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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 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
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Ukulandelana kwe-mRNA okugcwele -PacBio
Nakuba ukulandelana kwe-mRNA okusekelwe ku-NGS kuyithuluzi elisebenziseka kalula lokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuthembela kwayo ekufundeni okufushane kuvimbela ukusetshenziswa kwayo ekuhlaziyeni okuyinkimbinkimbi kwe-transcriptomic. Ngakolunye uhlangothi, ukulandelana kwe-PacBio (Iso-Seq) kusebenzisa ubuchwepheshe bokufunda isikhathi eside, okuvumela ukulandelana kwemibhalo ye-mRNA yobude obugcwele. Le ndlela yenza kube lula ukuhlola okuphelele kokuhlanganisa okunye, ukuhlanganiswa kwezakhi zofuzo, kanye ne-poly-adenylation. Kodwa-ke, kunezinye izinketho zokulinganisa ukubonakaliswa kwezakhi zofuzo ngenxa yenani elikhulu ledatha elidingekayo. Ubuchwepheshe bokulandelana kwe-PacBio buthembele ekulandeleni kwe-single-molecule, real-time (SMRT), okunikeza inzuzo ehlukile ekubambeni imibhalo ye-mRNA yobude obugcwele. Le ndlela entsha ihilela ukusebenzisa ama-waveguides e-zero-mode (ZMWs) kanye nemithombo emincane evumela ukubonwa kwesikhathi sangempela komsebenzi we-DNA polymerase ngesikhathi sokulandelelana. Ngaphakathi kwala ma-ZMW, i-DNA polymerase ye-PacBio ihlanganisa umucu we-DNA ohambisanayo, ikhiqiza ukufunda okude okuhlanganisa yonke imibhalo ye-mRNA. Ukusebenza kwe-PacBio kumodi yokulandelana kwe-Circular Consensus (CCS) kuthuthukisa ukunemba ngokulandelelanisa i-molecule efanayo ngokuphindaphindiwe. Ukufundwa kwe-HiFi okukhiqizwe kunokunemba okufana ne-NGS, okufaka isandla ekuhlaziyweni okuphelele nokuthembekile kwezici eziyinkimbinkimbi ze-transcriptomic.
I-Platform: I-PacBio Sequel II; I-PacBio Revio
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Ukulandelelana kwe-mRNA ye-Eukaryotic-NGS
Ukulandelana kwe-mRNA kuyindlela esebenzisa ukulandelana kwesizukulwane esilandelayo ukuze kwembulwe ubukhona kanye nobuningi bama-molecule e-RNA kusampula yezinto eziphilayo, okunikeza isithombe-skrini sokubonakaliswa kwezakhi zofuzo kusampula.
Ngezinhlelo zayo eziningi, leli thuluzi eliphambili lembula amaphrofayili ayinkimbinkimbi okubonakaliswa kwezakhi zofuzo, izakhiwo zezakhi zofuzo, kanye nezinqubo zama-molecule ezihlotshaniswa nezinqubo ezahlukahlukene zezinto eziphilayo.
Le ndlela isetshenziswa kabanzi ocwaningweni, ekuxilongweni kwezokwelapha, nasekuthuthukisweni kwemithi, njengoba inikeza ukuqonda ngobunzima be-dynamics yamaseli kanye nokulawulwa kwezakhi zofuzo, okuhlinzeka ngesisekelo esiqinile sokuvusa ilukuluku mayelana namandla ayo emikhakheni ehlukene.
Amapulatifomu atholakalayo: Illumina NovaSeq X; DNBSEQ-T7
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Ukulandelelana kwe-mRNA okusekelwe ku-Non-Reference
Ukulandelana kwe-mRNA kunika amandla ukuqoshwa okuphelele kwazo zonke izinguqulo ze-mRNA ngaphakathi kwamaseli ngaphansi kwezimo ezithile. Lobu buchwepheshe obusezingeni eliphezulu busebenza njengethuluzi elinamandla, lembula amaphrofayili okubonakaliswa kwezakhi zofuzo ayinkimbinkimbi, izakhiwo zezakhi zofuzo, kanye nezinqubo zama-molecule ezihlotshaniswa nezinqubo ezahlukahlukene zebhayoloji. Njengoba kusetshenziswa kabanzi ocwaningweni oluyisisekelo, ekuxilongweni kwezokwelapha, kanye nasekuthuthukisweni kwemithi, ukulandelana kwe-mRNA kunikeza ukuqonda ngobunzima be-dynamics yamaseli kanye nokulawulwa kwezakhi zofuzo.
Ipulatifomu: Illumina NovaSeq X; DNBSEQ-T7
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Ukulandelana Okude Okungafaki Amakhodi-Illumina
Ama-RNA amade angabhalisi (ama-lncRNA) amade kunama-nucleotide angu-200 anamandla amancane okubhala ikhodi futhi ayizinto ezibalulekile ngaphakathi kwe-RNA engabhalisi ikhodi. Atholakala ku-nucleus kanye ne-cytoplasm, lawa ma-RNA adlala indima ebalulekile ekulawulweni kwe-epigenetic, transcriptional, kanye ne-post-transcriptional, okugcizelela ukubaluleka kwawo ekwakheni izinqubo zamaseli kanye nama-molecule. Ukulandelana kwe-LncRNA kuyithuluzi elinamandla ekuhlukaniseni amaseli, i-Ontogenesis, kanye nezifo zabantu.
Ipulatifomu: I-Illumina NovaSeq X
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Ukulandelelana Kwe-RNA Encane-Illumina
I-RNA encane iyiqembu lama-molecule e-RNA afaka phakathi ama-microRNA (ama-miRNA), ama-RNA amancane aphazamisayo (ama-siRNA), kanye nama-RNA asebenzisana ne-piwi (ama-piRNA). Phakathi kwalawa, ama-miRNA, angaba ngu-18-25 nucleotide ubude, aphawuleka kakhulu ngezindima zawo ezibalulekile zokulawula ezinqubweni ezahlukahlukene zamaseli. Ngamaphethini okubonakaliswa aqondene nezicubu kanye nesigaba, ama-miRNA abonisa ukulondolozwa okuphezulu kuzo zonke izinhlobo ezahlukene.
Ipulatifomu: Illumina NovaSeq
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Ukulandelana kwe-CircRNA-Illumina
Ukulandelana kwe-circular RNA (circRNA-seq) kuphrofayili futhi kuhlaziye ama-RNA ayindilinga, uhlobo lwama-molecule e-RNA akha ama-loop avaliwe ngenxa yezehlakalo zokuhlanganisa ezingezona eze-canonical, okunikeza le RNA ukuzinza okwandisiwe. Ngenkathi amanye ama-circRNA ekhonjisiwe ukuthi asebenza njengeziponji ze-microRNA, ehlukanisa ama-microRNA futhi ewavimbela ekulawuleni ama-mRNA awo aqondiwe, amanye ama-circRNA angasebenzisana namaprotheni, aguqule ukubonakaliswa kwezakhi zofuzo, noma abe nezindima ezinqubweni zamaseli. Ukuhlaziywa kokubonakaliswa kwe-circRNA kunikeza ukuqonda ngezindima zokulawula zala ma-molecule kanye nokubaluleka kwawo ezinqubweni ezahlukahlukene zamaseli, izigaba zokukhula, kanye nezimo zezifo, okufaka isandla ekuqondeni okujulile kobunzima bokulawulwa kwe-RNA kumongo wokubonakaliswa kwezakhi zofuzo.
Ipulatifomu: Illumina Novaseq X
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Ukulandelana Okuphelele Kwe-Transcriptome - Illumina
Ukulandelana kwe-Wole transcriptome kunikeza indlela ephelele yokubhala ama-molecule e-RNA ahlukahlukene, ahlanganisa ukufaka amakhodi (i-mRNA) nama-RNA angabhalisi (i-lncRNA, i-circRNA, kanye ne-miRNA). Le ndlela ibamba yonke i-transcriptome yamaseli athile ngesikhathi esithile, okuvumela ukuqonda okuphelele kwezinqubo zamaseli.
Amapulatifomu atholakalayo: Illumina Novaseq X
