I-mRNA Sequencing-NGS esekwe kwiReferensi
Iimbonakalo
● Ukubanjwa kwe-poly mRNA ngaphambi kokulungiswa kwethala leencwadi
● Ayixhomekekanga kuyo nayiphi na i-genome yesalathiso: isekelwe kwi-de novo assembly of transcripts, ivelisa uluhlu lwee-unigenes ezichazwe ngeedathabheyisi ezininzi (NR, Swiss-Prot, COG, KOG, eggNOG, Pfam, GO, KEGG)
● Uhlalutyo olupheleleyo lwe-bioinformatic lwe-gene expression kunye nesakhiwo se-transcript
Iingenelo zeNkonzo
●Ubuchule Obubanzi: ngerekhodi lokucubungula iisampuli ezingaphezu kwama-600,000 kwi-BMKGENE, eziquka iintlobo ezahlukeneyo zeesampuli ezifana neenkcubeko zeeseli, izicubu, kunye nolwelo lomzimba, iqela lethu lizisa amava amaninzi kuyo yonke iprojekthi. Siphumelele ukuvala iiprojekthi ze-mRNA-Seq ezingaphezu kwe-100,000 kwiindawo ezahlukeneyo zophando.
●Ulawulo Oluqinileyo Lomgangatho: siphumeza amanqaku olawulo oluphambili kuzo zonke izigaba, ukusuka ekulungiseleleni isampuli kunye nethala leencwadi ukuya kulandelelwano kunye ne-bioinformatics. Olu hlolo olucokisekileyo luqinisekisa ukunikezelwa kweziphumo ezisemgangathweni ophezulu rhoqo.
● Ingcaciso epheleleyo: sisebenzisa iidathabheyisi ezininzi ukuze sichaze ngokusebenzayo iiGenes ezichazwe ngokuDifferentially Expressed Genes (DEGs) kwaye senze uhlalutyo oluhambelanayo lokufumisa, sinike ulwazi malunga neenkqubo zeseli kunye neemolekyuli eziphantsi kwempendulo ye-transcriptome.
●Inkxaso Emva koThengiso: ukuzibophelela kwethu kudlulela ngaphaya kokugqitywa kweprojekthi kunye nexesha lenkonzo emva kokuthengiswa kweenyanga ezi-3. Ngeli xesha, sinikezela ngokulandela iprojekthi, uncedo lokusombulula iingxaki, kunye neeseshoni ze-Q&A ukuphendula nayiphi na imibuzo enxulumene neziphumo.
Iimfuno zeSampuli kunye nokuHanjiswa
| Ithala leeNcwadi | Icebo lokulandelelanisa izinto | Idatha iyacetyiswa | Ulawulo lwemeko |
| I-Poly A etyebileyo | I-Illumina PE150 I-DNBSEQ-T7 | 6-10 Gb | Q30≥85% |
Iimfuno zeSampuli:
Iinukleotide:
| I-Conc.(ng/μl) | Ubungakanani (μg) | Ubunyulu | Ingqibelelo |
| ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Ungcoliseko lweproteni okanye lwe-DNA oluncinci okanye alubonakali kwijeli. | Kwizityalo: I-RIN≥4.0; Kwizilwanyana: I-RIN≥4.5; 5.0≥28S/18S≥1.0; ukuphakama okulinganiselweyo okanye okungekho siseko |
● Izityalo:
Ingcambu, Isiqu okanye iPetal: 450 mg
Igqabi okanye Imbewu: 300 mg
Isiqhamo: 1.2 g
● Isilwanyana:
Intliziyo okanye Amathumbu: 300 mg
I-Viscera okanye ubuchopho: 240 mg
Izihlunu: 450 mg
Amathambo, Iinwele okanye Ulusu: 1g
● Ii-arthropod:
Izinambuzane: 6g
I-Crustacea: 300 mg
● Igazi elipheleleyo: Ityhubhu e-1
● Iiseli: 106 iiseli
Ukuhanjiswa kweSampuli okuCetyisiweyo
Isikhongozeli: Ityhubhu ye-centrifuge ye-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
Iilebheli zesampulu: Iqela + ikopi umz. A1, A2, A3; B1, B2, B3.
Ukuthunyelwa:
1. Umkhenkce owomileyo: Iisampulu kufuneka zipakishwe kwiingxowa zize zingcwatywe kumkhenkce owomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwityhubhu yokuzinzisa i-RNA (umz. i-RNAstable®) kwaye zithunyelwe kubushushu begumbi.
Ukuhamba komsebenzi wenkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampulu
Ukukhutshwa kwe-RNA
Ukwakhiwa kweThala leeNcwadi
Ukulandelelana
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengisa
I-Bioinformatics
Ukuhlanganiswa kweTranscriptome kunye nokukhethwa kwe-unigene
Isichazi se-Unigene
Ulwalamano lwesampulu kunye novavanyo lweekopi zebhayoloji
IiGene ezichazwe ngokwahlukileyo (ii-DEG)
Ingcaciso esebenzayo yee-DEG
Ukuphuculwa kokusebenza kwe-DEGs
Hlola inkqubela phambili eququzelelwe ziinkonzo ze-BMKGene zokulandelelana kwe-eukaryotic NGS mRNA ngokusebenzisa ingqokelela yeempapasho ezikhethiweyo.
UShen, F. et al. (2020) 'Ukuhlanganiswa kwe-transcriptome entsha kunye nokubonakaliswa kwe-gene ecalucalulwa ngokwesini kwi-gonads ye-Amur catfish (Silurus asotus)', iGenomics, 112(3), iphe. 2603–2614. doi: 10.1016/J.YGENO.2020.01.026.
UZhang, C. et al. (2016) 'Uhlalutyo lwe-transcriptome ye-sucrose metabolism ngexesha lokudumba kwebhalbhu kunye nophuhliso kwi-anyanisi (Allium cepa L.)', Frontiers in Plant Science, 7(Septemba), p. 212763. doi: 10.3389/FPLS.2016.01425/BIBTEX.
UZhu, C. et al. (2017) 'Indibano entsha, ukuchazwa kunye nengcaciso ye-transcriptome yeSarcocheilichthys sinensis', PLoS ONE, 12(2). doi: 10.1371/JOURNAL.PONE.0171966.
UZou, L. et al. (2021) 'Uhlalutyo lwe-De novo transcriptome lubonelela ngengqiqo malunga nokunyamezela ityuwa yePodocarpus macrophyllus phantsi koxinzelelo lwetyuwa', iBMC Plant Biology, 21(1), iphe. 1–17. doi: 10.1186/S12870-021-03274-1/FIGURES/9.
