Ulandelelwano olupheleleyo lwe-mRNA -PacBio
Iinkonzo eziluncedo
● Ukufundwa ngokuthe ngqo kwimolekyuli ye-cDNA yobude obupheleleyo ukusuka kwi-3'- ekupheleni ukuya kwi-5'- ekupheleni
● Isisombululo senqanaba le-iso-form kwisakhiwo solandelelwano
● Imibhalo echaneke kakhulu kunye nemfezeko
● Iyahambelana kakhulu neentlobo ze-vaiours
● Umthamo omkhulu wokulandelelana kunye ne-4 PacBio Sequel II yokulandelelana kwamaqonga axhotyiswe
● Amava aphezulu ngeeprojekthi zokulandelelana kwe-RNA ezingaphezulu kwe-700
● Ukuhanjiswa kweziphumo ezisekelwe kwi-BMKCloud: Ukuchithwa kwedatha ngokwezifiso ezikhoyo kwiqonga.
● Iinkonzo zasemva kokuthengiswa zisebenza iinyanga ezi-3 ekugqityweni kweprojekthi
IiNgcaciso zeNkonzo
Iqonga: I-PacBio Sequel II
Ithala leencwadi lolandelelwano: Ithala leencwadi lePoly A-enriched mRNA
Imveliso yedatha ecetyiswayo: 20 Gb/isampulu (Kuxhomekeke kwiintlobo)
FLNC (%): ≥75%
*I-FLNC: Ubude obupheleleyo be-non-chimeric transcipt
Uhlalutyo lwe-Bioinformatics
● Ukusetyenzwa kwedatha ekrwada
● Ukuchongwa okubhaliweyo
● Isakhiwo solandelelwano
● Ubungakanani Belizwi
● Inkcazo yomsebenzi
IiMfuno zeSampulu kunye nokuhanjiswa
IiMfuno zeSampulu:
IiNucleotides:
| Conc.(ng/μl) | Imali (μg) | Ubunyulu | Ingqibelelo |
| ≥ 120 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Kwizityalo: RIN≥7.5; Kwizilwanyana: RIN≥8.0; 5.0≥ 28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
Izicubu: Ubunzima (bomileyo):≥1 g
*Kwithishu ezincinci kuno-5 mg, sicebisa ukuba uthumele ifleshi ekhenkcezisiweyo (kwinitrogen engamanzi) isampulu yethishu.
Ukumiswa kweeseli:Ubalo lweeseli = 3×106- 1×107
*Sicebisa ukuthumela i-lysate yeseli enomkhenkce.Kwimeko apho iseli ibala encinci kune-5 × 105, i-flash ekhenkcezisiweyo kulwelo lwenitrogen iyacetyiswa, ekhethwayo xa kutsalwa kancinci.
Iisampulu zegazi:Umthamo≥1 mL
I-Microorganism:Ubunzima ≥ 1 g
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli:
I-2 ml yetyhubhu ye-centrifuge (i-tin foil ayikhuthazwa)
Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3;B1, B2, B3... ...
Ukuthunyelwa ngenqanawa:
1. Umkhenkce owomileyo: Iisampulu kufuneka zipakishwe kwiingxowa kwaye zingcwatywe kumkhenkce owomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwi-tube ye-RNA yokuzinzisa (umzekelo, i-RNAstable®) kwaye ithunyelwe kwiqondo lokushisa.
Ukuhamba komsebenzi weNkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampuli
Ukukhutshwa kwe-RNA
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
1.FLNC ubude bokuhanjiswa
Ubude bobude obupheleleyo be-non-chimeric read(FLNC) bubonisa ubude be-cDNA kulwakhiwo lwethala leencwadi.Ukuhanjiswa kobude be-FLNC sisalathisi esibalulekileyo ekuvavanyeni umgangatho wokwakhiwa kwethala leencwadi.
Ukuhanjiswa kobude bokufunda kwe-FLNC
2.Gqibezela unikezelo lobude bommandla we-ORF
Sisebenzisa iTransDecoder ukuqikelela imimandla yekhowudi yeprotheyini kunye nokulandelelana kwe-amino acid ehambelanayo ukuvelisa iiseti ze-unigene, eziqulethe ulwazi olupheleleyo olubhaliweyo olungafunekiyo kuzo zonke iisampuli.
Gqibezela ubude bonikezelo lommandla we-ORF
3.KEGG indlela yohlalutyo lokutyebisa
Imibhalo ebhaliweyo echazwe ngokwahlukileyo (DETs) inokuchongwa ngokulungelelanisa i-NGS-based RNA data sequencing kwi-full-length transcript sets eveliswa yi-PacBio data sequencing.Ezi DET zinokuqhubekeka zisetyenzwa kuhlalutyo olwahlukeneyo olusebenzayo, umz. uhlalutyo lwendlela ye-KEGG yokutyebisa.
I-DET KEGG indlela yokutyebisa -Iploti yeDot
Ityala le-BMK
Amandla ophuhliso lwePopulus stem transcriptome
Ipapashiwe: Ijenali yeBiyoteknoloji yeZityalo, 2019
Iqhinga lokulandelelanisa:
Ingqokelela yesampulu:imimandla yesiqu: i-apex, i-internode yokuqala (IN1), i-internode yesibini (IN2), i-internode yesithathu (IN3), i-internode (IN4) kunye ne-internode (IN5) esuka e-Nanlin895
NGS-ulandelelwano:I-RNA yabantu abali-15 badityaniswa njengesampulu yebhayoloji enye.Ukuphindaphindwa kwezinto ezintathu zebhayoloji zamanqaku nganye ziye zaqhutyelwa kulandelelwano lwe-NGS
TGS-ulandelelwano:Imimandla ye-Stem yahlulwa yaba yimimandla emithathu, okt i-apex, IN1-IN3 kunye ne-IN4-IN5.Ummandla ngamnye wawucutshungulwa ngokulandelelana kwePacBio kunye neentlobo ezine zamathala eencwadi: 0-1 kb, 1-2 kb, 2-3 kb kunye ne-3-10 kb.
Iziphumo eziphambili
1. Itotali yemibhalo eshicilelweyo yobude obupheleleyo engama-87150 ichongiwe, apho, ii-isoforms zenoveli ezingama-2081 kunye ne-62058 yenoveli engenye yee-isoforms ezizezinye zachongwa.
I-2.1187 lncRNA kunye ne-356 yofuzo lwe-fusion ichongiwe.
3.Ukususela ekukhuleni okuphambili ukuya ekukhuleni kwesibini, i-15838 evezwe ngokwahlukileyo kwimibhalo evela kwi-995 evezwe ngokwahlukileyo kwiigenes zachongwa.Kuzo zonke ii-DEGs, i-1216 yayiyimiba yokukhutshelwa, uninzi lwazo olungekaxelwa.
Uhlalutyo lwe-4.GO lwe-erichment lubonise ukubaluleka kokwahlulwa kweseli kunye nenkqubo yokunciphisa i-oxidation ekukhuleni okuphambili kunye nesekondari.
Iziganeko ezizezinye ze-splicing kunye ne-isoforms ezahlukeneyo
Uhlalutyo lwe-WGCNA kwizinto ezikhutshelweyo
Isalathiso
Chao Q, Gao ZF, Zhang D, et al.Amandla ophuhliso lwePopulus stem transcriptome.Isityalo se-Biotechnol J. 2019; 17 (1): 206-219.doi:10.1111/pbi.12958
