PacBio 2+3 Full-Length mRNA Solution
Mawonekedwe
● Kapangidwe ka maphunziro:
Zitsanzo zophatikizidwa ndi PacBio kuti zizindikire ma isoforms olembedwa
Zitsanzo zosiyana (zobwereza ndi zomwe ziyenera kuyesedwa) zotsatizana nazoNGS kuwerengera mawu olembedwa
● Sequencing PacBio mu CCS mode, kupanga HiFi kuwerenga
● Kutsatizana kwa zolembedwa zazitali zonse
● Kusanthula sikufuna kuti munthu akhale ndi ma genome; komabe, ikhoza kugwiritsidwa ntchito
● Kusanthula kwa bioinformamatic sikumangotanthauza kufotokoza kwa jini ndi isoform-level komanso kusanthula kwa lncRNA, kuphatikizika kwa majini, poly-adenylation, ndi kapangidwe ka majini.
Ubwino wake
● Kulondola Kwambiri: HiFi imawerenga molondola> 99.9% (Q30), poyerekeza ndi NGS
● Kusanthula kwa Njira Zina: kutsata zolembedwa zonse kumathandizira kuzindikirika ndi mawonekedwe a isoform.
● Kuphatikiza kwa PacBio ndi NGS Mphamvu: kuthandizira kuchuluka kwa mawu pamlingo wa isoform, kuwulula kusintha komwe kumatha kubisika posanthula mawonekedwe onse a jini
● Luso Lambiri: Pokhala ndi mbiri yakukwaniritsa ma projekiti opitilira 1100 a PacBio aatali komanso kukonza zitsanzo zopitilira 2300, gulu lathu limabweretsa zokumana nazo zambiri pantchito iliyonse.
● Thandizo Pambuyo pa Kugulitsa: Kudzipereka kwathu kumapitilira kutha kwa projekiti ndi nthawi ya miyezi itatu mutagulitsa. Panthawiyi, timapereka zotsatila za polojekiti, chithandizo chazovuta, ndi magawo a Q&A kuti tiyankhe mafunso aliwonse okhudzana ndi zotsatira.
Zitsanzo Zofunika ndi Kutumiza
| Library | Njira yotsatirira | Deta yovomerezeka | Kuwongolera Kwabwino |
| Laibulale ya PolyA yolemeretsa mRNA CCS | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| Poly A yowonjezera | Chithunzi cha PE150 | 6-10 Gb | Q30≥85% |
Nucleotides
|
| Conc.(ng/μl) | Mtengo (μg) | Chiyero | Umphumphu |
| Illumina Library | ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Zochepa kapena zopanda mapuloteni kapena DNA kuipitsidwa komwe kumawonetsedwa pa gel. | Kwa zomera: RIN≥4.0; Kwa nyama: RIN≥4.5; 5.0≥28S/18S≥1.0; kukwera kochepa kapena kopanda koyambira |
| Library ya PacBio | ≥100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Zochepa kapena zopanda mapuloteni kapena DNA kuipitsidwa komwe kumawonetsedwa pa gel. | Zomera: RIN≥7.5 Zinyama: RIN≥8.0 5.0≥28S/18S≥1.0; kukwera kochepa kapena kopanda koyambira |
Kutumiza Kwachitsanzo Kovomerezeka
Chidebe: 2 ml centrifuge chubu (zojambula za malata ndizosavomerezeka)
Zitsanzo zolembera: Gulu+ lofanana mwachitsanzo A1, A2, A3; B1, B2, B3.
Kutumiza:
1. Owuma-ayezi:Zitsanzo ziyenera kupakidwa m'matumba ndikukwiriridwa mu ayezi wouma.
2. RNAstable chubu: RNA zitsanzo akhoza zouma mu RNA kukhazikika chubu (mwachitsanzo RNAstable®) ndi kutumizidwa mu firiji.
Mulinso zowunikira izi:
Kuwongolera khalidwe la data yaiwisi
Alternative Polyadenylation Analysis (APA)
Kusanthula zolemba za Fusion
Kusanthula kwa Njira Zina za Splicing
Benchmarking Universal Single-Copy Orthologs (BUSCO).
Kusanthula kwa zolemba za Novel: Kuneneratu za ma coding sequence (CDS) ndi ndemanga zogwira ntchito
Kusanthula kwa lncRNA: kulosera za lncRNA ndi zolinga
Chizindikiritso cha MicroSatelite (SSR)
Differentially Expressed Transcripts (DETs) kusanthula
Kusanthula kwa Mitundu Yosiyanasiyana (DEGs).
Ndemanga zogwira ntchito za DEGs ndi DETs
Kusanthula kwa BUSCO
Kusanthula kwa Njira Zina za Splicing
Alternative Polyadenylation Analysis (APA)
Mitundu Yosiyanasiyana (DEGs) ndi Zolemba (DETs9 anlaysis
Mapuloteni-mapuloteni olumikizana ndi ma DET ndi ma DEG
Onani kupita patsogolo kotsogozedwa ndi BMKGene's PacBio 2+3 mRNA yayitali motsatizana ndi zofalitsa zosanjidwa.
Chao, Q. et al. (2019) 'Kukula kwamphamvu kwa Populus stem transcriptome', Plant Biotechnology Journal, 17(1), pp. 206-219. doi: 10.1111/PBI.12958.
Deng, H. et al. (2022) 'Dynamic Changes in Ascorbic Acid Content during Fruit Development and Ripening of Actinidia latifolia (an Ascorbate-Rich Fruit Crop) and the Associated Molecular Mechanisms', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Kuneneratu mogwira mtima kwa majini amtundu wa biosynthetic omwe amaphatikizidwa mu bioactive polyphyllins ku Paris polyphylla', Communications Biology 2022 5: 1, 5 (1), pp. 1-10. doi: 10.1038/s42003-022-03000-z.
Liu, M. et al. (2023) 'Combined PacBio Iso-Seq ndi Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome ndi Cytochrome P450 Genes', Insects, 14 (4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'Kafukufuku wa zovuta zolembera pogwiritsa ntchito PacBio single-molecule real-time analysis kuphatikiza ndi Illumina RNA sequencing kuti amvetse bwino ricinoleic acid biosynthesis mu Ricinus communis', BMC Genomics, 20 (1), pp. 1-17. doi: 10.1186/S12864-019-5832-9/ZINTHU/7.
