PacBio 2+3 Full-Length mRNA Solution
Nta
● Kawm tsim:
Cov qauv sib xyaw ua ke nrog PacBio txhawm rau txheeb xyuas cov ntawv teev lus isoforms
Cais cov qauv (replicates thiab tej yam kev mob yuav tsum tau kuaj) sequenced nrogNGS txhawm rau txheeb xyuas cov ntaub ntawv sau tseg
● PacBio sequencing hauv CCS hom, tsim HiFi nyeem
● Sequence ntawm cov ntawv teev lus tag nrho
● Kev tshuaj xyuas tsis tas yuav tsum muaj cov genome siv; txawm li cas los xij, nws tuaj yeem ua haujlwm
● Bioinformatic tsom xam suav nrog tsis tsuas yog qhia ntawm noob thiab isoform-theem tab sis kuj tsom xam ntawm lncRNA, noob fusions, poly-adenylation, thiab noob qauv
Qhov zoo
● Cov neeg raug siab heev: HiFi nyeem nrog qhov tseeb> 99.9% (Q30), piv rau NGS
● Alternative Splicing Analysis: sequencing tag nrho cov transcripts enables isoform identification thiab characterization.
● Kev sib xyaw ntawm PacBio thiab NGS Lub Zog: ua kom muaj nuj nqis ntawm kev qhia ntawm qib isoform, nthuav tawm qhov kev hloov pauv uas yuav raug npog thaum txheeb xyuas tag nrho cov noob qhia
● Kev paub dav dav: nrog rau cov ntaub ntawv teev tseg ntawm kev ua tiav ntau dua 1100 PacBio cov haujlwm ua haujlwm puv sijhawm thiab ua tiav ntau dua 2300 tus qauv, peb pab pawg coj cov kev paub dhau los rau txhua qhov haujlwm.
● Kev them nyiaj yug tom qab muag: Peb qhov kev cog lus txuas ntxiv dhau qhov kev ua tiav nrog rau 3 lub hlis tom qab muag kev pabcuam. Nyob rau lub sijhawm no, peb muab cov phiaj xwm taug qab, kev pab daws teeb meem, thiab Q&A ntu los daws cov lus nug ntsig txog cov txiaj ntsig.
Sample Requirements thiab Delivery
| Lub tsev qiv ntawv | Sequencing tswv yim | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
| PolyA enriched mRNA CCS lub tsev qiv ntawv | PacBio Sequel II PacBio Revio: koj puas xav tau ntau tus thwjtim? | 20/40 Gb 5/10 M CCS | Q30≥85% |
| Poly A enriched | Illumina PE 150 | 6-10 Gb | Q30≥85% |
Nucleotides
|
| Conc. (ng / μl) | Tus nqi (μg) | Purity | Kev ncaj ncees |
| Lub tsev qiv ntawv Illumina | ≥ 10 | ≥ 0.2 | OD 260/280 = 1.7-2.5 OD 260/230 = 0.5-2.5 Txwv tsis pub lossis tsis muaj protein ntau lossis DNA kis tau pom ntawm gel. | Rau cov nroj tsuag: RIN≥4.0; Rau tsiaj: RIN≥4.5; 5.0 ≥28S / 18S≥1.0; txwv los yog tsis muaj qhov nce siab |
| PacBio lub tsev qiv ntawv | ≥100 | ≥ 1.0 | OD 260/280 = 1.7-2.5 OD 260/230 = 0.5-2.5 Txwv tsis pub lossis tsis muaj protein ntau lossis DNA kis tau pom ntawm gel. | Nroj tsuag: RIN ≥7.5 Tsiaj txhu: RIN≥8.0 5.0 ≥28S / 18S≥1.0; txwv los yog tsis muaj qhov nce siab |
Pom zoo coj mus kuaj
Ntim: 2 ml centrifuge tube (Tin foil tsis pom zoo)
Sample labeling: Pab pawg + replicate xws li A1, A2, A3; B1, B2, B3.
Kev xa khoom:
1. Qhuav-ice:Cov qauv yuav tsum tau ntim rau hauv hnab thiab muab faus rau hauv cov dej khov qhuav.
2. RNAstable raj: RNA cov qauv tuaj yeem qhuav hauv RNA stabilization raj (xws li RNAstable®) thiab xa mus rau hauv chav tsev kub.
Xws li cov kev txheeb xyuas hauv qab no:
Raw cov ntaub ntawv zoo tswj
Alternative Polyadenylation Analysis (APA)
Fusion transcript tsom xam
Alternative Splicing Analysis
Benchmarking Universal Single-Copy Orthologs (BUSCO) tsom xam
Novel transcript analysis: twv ua ntej ntawm coding sequences (CDS) thiab functional annotation
LncRNA tsom xam: kwv yees ntawm lncRNA thiab lub hom phiaj
MicroSatelite Identification (SSR)
Kev txheeb xyuas qhov sib txawv ntawm Kev Tshaj Tawm Tshaj Tawm (DETs).
Differentially Expressed Genes (DEGs) tsom xam
Cov lus piav qhia ua haujlwm ntawm DEGs thiab DETs
BUSCO tsom xam
Alternative Splicing Analysis
Alternative Polyadenylation Analysis (APA)
Cov Genes Sib txawv (DEGs) thiab Cov Ntawv Teev Npe (DETs9 anlaysis
Protein-Protein kev sib koom tes tes hauj lwm ntawm DETs thiab DEGs
Tshawb nrhiav cov kev nce qib uas tau pab txhawb los ntawm BMKGene's PacBio 2 + 3 tag nrho-ntev mRNA sequencing los ntawm kev sau cov ntawv tshaj tawm.
Chao, Q. et al. (2019) 'Txoj kev loj hlob ntawm Populus stem transcriptome', Plant Biotechnology Journal, 17(1), pp. 206–219. doi: 10.1111/PBI.12958 ib.
Deng, H. et al. (2022) 'Dynamic Changes in Ascorbic Acid Content during Fruit Development and Ripening of Actinidia latifolia (ib qho Ascorbate-Rich Fruit Crop) thiab Associated Molecular Mechanisms', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Kev kwv yees ua tau zoo ntawm biosynthetic pathway noob koom nrog bioactive polyphyllins hauv 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 thiab Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome and Cytochrome P450 Genes', Kab, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lij, et al. (2019) 'Ib daim ntawv ntsuam xyuas ntawm transcriptome complexity siv PacBio ib leeg-molecule tsom xam lub sij hawm tiag tiag ua ke nrog Illumina RNA sequencing rau kev nkag siab zoo ntawm ricinoleic acid biosynthesis nyob rau hauv Ricinus communis', BMC Genomics, 20(1), pp. 1-17. doi: 10.1186/S12864-019-5832-9/FIGURES/7.
