PacBio 2+3 Dherer buuxa mRNA Solution
Astaamaha
● Nakhshad waxbarasho:
Muunad la dajiyay oo lala daba socdo PacBio si loo aqoonsado soocyada qoraalka qoraalka ah
Muunado kala duwan (ku-noqodo iyo shuruudo la tijaabinayo) oo la isku xigxigayNGS si loo qiyaaso qoraalka qoraalka
● Taxanaha PacBio ee qaabka CCS, soona saaraya wax-akhrinta HiFi
● Isku xigxiga qoraalada dhererka buuxa
● Falanqaynta lagama maarmaanka ah genome tixraaceed; si kastaba ha ahaatee, waxaa laga yaabaa in la shaqaaleysiiyo
● Falanqaynta bioinformatic ka mid ma aha oo kaliya muujinta hidda-sidaha iyo heerka isoform-ka laakiin sidoo kale falanqaynta lncRNA, isku-dhafka hidda-wadaha, poly-adenylation, iyo qaab-dhismeedka hiddaha.
Faa'iidooyinka
● Saxnaanta sare: HiFi wuxuu u akhriyaa si sax ah> 99.9% (Q30), marka la barbar dhigo NGS
● Falanqaynta Kala-bax KaleIsku xigxiga qoraalada oo dhan waxay awood u siinaysaa aqoonsiga isoform iyo sifada.
● Isku darka PacBio iyo Xoogaga NGS: awood u yeelashada cabbirka hadalka ee heerka isoform, daaha ka qaadida isbeddelka laga yaabo in la daboolo marka la falanqeynayo dhammaan muujinta hidda-socodka
● Khibrad Balaadhan: iyada oo leh rikoor ah dhamaystirka in ka badan 1100 PacBio mashruucyo qoraal ah oo dherer buuxa ah iyo farsamaynta in ka badan 2300 muunado, kooxdeena waxay u keentaa khibrad badan mashruuc kasta.
● Taageerada iibka kadibBallanqaadkayaga waxa uu dhaafsiisan yahay dhamaystirka mashruuca oo leh 3 bilood oo adeega iibka kadib. Inta lagu jiro wakhtigan, waxaan bixinaa dabagalka mashruuca, caawinta cilad-raadinta, iyo fadhiyada Q&A si wax looga qabto su'aalo kasta oo la xidhiidha natiijooyinka.
Tusaalaha Shuruudaha iyo Bixinta
| Maktabadda | Istaraatiijiyada isku xigxiga | Xogta lagu taliyay | Xakamaynta tayada |
| PolyA tayaysan mRNA CCS maktabadda | Talaabada PacBio II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| Poly A waa la hodmay | Illumina PE150 | 6-10 Gb | Q30≥85% |
Nucleotides
|
| Conc.(ng/μl) | Qadarka (μg) | daahirnimo | Daacadnimo |
| Maktabadda Illumina | ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Xaddidan ama aan lahayn borotiin ama wasakh DNA oo lagu muujiyey jel. | Dhirta: RIN≥4.0; Xoolaha: RIN≥4.5; 5.0≥28S/18S≥1.0; xaddidan ama aan lahayn sare u qaadis aasaasi ah |
| Maktabada PacBio | ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Xaddidan ama aan lahayn borotiin ama wasakh DNA oo lagu muujiyey jel. | Dhirta: RIN≥7.5 Xayawaanka: RIN≥8.0 5.0≥28S/18S≥1.0; xaddidan ama aan lahayn sare u qaadis aasaasi ah |
Tusaalaha keenista lagu taliyay
Weel: 2 ml oo tuubo sentrifuge ah (Faylada qasacadaysan laguma talinayo)
Tusaalaha calaamadaynta: Kooxda+ku celcelinta tusaale A1, A2, A3; B1, B2, B3.
Raritaanka:
1. Barafka qallalan:Tijaabooyinku waxay u baahan yihiin in lagu xidho bac oo lagu aaso baraf qallalan.
2. Tuubooyinka RNAstable: Muunada RNA waxaa lagu qallajin karaa tuubada xasilinta RNA (tusaale RNAstable®) waxaana lagu rari karaa heerkulka qolka.
Waxa ku jira falanqaynta soo socota:
Xakamaynta tayada xogta ceeriin
Falanqaynta Polyadenylation Beddelka ah (APA)
Falanqaynta qoraalka Fusion
Falanqaynta Kala-bax Kaduwan
Benchmarking Universal Single-Copy Orthologs (BUSCO) falanqaynta
Falanqaynta qoraalka cusub: saadaalinta taxanaha koodhaynta (CDS) iyo sharraxaadda shaqaynaysa
Falanqaynta lncRNA: saadaalinta lncRNA iyo bartilmaameedyada
Aqoonsiga MicroSatelite (SSR)
Falanqaynta Qoraalada Kala Duwan ee La Soo Bandhigay (DETs).
Falanqaynta Hidde-sidayaasha Kala Duwan (DEGs).
Sharaxaada hawleed ee DEGs iyo DETs
Falanqaynta BUSCO
Falanqaynta Kala-bax Kaduwan
Falanqaynta Polyadenylation Beddelka ah (APA)
Hiddo-sidaha si ka duwan loo soo bandhigay (DEGs) iyo Qoraal-qoraal (DETs9 falanqeyn
Shabakadaha is dhexgalka borotiinka-Protein ee DETs iyo DEGs
Sahami horumarada uu fududeeyay BMKGene's PacBio 2+3 dhererka buuxa ee mRNA isku xigxiga iyada oo loo marayo ururinta daabacaado la soo xulay.
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Hua, X. iyo al. (2022) 'Saadaasha wax ku oolka ah ee hiddo-wadaha jidka biosynthetic ee ku lug leh polyphyllins bioactive ee Paris polyphylla', Bayoolojiga Isgaarsiinta 2022 5: 1, 5(1), pp. 1-10. doi: 10.1038/s42003-022-03000-z.
Liu, M. iyo al. (2023) 'Isku-dhafka PacBio Iso-Seq iyo Illumina RNA-Seq Falanqaynta Tuta absoluta (Meyrick) Transcriptome iyo Cytochrome P450 Genes', Cayayaanka, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun iyo al. (2019) 'Sahaminta kakanaanta transcriptome iyadoo la adeegsanayo falanqaynta PacBio hal-molecule-waqtiga-dhabta ah oo ay weheliso taxanaha Illumina RNA si loo fahmo sii wanagsan ee biosynthesis ricinoleic acid ee Ricinus communis', BMC Genomics, 20(1), pp. 1-17. doi: 10.1186/S12864-019-5832-9/SAWIRRO/7.
