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Cikakkun Tsawon mRNA Sequencing-Nanopore

Yayin da tsarin mRNA na tushen NGS kayan aiki ne mai dacewa don ƙididdige maganganun kwayoyin halitta, dogaro da gajeriyar karantawa yana taƙaita tasirin sa a cikin hadaddun nazarin kwafin rubutu. A gefe guda, jerin nanopore yana amfani da fasahar dogon karantawa, yana ba da damar jeri cikakken kwafin mRNA. Wannan tsarin yana sauƙaƙe bincike mai zurfi na madadin splicing, gene fusions, poly-adenylation, da ƙididdiga na mRNA isoforms.

Tsarin Nanopore, hanyar da ta dogara da siginar lantarki na nanopore guda ɗaya na ainihin lokacin, yana ba da sakamako a cikin ainihin lokaci. Jagoran sunadaran motoci, DNA mai ɗaure biyu yana ɗaure zuwa sunadaran nanopore da aka saka a cikin biofilm, buɗewa yayin da yake wucewa ta tashar nanopore a ƙarƙashin bambancin ƙarfin lantarki. Ana gano siginonin lantarki na musamman waɗanda aka samar ta tushe daban-daban akan madaidaicin DNA kuma ana rarraba su a cikin ainihin lokaci, suna sauƙaƙe daidaitaccen jerin abubuwan nucleotide. Wannan sabuwar dabarar ta shawo kan iyakoki na ɗan gajeren karantawa kuma tana ba da dandamali mai ƙarfi don ƙididdigewar nazarin halittu, gami da haɗaɗɗun nazarin fassarar, tare da sakamako nan take.

Dandalin: Nanopore PromethION 48

Tuntube Mu

Cikakkun Sabis

Bioinformatics

Sakamakon Demo

Fitattun wallafe-wallafe

Siffofin

● Ɗaukar poly-A mRNA tare da haɗin cDNA da shirye-shiryen ɗakin karatu

● Taswirar cikakken tsawon rubutun

● Binciken bioinformatic dangane da daidaitawa zuwa kwayoyin halitta

● Binciken bioinformatic ya haɗa da ba kawai magana a jinsin halitta da matakin isoform ba amma har ma nazarin lncRNA, fusions gene, poly-adenylation da tsarin kwayoyin halitta.

Amfanin Sabis

●Ƙimar magana a matakin isoform: ba da damar cikakken bayani da cikakken bincike na magana, bayyana canjin da za a iya rufewa yayin da ake nazarin dukkan maganganun kwayoyin halitta.

●Rage Buƙatun Bayanai:Idan aka kwatanta da Sequencing-Generation Sequencing (NGS), jerin Nanopore yana nuna ƙananan buƙatun bayanai, yana ba da damar daidaitattun matakan ƙididdige ƙididdige jinsi tare da ƙaramin bayanai.

●Mafi girman daidaito na ƙididdigar magana: duka a matakin kwayoyin halitta da kuma isoform

●Gane ƙarin bayanan kwafi: madadin polyadenylation, fusion genes da lcnRNA da kwayoyin da aka yi niyya

●Ƙwararren Ƙwararru: Ƙungiyarmu tana kawo kwarewa mai yawa ga kowane aikin, bayan kammala aikin 850 Nanopore na cikakken tsawon ayyukan da aka sarrafa da kuma sarrafa samfurori sama da 8,000.

●Tallafin Bayan Talla: alkawarinmu ya wuce bayan kammala aikin tare da lokacin sabis na watanni 3 bayan-sayar. A wannan lokacin, muna ba da bibiyar aikin, taimako na warware matsala, da kuma zaman Q&A don magance duk wata tambaya da ta shafi sakamakon.

Samfuran Bukatun da Bayarwa

Laburare	Dabarun jeri	An ba da shawarar bayanai	Kula da inganci
Poly A ya wadata	Farashin PE150	6/12 GB	Matsakaicin ƙimar inganci: Q10

Samfuran Bukatun:

Nucleotides:

Conc.(ng/μl)

Adadin (μg)

Tsafta

Mutunci

≥ 100

≥ 1.0

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Iyakance ko babu furotin ko gurɓataccen DNA da aka nuna akan gel.

Don tsire-tsire: RIN≥7.0;

Na dabbobi: RIN≥7.5;

5.0≥28S/18S≥1.0;

iyakance ko babu hawan tushe

● Shuke-shuke:

Tushen, kara ko petal: 450 MG

Leaf ko iri: 300 MG

'Ya'yan itace: 1.2 g

● Dabbobi:

Zuciya ko hanji: 300 MG

Viscera ko Brain: 240 MG

tsoka: 450 MG

Kasusuwa, Gashi ko Fata: 1g

● Arthropods:

Kwari: 6g

Crustacea: 300 MG

● Jini dukaku: 1 tube

● Kwayoyin: 10⁶Kwayoyin

Isar da Samfurin Nasiha

Kwantena: 2 ml bututu centrifuge (Ba a ba da shawarar foil tin ba)

Alamar samfur: Ƙungiya+ kwafi misali A1, A2, A3; B1, B2, B3.

Kawo:

1. Busasshen ƙanƙara: Ana buƙatar samfurori a cikin jaka kuma a binne su a bushe-kankara.

2. RNAstable tubes: Ana iya bushe samfuran RNA a cikin bututun daidaitawar RNA (misali RNAstable®) kuma a tura su cikin zafin jiki.

Gudun Aikin Sabis

Nucleotides:

Samfurin bayarwa

Gina ɗakin karatu

Jeri

Binciken bayanai

Bayan-sayar da sabis

Gudun Aikin Sabis

Nama:

Gwajin ƙira

Samfurin bayarwa

RNA cirewa

Gina ɗakin karatu

Jeri

Binciken bayanai

Bayan-sayar da sabis

Na baya: Tsarin mRNA mai cikakken tsayi -PacBio

Na gaba: 10x Genomics Visium Spatial Transcriptome

● Aikin sarrafa danyen bayanai

● Ƙirar rubutu

● Madadin splicing

● Ƙididdigar ƙididdigewa a matakin kwayoyin halitta da matakin isoform

● Nazarin magana daban-daban

● Bayanin Aiki da haɓakawa (DEGs da DETs)

Madadin bincike splicing Alternative Polyadenylation Analysis (APA)

lncRNA tsinkaya

Annotation na novel genes

Tarin DETs

Protein-Protein Networks a DEGs

Bincika ci gaban da BMKGene's Nanopore cikakken tsawon sabis na jerin abubuwan mRNA ya sauƙaƙe ta hanyar tarin wallafe-wallafe.

Gong, B. et al. (2023) 'Epigenetic da transcriptional kunnawa na secretory kinase FAM20C a matsayin oncogene a glioma', Journal of Genetics and Genomics, 50(6), shafi 422-433. doi: 10.1016/J.JGG.2023.01.008.

Shi, Z. et al. (2023) 'cikakkar nau'in rubutun ƙwayoyin lymphocytes suna amsawa ga IFN-γ yana nuna amsawar rigakafin Th1-skewed a cikin flounder (Paralichthys olivaceus)', Kifi & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.

Ma, Y. et al. (2023) 'Binciken kwatankwacin hanyoyin PacBio da ONT RNA don gano dafin Nemopilema Nomurai', Genomics, 115(6), shafi. 110709. doi: 10.1016/J.YGENO.2023.110709.

Yau, D. et al. (2023) 'Binciken Nano-seq yana nuna halaye daban-daban na aiki tsakanin exosomes da microvesicles da aka samo daga hUMSC', Binciken Kwayoyin Halitta da Farfa, 14 (1), shafi 1-13. doi: 10.1186/S13287-023-03491-5/TABLES/6.