Ngwọta mRNA PacBio 2+3 zuru oke
Atụmatụ
● Nhazi ọmụmụ:
Ihe nlere nke agbakọtara na PacBio iji chọpụta isoforms transcript
Ụdị dị iche iche (nsụgharị na ọnọdụ a ga-anwale) na usoroNGS iji wepụta okwu transcript
● Usoro PacBio na ọnọdụ CCS, na-emepụta HiFi na-agụ
● Usoro nke ederede zuru oke
● Nyocha adịghị mkpa ka genome ntụaka; Otú ọ dị, enwere ike iji ya rụọ ọrụ
● Nyocha bioinformatic na-agụnye ọ bụghị nanị okwu dị na mkpụrụ ndụ ihe nketa na ọkwa isoform kamakwa nyocha nke lncRNA, mkpụrụ ndụ ihe nketa, poly-adenylation, na usoro mkpụrụ ndụ ihe nketa.
Uru
● Ịdị nnọọ mma: HiFi na-agụ nke ọma> 99.9% (Q30), iji tụnyere NGS
● Ntụle Splicing ọzọ: ịdekọ transcript niile na-enyere njirimara na njirimara isoform aka.
● Nchikota nke PacBio na ike NGS: na-eme ka ọnụọgụ okwu dị na ọkwa isoform, mkpughe mgbanwe nke enwere ike kpuchie mgbe ị na-enyocha okwu mkpụrụ ndụ niile.
● Ọkachamara dị ukwuu: na ndekọ egwu nke imecha ihe karịrị 1100 PacBio zuru ezu transcriptome oru na nhazi n'elu 2300 samples, anyị otu na-eweta a ọgaranya ahụmahụ na ọ bụla oru ngo.
● Nkwado maka ire ere: nkwa anyị ga-agafe karịa mmecha ọrụ yana oge ọrụ ọnwa 3 gachara. N'ime oge a, anyị na-enye nlebanya ọrụ, enyemaka nchọpụta nsogbu, yana oge ajụjụ & A iji zaa ajụjụ ọ bụla metụtara nsonaazụ ya.
Ihe nlele chọrọ na nnyefe
| Ụlọ akwụkwọ | Usoro usoro | akwadoro data | Njikwa ogo |
| Ọbá akwụkwọ mRNA CCS nke PolyA emelitere | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
| Poly A bara ụba | Ilumina PE150 | 6-10 Gb | Q30≥85% |
Nucleotides
|
| Conc.(ng/μl) | Ọnụ ego (μg) | Ịdị ọcha | Iguzosi ike n'ezi ihe |
| Illumina Library | ≥ 10 | ≥ 0.2 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Enwere oke ma ọ bụ enweghị protein ma ọ bụ mmetọ DNA nke egosiri na gel. | Maka osisi: RIN≥4.0; Maka anụmanụ: RIN≥4.5; 5.0≥28S/18S≥1.0; nwere oke ma ọ bụ enweghị mgbago elu |
| Ụlọ akwụkwọ PacBio | ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Enwere oke ma ọ bụ enweghị protein ma ọ bụ mmetọ DNA nke egosiri na gel. | Osisi: RIN≥7.5 Anụmanụ: RIN≥8.0 5.0≥28S/18S≥1.0; nwere oke ma ọ bụ enweghị mgbago elu |
Nnyefe nlele akwadoro
Akpa: 2 ml centrifuge tube (Tin foil adịghị akwadoro)
Ịkpọ aha nlele: Otu+ megharịa dịka A1, A2, A3; B1, B2, B3.
Mbupu:
1. Akpụrụ akpụ:A ga-akwakọba ihe nlele n'ime akpa ma lie ya na ice-akọrọ.
2. tubes RNAstable: Enwere ike ịkpọsa ihe nlele RNA na tube stabilization RNA (dịka ọmụmaatụ RNAstable®) ma bufee ya n'ime ụlọ okpomọkụ.
Ọ gụnyere nyocha ndị a:
Njikwa ogo data raw
Nhọrọ Polyadenylation Analysis (APA)
Nyocha transcript Fusion
Nyocha Splicing ọzọ
Nyocha Benchmarking Universal Single-Copy Orthologs (BUSCO).
Ntụle transcript ọhụrụ: amụma usoro nzuzo (CDS) na nkọwa ọrụ
Nyocha lncRNA: amụma nke lncRNA na ebumnuche
Nchọpụta MicroSatelite (SSR)
Nyocha ederede dị iche iche (DETs).
Nyocha mkpụrụ ndụ ihe nketa dị iche iche (DEGs).
Nkọwa ọrụ nke DEGs na DETs
Nyocha BUSCO
Nyocha Splicing ọzọ
Nhọrọ Polyadenylation Analysis (APA)
Mkpụrụ ndụ ihe nketa egosipụtara dị iche iche (DEGs) na transcripts (ndepụta DETs9
Netwọk mmekọrịta Protein-Protein nke DETs na DEGs
Nyochaa ọganihu nke BMKGene's PacBio 2+3 na-akwado usoro mRNA ogologo site na nchịkọta mbipụta.
Chao, Q. et al. (2019) 'Mkpụrụ ndụ mmepe nke Populus stem transcriptome', Plant Biotechnology Journal, 17(1), p. 206-219. doi: 10.1111/PBI.12958.
Deng, H. et al. (2022) 'Mgbanwe mgbanwe na ọdịnaya ascorbic acid n'oge mmepe mkpụrụ osisi na mfri nke Actinidia latifolia (ihe ubi mkpụrụ osisi Ascorbate-Rich) na Associated Molecular Mechanisms', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Amụma dị irè nke mkpụrụ ndụ ihe nketa ụzọ biosynthetic metụtara na bioactive polyphyllins na Paris polyphylla', Communications Biology 2022 5:1, 5(1), p. 1-10. doi: 10.1038/s42003-022-03000-z.
Liu, M. et al. (2023) 'Combined PacBio Iso-Seq na Illumina RNA-Seq Analysis of Tuta absoluta (Meyrick) Transcriptome na Cytochrome P450 Genes', Insects, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'Nnyocha nke transcriptome complexity site na iji PacBio single-molecule real-time analysis jikọtara ya na Illumina RNA sequencing maka nghọta ka mma nke ricinoleic acid biosynthesis na Ricinus communis', BMC Genomics, 20(1), p. 1-17. doi: 10.1186/S12864-019-5832-9/ỌFỌDỤ/7.
