQ kuhilela ukuhlukaniswa kwe-DNA, ukulungiswa kwe-ssDNA, kanye nokukhulisa i-rolling circle ukuthola ama-nanoball e-DNA (DNB). Lawa abese elayishwa endaweni eqinile bese elandelaniswa yi-combinatorial Probe-Anchor Synthesis (cPAS). Ubuchwepheshe be-DNBSEQ buhlanganisa izinzuzo zokuba nesilinganiso samaphutha esiphansi sokukhulisa nokusebenzisa amaphethini amaphutha aphezulu ngama-nanoball, okuholela ekulandeleni umthamo ophezulu kanye nokunemba.
Insizakalo yethu yokulandelelanisa amalabhulali eyenziwe ngaphambilini ivumela amakhasimende ukuthi alungiselele amalabhulali okulandelelanisa ama-Illumina avela emithonjeni eyahlukahlukene (i-mRNA, i-genome ephelele, i-amplicon, amalabhulali angu-10x, phakathi kwabanye), aguqulwa abe amalabhulali e-MGI emalabhorethri ethu ukuze ahlelwe ngokulandelana ku-DNBSEQ-T7, okuvumela inani eliphezulu ledatha ngezindleko eziphansi.
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Ukusebenzisana kwe-Chromatin okusekelwe ku-Hi-C
I-Hi-C iyindlela eyenzelwe ukubamba ukwakheka kwezakhi zofuzo ngokuhlanganisa ukusebenzisana okusekelwe ekuhloleni ukusondelana kanye nokulandelana okuphezulu kokuphuma. Le ndlela isekelwe ekuxhumeni kwe-chromatin ne-formaldehyde, kulandelwe ukugaya kanye nokuhlanganiswa kabusha ngendlela yokuthi izingcezu ezixhumene ndawonye kuphela ezizokwakha imikhiqizo yokuhlanganiswa. Ngokulandelanisa le mikhiqizo yokuhlanganiswa, kungenzeka ukutadisha inhlangano ye-3D ye-genome. I-Hi-C ivumela ukutadisha ukusatshalaliswa kwezingxenye ze-genome ezipakishwe kancane (ama-compartments e-A, i-euchromatin) futhi okungenzeka ukuthi zisebenza ngokubhala, kanye nezifunda ezipakishwe kakhulu (ama-compartments e-B, i-Heterochromatin). I-Hi-C ingasetshenziswa futhi ukukhomba ama-Topologically Associated Domains (ama-TAD), izifunda ze-genome ezinezakhiwo ezigoqiwe futhi cishe zinezindlela ezifanayo zokuveza, kanye nokuhlonza ama-chromatin loops, izifunda ze-DNA ezihlanganiswe ndawonye ngamaprotheni futhi ezivame ukucebiswa ezintweni ezilawulayo. Insizakalo yokulandelelana kwe-BMKGene ye-Hi-C inika abacwaningi amandla okuhlola ubukhulu bendawo ye-genomics, ivula izindlela ezintsha zokuqonda ukulawulwa kwe-genome kanye nemiphumela yayo empilweni nasezifweni.
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Isixazululo se-mRNA esigcwele ubude be-PacBio 2+3
Nakuba ukulandelana kwe-mRNA okusekelwe ku-NGS kuyithuluzi elisebenziseka kalula lokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuthembela kwayo ekufundeni okufushane kunciphisa ukusebenza kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic. Ngakolunye uhlangothi, ukulandelana kwe-PacBio (Iso-Seq) kusebenzisa ubuchwepheshe bokufunda isikhathi eside, okuvumela ukulandelana kwemibhalo ye-mRNA egcwele ubude. Le ndlela yenza kube lula ukuhlola okuphelele kokuhlanganisa okunye, ukuhlanganiswa kwezakhi zofuzo, kanye ne-poly-adenylation, yize kungeyona inketho eyinhloko yokulinganisa ukubonakaliswa kwezakhi zofuzo. Inhlanganisela engu-2+3 ivala igebe phakathi kwe-Illumina ne-PacBio ngokuthembela ku-PacBio HiFi reads ukuhlonza isethi ephelele ye-transcript isoforms kanye ne-NGS sequencing ukuze kulinganiswe ama-isoforms afanayo.
Amapulatifomu: I-PacBio Revio kanye ne-Illumina NovaSeq
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Ukuhlaziywa Kobudlelwano Bomhlaba Wonke be-Genome
Inhloso ye-Genome-Wide Association Studies (GWAS) ukuhlonza izinhlobo zezakhi zofuzo (izinhlobo zezakhi zofuzo) ezixhunyaniswe nezici ezithile (izinhlobo ze-phenotype). Ngokuhlola izimpawu zezakhi zofuzo kulo lonke i-genome kubantu abaningi, i-GWAS ikhipha izinhlangano ze-genotype-phenotype ngokusebenzisa ukuhlaziywa kwezibalo ezingeni labantu. Le ndlela ithola ukusetshenziswa okubanzi ekucwaningeni izifo zabantu nasekuhloleni izakhi zofuzo ezisebenzayo ezihlobene nezici eziyinkimbinkimbi ezilwaneni noma ezitshalweni.
Kwa-BMKGENE, sinikeza izindlela ezimbili zokwenza i-GWAS kubantu abaningi: ukusebenzisa i-Whole-Genome Sequencing (WGS) noma ukukhetha indlela yokulandelela i-genome encishisiwe, i-Specific-Locus Amplified Fragment (SLAF) ethuthukiswe ngaphakathi endlini. Ngenkathi i-WGS ifanela ama-genome amancane, i-SLAF ivela njengendlela engcono kakhulu yokufunda abantu abaningi abanama-genome amade, inciphisa ngempumelelo izindleko zokulandelela, ngenkathi iqinisekisa ukusebenza kahle kokuthola uphawu lwezakhi zofuzo.
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Ukulandelana kwe-RNA ye-nuclei eyodwa
Ukuthuthukiswa kobuchwepheshe bokubamba iseli elilodwa kanye nokwakha umtapo wolwazi ngokwezifiso, kuhlanganiswe nokulandelana okuphezulu, kushintshe izifundo zokubonakaliswa kwezakhi zofuzo ezingeni leseli. Lokhu kuphumelela kuvumela ukuhlaziywa okujulile nokuphelele kwenani lamaseli ayinkimbinkimbi, ukunqoba imikhawulo ehlotshaniswa nokulinganiswa kokubonakaliswa kwezakhi zofuzo kuwo wonke amaseli nokugcina ukungafani kwangempela ngaphakathi kwala maqembu. Ngenkathi ukulandelana kwe-RNA yeseli elilodwa (scRNA-seq) kunezinzuzo ezingenakuphikwa, kuhlangana nezinselele ezicutshini ezithile lapho ukudalwa kokumiswa kweseli elilodwa kuba nzima futhi kudinga amasampula amasha. Kwa-BMKGene, sibhekana nalesi sithiyo ngokunikeza ukulandelana kwe-RNA ye-nucleus eyodwa (snRNA-seq) sisebenzisa ubuchwepheshe be-10X Genomics Chromium obusezingeni eliphezulu. Le ndlela yandisa ububanzi bamasampula afanelekela ukuhlaziywa kwe-transcriptome ezingeni leseli elilodwa.
Ukuhlukaniswa kwama-nuclei kufezwa nge-chip entsha ye-10X Genomics Chromium, equkethe uhlelo lwe-microfluidics oluneziteshi eziyisishiyagalombili oluneziphambano ezimbili. Ngaphakathi kwalolu hlelo, ama-gel beads afaka ama-barcode, ama-primer, ama-enzyme, kanye ne-nucleus eyodwa afakwe emaconsini kawoyela anobukhulu be-nanoliter, akha i-Gel Bead-in-Emulsion (GEM). Ngemva kokwakheka kwe-GEM, i-cell lysis kanye nokukhululwa kwe-barcode kwenzeka ngaphakathi kwe-GEM ngayinye. Ngemva kwalokho, ama-molecule e-mRNA adlula ekubhalweni okuphambene abe ama-cDNA, afaka ama-barcode ayi-10X kanye nama-Unique Molecular Identifiers (UMIs). Lawa ma-cDNA abe esefakwa ekwakhiweni komtapo wolwazi ojwayelekile, okwenza kube lula ukuhlola okuqinile nokuphelele kwamaphrofayili okubonakaliswa kwezakhi zofuzo ezingeni leseli elilodwa.
Ipulatifomu: Ipulatifomu ye-10 × Genomics Chromium kanye ne-Illumina NovaSeq
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Ukulandelana Kwezakhi Zofuzo Zezitshalo/Zezilwane
I-Whole Genome Sequencing (WGS) inqubo esetshenziswa ukunquma ngokuphelele ukulandelana kwe-DNA ye-genome yesidalwa ngesikhathi esisodwa.
Ngokuvamile, le nsizakalo ihlukaniswe ngamaqembu amabili ahlukene kuye ngokuthi kukhona i-genome ebhekisela kuyo:
- De novoukulandelana kwe-genome ephelele.Kulesi simo, i-genome ezolandelaniswa ayinayo i-genome yokubhekisela etholakalayo, futhi ngenxa yaleso sizathu, inhloso yalokhu kulandelana ukuyikhiqiza (noma ukuthuthukisa ekhona). Le ndlela idinga ukusebenzisa kokubili, idatha ye-Illumina kanye nokulandelana kokufunda isikhathi eside ukuthuthukisa ukuhlanganiswa kwe-genome ngokudala ukufana phakathi kokufundwa.
- Ukulandelanisa kabusha.Kubhekisela ekulandeleni konke kwezakhi zofuzo zabantu abahlukene bezinhlobo ngezinhlobo zofuzo ezaziwa ngokuthi ama-genome. Ngalesi sisekelo, umehluko wezakhi zofuzo wabantu noma abantu ungabonakala kabanzi.
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Ukulandelana kwe-mRNA okugcwele ubude
Nakuba ukulandelana kwe-mRNA okusekelwe ku-NGS kuyithuluzi elisebenziseka kalula lokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuthembela kwayo ekufundeni okufushane kunciphisa ukusebenza kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic. Ngakolunye uhlangothi, ukulandelana kwe-nanopore kusebenzisa ubuchwepheshe bokufunda isikhathi eside, okuvumela ukulandelana kwemibhalo ye-mRNA egcwele ubude. Le ndlela yenza kube lula ukuhlola okuphelele kokuhlanganisa okunye, ukuhlanganiswa kwezakhi zofuzo, i-poly-adenylation, kanye nokulinganisa kwe-mRNA isoforms.
Ukulandelana kwe-nanopore, indlela encike ezimpawini zikagesi ze-nanopore single-molecule real-time, inikeza imiphumela ngesikhathi sangempela. Iqondiswa amaprotheni emoto, i-DNA enezintambo ezimbili ibopha amaprotheni e-nanopore afakwe ku-biofilm, ikhululeke njengoba idlula esiteshini se-nanopore ngaphansi komehluko we-voltage. Izimpawu zikagesi ezihlukile ezikhiqizwa yizisekelo ezahlukene ku-DNA strand ziyatholwa futhi zihlukaniswe ngesikhathi sangempela, zenza kube lula ukulandelana kwe-nucleotide okunembile nokuqhubekayo. Le ndlela entsha inqoba imikhawulo yokufunda okufushane futhi inikeza ipulatifomu enamandla yokuhlaziywa kwe-genomic okuyinkimbinkimbi, kufaka phakathi izifundo eziyinkimbinkimbi ze-transcriptomic, nemiphumela esheshayo.
Ipulatifomu: I-Nanopore PromethION 48
