MRNA fa'asologa atoa -PacBio
Vaega
● fa'asologa o le cDNA mai le poly-A mRNA soso'o ai ma tapenaga faletusi
● Fa'asologa i le CCS mode, fa'atupu fa'amatalaga HiFi
● Fa'asologa o fa'amaumauga atoa
● O le suʻesuʻega e le manaʻomia se genome faʻasino; ae peitai, e mafai ona faafaigaluegaina
● Bioinformatic su'esu'ega e mafai ai ona su'esu'eina fa'amaumauga o le isoform lncRNA, fusuaga o kenera, poly-adenylation, ma le fa'atulagaina o kene.
Tulaga Auaunaga
●Maualuga Maualuga: HiFi faitau ma le saʻo> 99.9% (Q30), faʻatusatusa i le NGS
● Alternative Splicing Analysis: fa'asologa o fa'amaumauga atoa e mafai ai ona fa'ailoaina ma fa'ailoga
●Tomai Tele: fa'atasi ai ma se fa'amaumauga o le fa'amae'aina o le sili atu i le 1100 PacBio transcriptome galuega fa'atino atoa ma le fa'agaioiina i luga ole 2300 fa'ata'ita'iga, o la matou 'au e aumaia le tele o le poto masani i galuega uma.
●Lagolago Post-Fa'atau: o la matou tautinoga e faʻalautele atu i tua atu o le faʻamaeʻaina o galuega faʻatasi ma le 3-masina pe a maeʻa le faʻatau atu taimi auaunaga. I le taimi lea, matou te ofoina atu le tulitatao o poloketi, fesoasoani faʻafitauli, ma Q&A sauniga e faʻafeiloaʻi ai soʻo se fesili e fesoʻotaʻi ma iʻuga.
Fa'ata'ita'iga Mana'oga ma le Tu'uina atu
| Faletusi | Fuafuaga fa'asologa | Fa'amatalaga fautuaina | Puleaina lelei |
| PolyA fa'atamaoaigaina mRNA CCS faletusi | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
Fa'ata'ita'iga Mana'oga:
Nucleotides:
● La'au:
A'a, Po'o Po'o Petal: 450 mg
Laulaau po'o Fatu: 300 mg
Fualaau: 1.2 g
● Manu:
FOTO po'o le Tonu: 300 mg
Viscera po'o Fai'ai: 240 mg
Muso: 450 mg
Ivi, Laulu po'o Pa'u: 1g
● Arthropods:
Iniseti: 6g
Crustacea: 300 mg
● toto atoa: 1 paipa
● Selula: 106 sela
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Fa'amaoni |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Fa'atapula'a pe leai se polotini po'o le fa'aleagaina o DNA o lo'o fa'aalia ile gel. | Mo laau toto: RIN≥7.5; Mo manu: RIN≥8.0; 5.0≥ 28S/18S≥1.0; fa'atapula'a pe leai se maualuga maualuga |
Fautuaina Fa'ata'ita'iga Tu'uina atu
pusa: 2 ml centrifuge paipa (E le fautuaina le pepa apa)
Fa'ailoga fa'ata'ita'iga: Vaega+fa'atusa eg A1, A2, A3; B1, B2, B3.
utaina:
1. Fa'amamago-aisa: Fa'ata'ita'iga e mana'omia ona fa'apipi'i i taga ma tanu i le aisa-mago.
2. RNAstable tubes: RNA samples e mafai ona fa'amago ile RNA stabilization tube(eg RNAstable®) ma lafo ile potu vevela.
Auala o Galuega Au'aunaga
Fa'ata'ita'iga mamanu
Fa'ata'ita'iga tu'uina atu
RNA su'ega
Fausia faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
E aofia ai le au'ili'iliga nei:
● Pulea lelei o fa'amaumauga mata'utia
● Alternative Polyadenylation Analysis (APA)
● Fusion transcript analysis
● Alternative Splicing Analysis
● Su'esu'ega ole Universal Single-Copy Orthologs (BUSCO).
● Su'esu'ega tala fou: va'aiga o fa'asologa fa'asologa (CDS) ma fa'amatalaga fa'atino
● su'esu'ega lncRNA: va'aiga o lncRNA ma fa'amoemoe
● MicroSatelite Identification (SSR)
su'esu'ega BUSCO
Alternative Splicing Analysis
Alternative Polyadenylation Analysis (APA)
Fa'amatalaga aoga o tusitusiga fou
Su'esu'e le aga'i i luma na fa'afaigofieina e le BMKGene's Nanopore mRNA fa'asologa atoa auaunaga i totonu o lenei lomiga fa'aalia.
Ma, Y. et al. (2023) 'Su'esu'ega fa'atusatusa o PacBio ma ONT RNA fa'asologa o metotia mo le fa'ailoaina o mea oona a Nemopilema Nomurai', Genomics, 115(6), i. 110709. doi: 10.1016/J.YGENO.2023.110709.
Chao, Q. et al. (2019) 'O le atina'e malosi o le Populus stem transcriptome', Plant Biotechnology Journal, 17(1), itulau 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 (o Ascorbate-Rich Fruit Crop) ma le Associated Molecular Mechanisms', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Vailoaiga lelei o kenera ala biosynthetic o loʻo aʻafia i polyphyllins bioactive i Paris polyphylla', Communications Biology 2022 5: 1, 5(1), itulau 1–10. doi: 10.1038/s42003-022-03000-z.
Liu, M. et al. (2023) 'Combined PacBio Iso-Seq and Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome and Cytochrome P450 Genes', Iniseti, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'O se suʻesuʻega o le lavelave faʻasalalau e faʻaaoga ai le PacBio tasi-molole suʻesuʻega faʻatasi ma le Illumina RNA faʻasologa mo se malamalama sili atu i le biosynthesis o le ricinoleic acid i Ricinus communis', BMC Genomics, 20(1), itulau 1–17. doi: 10.1186/S12864-019-5832-9.
