PacBio 2+3 Fofo mRNA atoa
Vaega
● Fuafuaga su'esu'e:
Fa'asoa fa'ata'ita'iga fa'asologa fa'atasi ma PacBio e fa'ailoa ai fa'asologa o fa'amaumauga
Tu'ufa'atasi fa'ata'ita'iga (fa'atusa ma tu'utu'uga e fa'ata'ita'i) fa'asolosolo fa'atasiNGS e fa'atatauina fa'amatalaga fa'amatalaga
● Fa'asologa o PacBio ile CCS mode, fa'atupuina le faitau HiFi
● Fa'asologa o fa'amaumauga atoa
● E lē manaʻomia se suʻesuʻega o le genome; ae peitai, e mafai ona faafaigaluegaina
● Bioinformatic suʻesuʻega e aofia ai e le gata o faʻamatalaga ile gene ma le isoform-level ae faʻapea foʻi le suʻesuʻeina o lncRNA, faʻasologa o kene, poly-adenylation, ma le faʻatulagaina o kene.
Tulaga lelei
● Saʻo Maualuga: HiFi faitau ma le saʻo> 99.9% (Q30), faʻatusatusa i le NGS
● Alternative Splicing Analysis: fa'asologa o fa'amaumauga uma e mafai ai ona fa'ailoaina ma fa'ailoga.
● Tuufaatasiga o PacBio ma NGS Malosi: fa'ataga le fa'avasegaina o fa'amatalaga ile tulaga fa'aisoform, fa'aalia suiga e ono ufiufi pe a su'esu'eina le fa'aaliga atoa o kene.
● 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.
● Post-Fa'atau Lagolago: 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% |
| Poly A fa'atamaoaigaina | Illumina PE150 | 6-10 Gb | Q30≥85% |
Nucleotides
|
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Amiotonu |
| Illumina Library | ≥ 10 | ≥ 0.2 | 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≥4.0; Mo manu: RIN≥4.5; 5.0≥28S/18S≥1.0; fa'atapula'a pe leai se maualuga maualuga |
| PacBio faletusi | ≥ 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. | La'au: RIN≥7.5 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
Containe: 2 ml centrifuge tube (E le fautuaina le pepa apa)
Fa'ailoga fa'ata'ita'iga: Vaega+fa'atusa eg A1, A2, A3; B1, B2, B3.
utaina:
1. Aisa mago:O fa'ata'ita'iga e mana'omia ona teu 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.
E aofia ai le au'ili'iliga nei:
Pulea lelei fa'amatalaga mata'utia
Alternative Polyadenylation Analysis (APA)
Fusion transcript analysis
Alternative Splicing Analysis
Su'esu'ega o le Universal Single-Copy Orthologs (BUSCO).
Su'esu'ega o tusitusiga fou: va'aiga o fa'asologa fa'asologa (CDS) ma fa'amatalaga fa'atino
lncRNA su'esu'ega: va'aiga o lncRNA ma fa'amoemoe
MicroSatelite Identification (SSR)
Su'esu'ega o Fa'amatalaga Eseese (DETs).
Su'esu'ega o Genes Eseese (DEGs).
Fa'amatalaga fa'atino o DEGs ma DETs
su'esu'ega BUSCO
Alternative Splicing Analysis
Alternative Polyadenylation Analysis (APA)
Genes Fa'aalia Eseese (DEGs) ma Transcripts (DETs9 anlaysis
Polotini-Polotini feso'ota'iga feso'ota'iga o DETs ma DEGs
Su'esu'e le aga'iga'i i luma na fa'afaigofieina e le BMKGene's PacBio 2+3 fa'asologa mRNA atoa e ala i le aoina o fa'asalalauga.
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/ATA/7.
