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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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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
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Ulandelelwano olupheleleyo lwe-mRNA -PacBio
Nangona ukulandelelana kwe-mRNA okusekwe kwi-NGS kusisixhobo esiguquguqukayo sokulinganisa ukubonakaliswa kwezakhi zofuzo, ukuxhomekeka kwayo kwiindlela ezimfutshane zokufunda kuthintela ukusetyenziswa kwayo kuhlalutyo oluntsonkothileyo lwe-transcriptomic. Kwelinye icala, ukulandelelana kwe-PacBio (Iso-Seq) kusebenzisa iteknoloji yokufunda ixesha elide, okuvumela ukulandelelana kwee-transcript ze-mRNA ezipheleleyo. Le ndlela yenza kube lula ukuphononongwa ngokubanzi kokudibanisa okuhlukeneyo, ukuhlanganiswa kwezakhi zofuzo, kunye ne-poly-adenylation. Nangona kunjalo, kukho ezinye iindlela zokulinganisa ukubonakaliswa kwezakhi zofuzo ngenxa yenani elikhulu ledatha efunekayo. Itekhnoloji yokulandelelana kwe-PacBio ixhomekeke kulandelelwano lwe-single-molecule, real-time (SMRT), ebonelela ngenzuzo eyahlukileyo ekubambeni ii-transcript ze-mRNA ezipheleleyo. Le ndlela intsha ibandakanya ukusebenzisa ii-waveguides ze-zero-mode (ZMWs) kunye ne-microfabricated wells ezivumela ukujongwa kwexesha langempela komsebenzi we-DNA polymerase ngexesha lokulandelelana. Ngaphakathi kwezi ZMW, i-DNA polymerase ye-PacBio idibanisa umsonto we-DNA oncedisayo, ivelisa ukufundwa okude okugubungela yonke i-transcripts ye-mRNA. Ukusebenza kwePacBio kwimo yeCircular Consensus sequencing (CCS) kuphucula ukuchaneka ngokulandelelanisa ngokuphindaphindiweyo imolekyuli efanayo. IiHiFi reads ezenziweyo zichanekile ngokufana ne-NGS, nto leyo enegalelo ngakumbi kuhlalutyo olupheleleyo noluthembekileyo lweempawu ze-transcriptomic ezintsonkothileyo.
Iqonga: I-PacBio Sequel II; I-PacBio Revio
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Ukulandelelana kwe-mRNA ye-Eukaryotic-NGS
Ukulandelelana kwe-mRNA yindlela esebenzisa ulandelelwano lwesizukulwana esilandelayo ukubonisa ubukho kunye nobungakanani beemolekyuli ze-RNA kwisampulu yebhayoloji, ukubonelela ngemifanekiso emfutshane yendlela ezivezwa ngayo iijini kwisampulu.
Ngezicelo zayo ezibanzi, esi sixhobo siphambili sityhila iiprofayili ezintsonkothileyo zokubonakaliswa kwezakhi zofuzo, izakhiwo zezakhi zofuzo, kunye neendlela zemolekyuli ezinxulumene neenkqubo ezahlukeneyo zebhayoloji.
Le ndlela isetyenziswa kakhulu kuphando, ukuxilongwa kwezonyango, kunye nophuhliso lwamayeza, kuba inika ulwazi malunga nobunzima be-dynamics yeseli kunye nolawulo lwezakhi zofuzo, inika isiseko esiqinileyo sokuvusa umdla malunga namandla ayo kwiindawo ezahlukeneyo.
Iiplatfomu ezikhoyo: Illumina NovaSeq X; DNBSEQ-T7
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I-mRNA Sequencing-NGS esekwe kwiReferensi
Ukulandelelana kwe-mRNA kunika amandla ukubhalwa ngokubanzi kwazo zonke ii-mRNA transcripts ngaphakathi kweeseli phantsi kweemeko ezithile. Le teknoloji intsha isebenza njengesixhobo esinamandla, ityhila iiprofayili ezintsonkothileyo zokubonakaliswa kwezakhi zofuzo, izakhiwo zezakhi zofuzo, kunye neendlela zemolekyuli ezinxulumene neenkqubo ezahlukeneyo zebhayoloji. Yamkelwe ngokubanzi kuphando olusisiseko, ukuxilongwa kwezonyango, kunye nophuhliso lwamayeza, ukulandelelana kwe-mRNA kunika ukuqonda kwiingxaki ze-dynamics zeseli kunye nolawulo lwezakhi zofuzo.
Iqonga: Illumina NovaSeq X; DNBSEQ-T7
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Ulandelelwano olude olungasebenzisi iikhowudi-Illumina
IiRNA ezinde ezingabhalisiyo iikhowudi (ii-lncRNA) zinde kunee-nucleotides ezingama-200 ezinamandla amancinci okubhala iikhowudi kwaye zizinto ezibalulekileyo kwi-RNA engabhalisiyo iikhowudi. Ezi RNA zifumaneka kwi-nucleus nakwi-cytoplasm, zidlala indima ebalulekileyo kulawulo lwe-epigenetic, transcriptional, kunye ne-post-transcriptional, nto leyo egxininisa ukubaluleka kwazo ekubumbeni iinkqubo zeseli kunye neemolekyuli. Ukulandelelana kwe-LncRNA sisixhobo esinamandla ekuhlukaniseni iiseli, i-Ontogenesis, kunye nezifo zabantu.
Iqonga: Illumina NovaSeq X
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Ukulandelelana kwe-RNA encinci-Illumina
I-RNA encinci liqela lee-molecule ze-RNA eziquka ii-microRNA (ii-miRNA), ii-RNA ezincinci eziphazamisayo (ii-siRNA), kunye nee-RNA ezisebenzisana ne-piwi (ii-piRNA). Phakathi kwezi, ii-miRNA, ezimalunga ne-18-25 nucleotides ubude, ziphawuleka kakhulu ngendima yazo ebalulekileyo yokulawula kwiinkqubo ezahlukeneyo zeseli. Ngeepateni zokubonakalisa ezithile kwizicubu kunye nesigaba esithile, ii-miRNA zibonisa ulondolozo oluphezulu kwiintlobo ezahlukeneyo.
Iqonga: Illumina NovaSeq
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Ukulandelelana kwe-CircRNA-Illumina
Ukulandelelana kwe-RNA ejikelezayo (circRNA-seq) kuchaza kwaye kuhlalutye ii-RNA ezijikelezayo, udidi lwee-molecule ze-RNA ezenza ii-loops ezivaliweyo ngenxa yeziganeko ze-non-canonical splicing, ezinika le RNA uzinzo olongezelelekileyo. Ngelixa ezinye ii-circRNA zibonakalisiwe ukuba zisebenza njengeziponji ze-microRNA, ziqokelela ii-microRNA kwaye zithintele ekulawuleni ii-mRNA zazo ekujoliswe kuzo, ezinye ii-circRNA zinokusebenzisana neeproteni, ziguqule ukubonakaliswa kwezakhi zofuzo, okanye zibe neendima kwiinkqubo zeseli. Uhlalutyo lokubonakaliswa kwe-circRNA lubonelela ngengqiqo kwiindima zokulawula zezi molekyuli kunye nokubaluleka kwazo kwiinkqubo ezahlukeneyo zeseli, amanqanaba ophuhliso, kunye neemeko zesifo, okufaka isandla ekuqondeni okunzulu ubunzima bokulawulwa kwe-RNA kumxholo wokubonakaliswa kwezakhi zofuzo.
Iqonga: Illumina Novaseq X
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Ulandelelwano lweTranscriptome epheleleyo – Illumina
Ukulandelelana kwe-Wole transcriptome kunika indlela ebanzi yokuchonga ii-molecules ze-RNA ezahlukeneyo, eziquka i-coding (mRNA) kunye ne-non-coding RNAs (lncRNA, circRNA, kunye ne-miRNA). Le ndlela ibamba yonke i-transcriptome yeeseli ezithile ngexesha elithile, ivumela ukuqonda okupheleleyo kweenkqubo zeseli.
Iiplatfomu ziyafumaneka: Illumina Novaseq X
