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Kutsata kutalika kwa mRNA -PacBio

Kutsata kwa mRNA kwautali -PacBio Chithunzi Chowonetsedwa

Ngakhale kutsatizana kwa mRNA kochokera ku NGS ndi chida chogwiritsa ntchito mosiyanasiyana pakuwerengera ma jini, kudalira kwake pamawerengedwe afupikitsa kumalepheretsa kugwiritsidwa ntchito kwake pakuwunikira zovuta zolembera. Kumbali ina, PacBio sequencing (Iso-Seq) imagwiritsa ntchito ukadaulo wowerengera nthawi yayitali, zomwe zimathandizira kutsatizana kwa zolembedwa zazitali za mRNA. Njirayi imathandizira kufufuza mwatsatanetsatane njira zina zophatikizira, kuphatikiza ma jini, ndi poly-adenylation. Komabe, pali zisankho zina za gene expression quantification chifukwa cha kuchuluka kwa deta yofunikira. Ukadaulo wotsatizana wa PacBio umadalira kutsatizana kwa molekyulu imodzi, nthawi yeniyeni (SMRT), kupereka mwayi wapadera wojambula zolemba zazitali za mRNA. Njira yatsopanoyi imaphatikizapo kugwiritsa ntchito zero-mode waveguides (ZMWs) ndi zitsime zokhala ndi microfabricated zomwe zimathandiza kuti nthawi yeniyeni ya DNA polymerase iwonetsedwe panthawi yotsatizana. Mkati mwa ma ZMW awa, PacBio's DNA polymerase imapanga ulusi wowonjezera wa DNA, ndikupanga zowerengera zazitali zomwe zimatenga zolemba zonse za mRNA. Opaleshoni ya PacBio mumayendedwe a Circular Consensus sequencing (CCS) imakulitsa kulondola mwa kutsata mobwerezabwereza molekyulu yomweyo. Mawerengedwe a HiFi omwe amapangidwa ali ndi kulondola kofanana ndi NGS, zomwe zimathandizira pakuwunika kwatsatanetsatane komanso kodalirika kwa zovuta zolembera.

Platform: PacBio Sequel II; PacBio Revio

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Bioinformatics

Zotsatira za Demo

Zofalitsa Zowonetsedwa

Mawonekedwe

● cDNA synthesis kuchokera ku poly-A mRNA yotsatiridwa ndi kukonzekera laibulale

● Kutsata mu CCS mode, kupanga HiFi kuwerenga

● Kutsatizana kwa zolembedwa zazitali zonse

● Kusanthula sikufuna kuti munthu akhale ndi ma genome; komabe, ikhoza kugwiritsidwa ntchito

● Kusanthula kwa bioinformamatic kumathandiza kusanthula zolembedwa za isoform lncRNA, kuphatikiza kwa majini, poly-adenylation, ndi kapangidwe ka majini.

Ubwino wa Utumiki

●Kulondola Kwambiri: HiFi imawerenga molondola> 99.9% (Q30), poyerekeza ndi NGS

● Kusanthula kwa Njira Zina: kutsatizana kwa zolembedwa zonse kumathandizira kuzindikirika ndi mawonekedwe a isoform

●Katswiri Wazambiri: Pokhala ndi mbiri yakukwaniritsa ma projekiti opitilira 1100 a PacBio aatali komanso kukonza zitsanzo zopitilira 2300, gulu lathu limabweretsa zokumana nazo zambiri pantchito iliyonse.

●Thandizo la Post-Sales: Kudzipereka kwathu kumapitilira kutha kwa projekiti ndi nthawi ya miyezi itatu mutagulitsa. Panthawiyi, timapereka zotsatila za polojekiti, chithandizo chazovuta, ndi magawo a Q&A kuti tiyankhe mafunso aliwonse okhudzana ndi zotsatira.

Zitsanzo Zofunika ndi Kutumiza

Library

Njira yotsatirira

Deta yovomerezeka

Kuwongolera Kwabwino

Laibulale ya PolyA yolemeretsa mRNA CCS

PacBio Sequel II

PacBio Revio

20/40 Gb

5/10 M CCS

Q30≥85%

Zitsanzo Zofunika:

Nucleotides:

● Zomera:

Muzu, tsinde kapena petal: 450 mg

Tsamba kapena Mbewu: 300 mg

Chipatso: 1.2 g

● Chinyama:

Mtima kapena matumbo: 300 mg

Viscera kapena Ubongo: 240 mg

Minofu: 450 mg

Mafupa, Tsitsi kapena Khungu: 1g

● Matenda a nyamakazi:

Tizilombo: 6g

Crustacea: 300 mg

● Mwazi wonse:1 chubu

● Maselo: 10⁶maselo

Conc.(ng/μl)

Mtengo (μg)

Chiyero

Umphumphu

≥100

≥ 1.0

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Zochepa kapena zopanda mapuloteni kapena DNA kuipitsidwa komwe kumawonetsedwa pa gel.

Kwa zomera: RIN≥7.5;

Kwa nyama: RIN≥8.0;

5.0≥ 28S/18S≥1.0;

kukwera kochepa kapena kopanda koyambira

Kutumiza Kwachitsanzo Kovomerezeka

Chotengera: 2 ml centrifuge chubu (zojambula za malata ndizosavomerezeka)

Zitsanzo zolembera: Gulu+ lofanana mwachitsanzo A1, A2, A3; B1, B2, B3.

Kutumiza:

1. Madzi owuma: Zitsanzo ziyenera kupakidwa m'matumba ndikukwiriridwa mu ayezi wouma.

2. RNAstable chubu: RNA zitsanzo akhoza zouma mu RNA kukhazikika chubu (mwachitsanzo RNAstable®) ndi kutumizidwa mu firiji.

Kuyenda kwa Ntchito Yantchito

Kuyesera kupanga

Kupereka zitsanzo

Kusintha kwa RNA

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Zam'mbuyo: Eukaryotic mRNA Sequencing-NGS

Ena: Kutalika Kwathunthu kwa mRNA Sequencing-Nanopore

Mulinso zowunikira izi:

● Kuwongolera khalidwe la data yaiwisi

● Alternative Polyadenylation Analysis (APA)

● Kusanthula zolemba za Fusion

● Kusanthula kwa Njira Zina

● Benchmarking Universal Single-Copy Orthologs (BUSCO).

● Kusanthula kwa zolemba zatsopano: kuneneratu za kutsatizana kwa ma coding (CDS) ndi ndemanga zogwira ntchito

● Kusanthula kwa lncRNA: kuneneratu za lncRNA ndi zolinga

● MicroSatelite Identification (SSR)

Kusanthula kwa BUSCO

Kusanthula kwa Njira Zina za Splicing

Alternative Polyadenylation Analysis (APA)

Kafotokozedwe ka ntchito zamanovel zolembedwa

Onani kupita patsogolo kotsogozedwa ndi BMKGene's Nanopore yotsatizanatsatizana za mRNA m'bukuli.

Ma, Y. et al. (2023) 'Kuyerekeza kusanthula kwa PacBio ndi njira zotsatizana za ONT RNA za Nemopilema Nomurai venom identification', Genomics, 115 (6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.

Chao, Q. et al. (2019) 'Kukula kwamphamvu kwa 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 (an 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) 'Kuneneratu mogwira mtima kwa majini amtundu wa biosynthetic omwe amaphatikizidwa mu bioactive polyphyllins ku 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 ndi Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome ndi Cytochrome P450 Genes', Insects, 14 (4), p. 363. doi: 10.3390/INSECTS14040363/S1.

Wang, Lijun et al. (2019) 'Kafukufuku wa zovuta zolembera pogwiritsa ntchito PacBio single-molecule real-time analysis kuphatikiza ndi Illumina RNA sequencing kuti amvetse bwino ricinoleic acid biosynthesis mu Ricinus communis', BMC Genomics, 20 (1), pp. 1-17. doi: 10.1186/S12864-019-5832-9.