I-PacBio 2+3 Isisombululo esipheleleyo se-mRNA sobude obupheleleyo
Iimbonakalo
● Uyilo lokufunda:
Isampulu edibeneyo elandelelaniswe kunye ne-PacBio ukuchonga i-isoforms yokubhaliweyo
Iisampulu ezahlukeneyo (eziphindaphindwayo kunye neemeko eziza kuvavanywa) zilandelelaniswe kunyeI-NGS ukulinganisa inkcazo yokukhutshelwa
● Ukulandelelana kwePacBio kwimodi yeCCS, ukuvelisa ukufundwa kwe-HiFi
● Ulandelelwano lwemibhalo ekhutshelweyo yobude obupheleleyo
● Uhlalutyo aluyifuni i-genome yesalathiso; nangona kunjalo, ingasetyenziswa
● Uhlalutyo lwe-Bioinformatiki alubandakanyi kuphela ukuchazwa kwejene kunye ne-isoform-level kodwa kunye nohlalutyo lwe-lncRNA, ukuxubana kwemfuza, i-poly-adenylation, kunye nolwakhiwo lwemfuza.
Iingenelo
● Ukuchaneka Okuphakamileyo: I-HiFi ifundeka ngokuchanekileyo> 99.9% (Q30), xa ithelekiswa ne-NGS
● Uhlalutyo olulolunye lokwahlulwahlulwa: Ukulandelelanisa yonke imibhalo ekhutshelweyo yenza ukuchongwa kwe-isoform kunye nophawu.
● Ukudibanisa kwe-PacBio kunye ne-NGS Amandla: Yenza ubungakanani bokuchazwa kwinqanaba le-isoform, ityhila utshintsho olunokuthi lugqunywe xa kuhlalutywa yonke imbonakalo yemfuza.
● Ubungcaphephe: Sisebenze ngaphezulu kweesampulu ezingama-2300, iqela lethu lizisa ubutyebi bamava kuyo yonke iprojekthi.
● Inkxaso yePost-Sales: Ukuzinikela kwethu kunabela ngaphaya kokugqitywa kweprojekthi kunye neenyanga ezi-3 emva kokuthengiswa kwenkonzo. Ngeli xesha, sinikezela ngokulandelelana kweprojekthi, uncedo lokusombulula ingxaki, kunye neeseshoni ze-Q&A ukujongana nayo nayiphi na imibuzo enxulumene neziphumo.
IiMfuno zeSampulu kunye nokuhanjiswa
| Ithala leencwadi | Isicwangciso sokulandelelana | Idatha iyacetyiswa | Ulawulo lwemeko |
| Ithala leencwadi lePolyA liphucule ithala leencwadi le-mRNA CCS | I-PacBio Sequel II I-PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| UPoly A utyebile | Illumina PE150 | 6-10 Gb | Q30≥85% |
IiNucleotides
|
| Conc.(ng/μl) | Imali (μg) | Ubunyulu | Ingqibelelo |
| Ithala leencwadi le-Illumina | ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Kwizityalo: RIN≥4.0; Kwizilwanyana: RIN≥4.5; 5.0≥28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
| Ithala leencwadi lePacBio | ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Izityalo: RIN≥7.5 Izilwanyana: RIN≥8.0 5.0≥28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli: 2 ml centrifuge ityhubhu (Ifoyile yeTin ayikhuthazwa)
Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3; B1, B2, B3.
Ukuthunyelwa ngenqanawa:
1. Umkhenkce owomileyo:Iisampulu kufuneka zipakishwe kwiingxowa kwaye zingcwatywe kwindawo eyomileyo.
2. Iityhubhu ze-RNAstable: Iisampulu ze-RNA zingomiswa kwi-tube ye-RNA yokuzinzisa (umzekelo, i-RNAstable®) kwaye ithunyelwe kwiqondo lokushisa.
Ibandakanya uhlalutyo olulandelayo:
- Idatha ekrwada
- Ulawulo lwemeko
- Uhlalutyo olulolunye lwePolyadenylation (APA)
- Uhlalutyo lwe-Fusion transcript
- Uhlalutyo olulolunye lokwahlulwa
- Uhlalutyo lwe-Othologi yeKopi eNye kwi-Universal (BUSCO).
- Uhlalutyo lokubhaliweyo kweNoveli: ukuqikelelwa kolandelelwano lweekhowudi (CDS) kunye nochazo olusebenzayo
- Uhlalutyo lwe-lncRNA: ukubikezelwa kwe-lncRNA kunye neethagethi
- Ukuchongwa kweMicroSatelite (SSR)
- Uhlalutyo lwe-Differentially Expressed Transcripts (DETs).
- Uhlalutyo lwe-Gene echazwe ngokwahlukileyo (DEGs).
- Inkcazo esebenzayo yee-DEGs kunye nee-DET
Uhlalutyo lweBUSCO
Uhlalutyo olulolunye lokwahlulwa
Uhlalutyo olulolunye lwePolyadenylation (APA)
IiGene ezivezwe ngokwahlukileyo (DEGs) kunye neeTranscript (DETs9 anlaysis
Uthungelwano lweeProtheyini zeProtheyini zeDET kunye neeDEGs
Phonononga inkqubela phambili eququzelelwa yi-BMKGene's PacBio 2+3 yobude obugcweleyo be-mRNA elandelelanayo ngengqokelela egcinwe yopapasho.
Chao, Q. et al. (2019) 'Amandla ophuhliso lwePopulus stem transcriptome',Ijenali yeBiyoteknoloji yeZityalo, 17(1), iphepha 206–219. doi: 10.1111/PBI.12958.
Deng, H. et al. (2022) 'Iinguqu eziNgqongileyo kwiSiqulatho se-Ascorbic Acid ngexesha loPhuhliso lweZiqhamo kunye nokuVuthwa kwe-Actinidia latifolia (iSivumo seSiqhamo se-Ascorbate-Sityebi) kunye neNdlela eziManyeneyo ze-Molecular',Ijenali yaMazwe ngaMazwe yeNzululwazi yeMolekyuli, 23(10), iphe. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Ukuqikelelwa okusebenzayo kweendlela ze-biosynthetic ezibandakanyekayo kwi-polyphyllins ye-bioactive e-Paris polyphylla',Ibhayoloji yoNxibelelwano2022 5:1, 5(1), iphepha 1–10. doi: 10.1038/s42003-022-03000-z.
ULiu, M. et al. (2023) 'I-PacBio Iso-Seq edibeneyo kunye ne-Illumina RNA-Seq Uhlalutyo lwe-Tuta absoluta (Meyrick) Transcriptome kunye ne-Cytochrome P450 Genes',Izinambuzane, 14(4), iphe. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'Uvavanyo lwe-transcriptome complexity usebenzisa i-PacBio-molekyuli enye ye-real-time analysis edityaniswe nolandelelwano lwe-Illumina RNA ukuze kuqondwe ngcono i-ricinoleic acid biosynthesis kwi-Ricinus communis',I-BMC Genomics, 20(1), iphepha 1–17. doi: 10.1186/S12864-019-5832-9/AMAFANI/7.
