Kev txheeb xyuas mRNA tag nrho -PacBio
Cov yam ntxwv
● Kev tsim cDNA los ntawm poly-A mRNA ua raws li kev npaj tsev qiv ntawv
● Kev txheeb xyuas cov ntaub ntawv hauv hom CCS, tsim cov ntawv nyeem HiFi
● Kev txheeb xyuas cov ntawv sau tag nrho
● Qhov kev tshuaj xyuas tsis tas yuav tsum muaj cov noob caj noob ces siv ua piv txwv; txawm li cas los xij, nws yuav raug siv
● Kev tshuaj xyuas bioinformatic ua rau muaj kev tshuaj xyuas cov transcripts isoform lncRNA, gene fusions, poly-adenylation, thiab gene structure
Cov Kev Pabcuam Zoo
●Kev Raug SiabHiFi nyeem nrog qhov tseeb >99.9% (Q30), sib piv rau NGS
● Kev Tshawb Fawb Txog Kev Sib Txuas Lwm Txoj KevKev txheeb xyuas tag nrho cov ntawv sau ua ke ua rau muaj kev txheeb xyuas thiab piav qhia txog isoform
●Kev Paub Ntau Yam: nrog rau cov ntaub ntawv keeb kwm ntawm kev ua tiav ntau dua 1100 PacBio cov haujlwm transcriptome puv ntoob thiab ua ntau dua 2300 cov qauv, peb pab neeg coj ntau yam kev paub dhau los rau txhua qhov project.
●Kev Txhawb Nqa Tom Qab Muag Khoom: peb txoj kev cog lus txuas ntxiv mus dhau qhov ua tiav qhov project nrog rau 3 lub hlis tom qab muag kev pabcuam. Thaum lub sijhawm no, peb muab kev saib xyuas project, kev pab daws teeb meem, thiab cov lus nug thiab lus teb los teb cov lus nug ntsig txog cov txiaj ntsig.
Cov Qauv Yuav Tsum Tau Ua thiab Kev Xa Khoom
| Tsev qiv ntawv | Txoj kev npaj ua sequencing | Cov ntaub ntawv pom zoo | Kev Tswj Xyuas Zoo |
| PolyA enriched mRNA CCS tsev qiv ntawv | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
Cov Qauv Yuav Tsum Tau Ua:
Cov nucleotides:
● Cov Nroj Tsuag:
Cag, Qia los yog Paj: 450 mg
Nplooj los yog noob: 300 mg
Txiv hmab txiv ntoo: 1.2 g
● Tsiaj:
Lub plawv los yog hnyuv: 300 mg
Cov hnyuv lossis lub hlwb: 240 mg
Cov leeg: 450 mg
Pob txha, Plaub hau los yog Tawv Nqaij: 1g
● Cov kab mob Arthropod:
Kab: 6g
Cov ntses me me: 300 mg
● Cov ntshav tag nrho: 1 lub raj
● Cov Cell: 106 cov hlwb
| Conc.(ng/μl) | Tus nqi (μg) | Kev dawb huv | Kev ncaj ncees |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Muaj tsawg los yog tsis muaj protein lossis DNA uas pom ntawm gel. | Rau cov nroj tsuag: RIN≥7.5; Rau cov tsiaj: RIN≥8.0; 5.0≥ 28S / 18S≥1.0; txwv los yog tsis muaj qhov siab pib |
Kev Xa Qauv Pom Zoo
Thawv: Lub raj centrifuge 2 ml (tsis pom zoo kom siv ntawv ci)
Piv txwv kev sau npe: Pawg + theej piv txwv li A1, A2, A3; B1, B2, B3.
Kev Xa Khoom:
1. Dej khov qhuav: Cov qauv yuav tsum tau ntim rau hauv hnab thiab faus rau hauv dej khov qhuav.
2. Cov raj RNAstable: Cov qauv RNA tuaj yeem ziab hauv cov raj RNA stabilization (piv txwv li RNAstable®) thiab xa tuaj rau hauv chav tsev kub.
Kev Ua Haujlwm Pabcuam
Tsim qauv kev sim
Kev xa khoom qauv
Kev rho tawm RNA
Kev tsim kho tsev qiv ntawv
Kev txheeb xyuas kab ke
Kev tshuaj xyuas cov ntaub ntawv
Cov kev pab cuam tom qab muag
Muaj xws li cov kev tshuaj xyuas hauv qab no:
● Kev tswj xyuas cov ntaub ntawv raw zoo
● Kev Tshuaj Xyuas Polyadenylation Lwm Txoj (APA)
● Kev tshuaj xyuas cov ntawv sau sib xyaw
● Kev Tshawb Fawb Txog Kev Sib Txuas Lwm Txoj Kev
● Kev tshuaj xyuas Universal Single-Copy Orthologs (BUSCO) Benchmarking
● Kev tshuaj xyuas cov ntawv sau tshiab: kev kwv yees ntawm cov kab ke coding (CDS) thiab kev piav qhia ua haujlwm
● kev tshuaj xyuas lncRNA: kev kwv yees ntawm lncRNA thiab cov hom phiaj
● Kev Txheeb Xyuas MicroSatelite (SSR)
Kev tshuaj xyuas BUSCO
Kev Tshawb Fawb Txog Kev Sib Txuas Lwm Txoj Kev
Kev Tshuaj Xyuas Polyadenylation Lwm Txoj (APA)
Kev piav qhia ua haujlwm ntawm cov ntawv sau tshiab
Tshawb nrhiav cov kev nce qib uas tau pab txhawb los ntawm BMKGene's Nanopore full-length mRNA sequencing cov kev pabcuam hauv phau ntawv tshaj tawm tshwj xeeb no.
Ma, Y. et al. (2023) 'Kev tshuaj xyuas sib piv ntawm PacBio thiab ONT RNA sequencing txoj kev rau Nemopilema Nomurai venom kev txheeb xyuas', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
Chao, Q. et al. (2019) 'Kev loj hlob ntawm Populus stem transcriptome', Plant Biotechnology Journal, 17(1), pp. 206–219. doi: 10.1111/PBI.12958.
Deng, H. et al. (2022) 'Kev Hloov Pauv Dynamic hauv Ascorbic Acid Cov Ntsiab Lus thaum Lub Caij Txiv Hmab Txiv Ntoo Loj Hlob thiab Kev Loj Hlob ntawm Actinidia latifolia (ib qho qoob loo txiv hmab txiv ntoo nplua nuj Ascorbate) thiab Cov Txheej Txheem Molecular Txuas', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Kev kwv yees zoo ntawm cov noob caj noob ces biosynthetic uas koom nrog cov 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) 'Kev Tshawb Fawb Ua Ke ntawm PacBio Iso-Seq thiab Illumina RNA-Seq ntawm Tuta absoluta (Meyrick) Transcriptome thiab Cytochrome P450 Genes', Kab, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'Kev tshawb fawb txog qhov nyuaj ntawm cov ntawv sau siv PacBio ib leeg-molecule kev tshuaj xyuas lub sijhawm tiag tiag ua ke nrog Illumina RNA sequencing rau kev nkag siab zoo dua ntawm ricinoleic acid biosynthesis hauv Ricinus communis', BMC Genomics, 20(1), pp. 1–17. doi: 10.1186/S12864-019-5832-9.
