ʻO ke kaʻina mRNA lōʻihi piha -PacBio
Nā hiʻohiʻona
● cDNA synthesis mai ka poly-A mRNA i ukaliia e ka hoomakaukau waihona
● Ke kaʻina ʻana ma ke ʻano CCS, e hana ana i nā heluhelu HiFi
● Ke kaʻina ʻana o nā moʻolelo holoʻokoʻa
● ʻAʻole pono ke kālailai ʻana i kahi genome kuhikuhi; akā, hiki ke hoʻohana ʻia
● Hiki i ka loiloi bioinformatic ke kālailai ʻana o nā transcripts isoform lncRNA, gene fusions, poly-adenylation, a me ka gene structure
Pono lawelawe
●Pono Kiʻekiʻe: Heluhelu ʻo HiFi me ka pololei >99.9% (Q30), e like me NGS
● Nānā Hoʻopili ʻokoʻa: hiki i ke kaʻina ʻana o nā transcript holoʻokoʻa ke ʻike i ka isoform a me ke ʻano
●ʻIke Nui: me kahi moʻolelo o ka hoʻopau ʻana ma luna o 1100 PacBio nā papahana transcriptome holoʻokoʻa a me ka hana ʻana ma luna o 2300 mau laʻana, lawe kā mākou hui i ka waiwai o ka ʻike i kēlā me kēia papahana.
●Kākoʻo ma hope o ke kūʻai aku: ʻo kā mākou kūpaʻa e ʻoi aku ma mua o ka hoʻopau ʻana o ka papahana me kahi manawa lawelawe 3 mahina ma hope o ke kūʻai aku. I kēia manawa, hāʻawi mākou i ka hahai ʻana o ka papahana, kōkua hoʻoponopono, a me nā hālāwai Q&A e hoʻoponopono i nā nīnau pili i nā hopena.
Nā Koina Laʻana a me ka Hoʻouna ʻana
| Hale Waihona Puke | Hoʻolālā kaʻina | Manaʻo ʻia ka ʻikepili | Ka Mana Mana |
| Hoʻonui ʻia ʻo PolyA mRNA CCS waihona | PacBio Sequel II PacBio Revio | 20/40 Gb 5/10 M CCS | Q30≥85% |
Nā Koina Laʻana:
Nucleotides:
● Nā mea kanu:
Aʻa, ʻaʻa a i ʻole Petal: 450 mg
Lau a Hua paha: 300 mg
Hua: 1.2 g
● holoholona:
Puʻuwai a i ʻole ka ʻōpū: 300 mg
ʻO Viscera a i ʻole lolo: 240 mg
ʻiʻo: 450 mg
Iwi, lauoho a ʻili paha: 1g
● Nā ʻāʻī:
Nā Insect: 6g
Crustacea: 300 mg
● Ke koko holoʻokoʻa: 1 paipu
● Nā Pūnaewele: 106 pūnaewele
| Conc.(ng/μl) | Ka nui (μg) | Maemae | Pono pono |
| ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Hoʻopaʻa ʻia a ʻaʻole hoʻi i ka protein a i ʻole DNA contamination i hōʻike ʻia ma ka gel. | No nā mea kanu: RIN≥7.5; No nā holoholona: RIN≥8.0; 5.0≥ 28S/18S≥1.0; palena a ʻaʻole kiʻekiʻe kumu |
Manaʻo ʻia ka hāʻawi laʻana
ipu: 2 ml centrifuge paipu (ʻaʻole ʻōlelo ʻia ʻo Tin foil)
Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3; B1, B2, B3.
Hoʻouna:
1. Ka hau maloo: Pono e hoʻopili ʻia nā laʻana i loko o nā ʻeke a kanu ʻia i loko o ka hau maloʻo.
2. Nā paipu RNAstable: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA i loko o ka paipu hoʻopaʻa RNA (e laʻa me RNAstable®) a hoʻouna ʻia i loko o ka lumi wela.
Holo Hana Hana
Hoʻolālā hoʻokolohua
Hāʻawi laʻana
RNA unuhi
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
Loaʻa i kēia ka nānā ʻana:
● Ka mana o ka maikaʻi o ka ʻikepili maka
● Kānāwai Polyadenylation ʻē aʻe (APA)
● Hoʻopili transcript analysis
● Nānā Hoʻopili ʻokoʻa
● Ka nānā 'ana i nā Universal Single-Copy Orthologs (BUSCO).
● Nānā transcript hou: wanana o nā kaʻina hana coding (CDS) a me ka wehewehe hana
● lncRNA kālailai: wanana o lncRNA a me na pahu hopu
● MicroSatelite Identification (SSR)
ʻIkepili BUSCO
ʻAno Hoʻopili Hoʻopili ʻana
ʻIkepili Polyadenylation ʻē aʻe (APA)
ʻO ka hōʻike kikoʻī o nā transcript hou
E ʻimi i nā holomua i hoʻolako ʻia e BMKGene's Nanopore piha lōʻihi mRNA sequencing lawelawe i kēia paʻi hiʻona.
Ma, Y. et al. (2023) 'Ka hoʻohālikelike hoʻohālikelike o PacBio a me ONT RNA kaʻina hana no ka ʻike ʻana i ka mea make ʻo Nemopilema Nomurai', Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.
Chao, Q. et al. (2019) 'The developmental dynamics of the Populus stem transcriptome', Plant Biotechnology Journal, 17(1), pp. 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 (he Ascorbate-Rich Fruit Crop) and the Associated Molecular Mechanisms', International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.
Hua, X. et al. (2022) 'Wanaana kūpono o nā genes ala biosynthetic i komo i nā polyphyllins bioactive ma Paris polyphylla', Communications Biology 2022 5:1, 5(1), pp. 1–10. doi: 10.1038/s42003-022-03000-z.
Liu, M. et al. (2023) 'Hoʻohui ʻia ʻo PacBio Iso-Seq a me Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome and Cytochrome P450 Genes', Insects, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.
Wang, Lijun et al. (2019) 'He noiʻi o ka paʻakikī transcriptome me ka hoʻohana ʻana iā PacBio hoʻokahi-molele i ka manawa maoli i hui pū ʻia me ka Illumina RNA sequencing no ka ʻike maikaʻi ʻana i ka biosynthesis acid ricinoleic ma Ricinus communis', BMC Genomics, 20(1), pp. 1–17. doi: 10.1186/S12864-019-5832-9.
