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Heerka dhererka buuxa ee MRNA

MRNA-GRNA SIYAASADDA MRNAIONED -PACBIO Muuqaalka Muuqaalka

In kasta oo isku xigxiga Mrna uu ku saleysan yahay ee NGS-ka ah waa aalad isku-darka ah ee lagu muujiyo muujinta hiddaha, ku tiirsanaanta akhlaaqda gaagaaban ayaa xaddidaysa isticmaalkiisa falanqaynta Transcritomic. Dhanka kale, isku xirka Pacbio (ISO-SIQ) Waxay shaqaaleysiisaa tikniyoolajiyad aqrisooyin ah, taas oo awood u siisa taxanaha sheekooyinka guud ee MRNA. Qaabkan ayaa fududeeya sahaminta dhameystiran ee kala goynta kale, dhibcaha hidda-wadaha, iyo caadeenka poly-anoylation. Si kastaba ha noqotee, waxaa jira xulashooyin kale oo loogu talagalay muujinta fikradaha hidda-wadaha oo ay sabab u tahay xaddiga xogta loo baahan yahay. Tiknolojiyadda isku-darka Pacbio waxay ku tiirsan tahay hal-molecule, waqtiga-dhabta ah (SMRT) isku xirka, iyadoo la siinayo faa'iido gaar ah oo ku saabsan qabsashada waraaqaha dhexdhexaadka ah ee Mrna-dheregsan. Hababkan hal-abuurka leh wuxuu ku lug leeyahay isticmaalka mowqifka mowqifyada eber-pero-mowjadaha (zmws) iyo ceelasha microfabricy ee u saamaxaya kormeerka waqtiga-dhabta ah ee dhaqdhaqaaqa polymoother-ka DNA-ga inta lagu gudajiro. Gudaha zmws-ka gudaha, pacbio's polymyrase-ka DNA ayaa isku darka ah in la dhammaystiro oo DNA ah, oo muddo dheer akhrin kara guud ahaan dhameystiran qoraallada MRNA. Hawlgalka PACBo ee isku-darka isugeynta (CCS) wuxuu kor u qaadaa sax saxnaanta isagoo isdaba joog ah isku xidhka isla molecule. The HIFI-ga ayaa akhrinaya si sax ah oo u dhigma NGS, oo sii kordhineysa falanqayn dhammaystiran oo la isku halleyn karo oo ku saabsan astaamaha Transcritomic ee adag.

MUQDISHO: Pacbio Sequel II; PACBIO Revio

Nala soo xiriir

Faahfaahinta adeegga

Bioinformatics

Natiijooyinka Demo

Daabacaadda Feejignaanta

Astaamaha

● Synthesis-ka CDNA Stresis ka timid POLY-MRNA waxaa ku xigay diyaarinta maktabadda

● Isku xigxiga xadka CCS, abuurista Hibi ayaa akhrisa

● Isku xigxiga waraaqaha dhererka buuxa

Fal -la-soolaynta looma baahna tixraac tixraac tixraac; Si kastaba ha noqotee, waa la shaqaysan karaa

● Balancilitaanka bioinfflemfflem-ka ayaa u sahleysa falanqaynta qoraalka qoraalka ah ee Isoform LNCRIN, Foolalka hidda-wadaha, poly-odaynta, iyo qaab-dhismeedka hiddaha

Faa'iidooyinka adeegga

●Sax ah sare: Hifi wuxuu akhriyaa sax ahaanta> 99.9% (Q30), u dhigma NGS

● Falanqaynta kala-goynta kaladuwan: Isku-darka qoraallada oo dhan wuxuu awood u siinayaa aqoonsiga iyo astaamaha

●Khibrad ballaaran: Iyada oo la raacayo rikoor wado oo ah in la dhammeeyo wax ka badan 1100 oo pacbio-dherer ah oo pacbio ah oo buuxa iyo ka shaqeynta in ka badan 2300 saamba, kooxdeena waxay keentaa khibrad badan oo ku saabsan mashruuc kasta.

●Taageerada iibka ka dib: Balankeenna wuxuu ku fidsan yahay dhameystirka mashruuca ee leh muddo 3 bilood iibka iibka ah ka dib. Inta lagu jiro waqtigan, waxaan bixinaa dabagal dabagal mashruuc, gargaar dhibaatayn, iyo Q & SS & Kalfadhiyo si wax looga qabto wixii su'aalo ah ee la xiriira natiijooyinka.

Shuruudaha muunadda iyo dhalmada

Maktabad

Istaraatiijiyadda isku xigta

Xogta lagu taliyay

Xakamaynta Tayada

POLYA waxay kobcisay maktabada Mrna CCS

Pacbio sequel II

PACBIO Revio

20/40 GB

5/10 m ccs

Q3t85%

Shuruudaha muunadda:

Nucleotatides:

Dhirta:

Xidid, asliga ah ama petal: 450 mg

Caleen ama abuur: 300 mg

Miraha: 1.2 g

● Xayawaanka:

Wadnaha ama xiidmaha: 300 mg

Viscera ama maskaxda: 240 mg

Murqaha: 450 mg

Lafaha, timaha ama maqaarka: 1g

● Arthropods:

Cayayaanka: 6g

Crustacea: 300 mg

Dhiiga oo dhan: 1 tuubbo

● Unugyada: 10⁶unugyada

CODKA. (NG / μL)

Tirada (μg)

Daahir

Sharaf

≥ 100

≥ 1.0

OD260 / 280 = 1.7-2.5

OD260 / 230 = 0.5-2.5

Xadidan ama aan lahayn proteotiin ama wasakhda DNA ee lagu muujiyey jel.

Dhirta: Rin≥7.5;

Xayawaanka: Rin≥8.0;

5.0≥ 28s / 18s≥1.0;

Xaddidan ama kor u qaadis ah

Keenista muunad

Weelka: 2 ml centrifuge tuubbada (foil Tin laguma talinayo)

Calaamadaha muujinta: Kooxda + waxay ku daydaa tusaale ahaan a1, A2, A3; B1, B2, B3.

Shixnad:

1. Ice-ICE: Saamiyada ayaa loo baahan yahay in lagu buuxiyo bacaha oo lagu aasay baraf qalalan.

2. Tuunnada lagu qurxiyo

Socodka shaqada adeegga

Naqshadeynta Tijaabada

Keenista muunad

Soo saarida RNA

Dhismaha Maktabadda

Kala xidowdo

Falanqaynta Xogta

Adeegyada ka dib

Hore: Eukaryotic Mrna waa isku xigxiga-ngs

Xiga: Xilliga dhererka buuxa ee MRNA

Waxaa ku jira falanqaynta soo socota:

● Xakamaynta tayada tayada leh ee xogta ceeriin

● Falanqaynta kale ee plandeyadenlation (APA)

Falanqaynta Transiction Transcript Falanqaynta

● Falanqaynta kala-goynta kaladuwan

● Astaan u dhigista guud ee koobi-koobi-koobi-koobi ah (balanta balanta)

Plan Falanqaynta Transcript Cuntada ah: saadaalinta isku xirka isku xirka (CD-yada) iyo faahfaahinta shaqeynaya

● Falanqaynta Lancna: saadaalinta Lancna iyo yoolalka

● Aqoonsiga microsatit (SSR)

Falanqaynta Busco

Falanqaynta Kala-sooca Kala-soocidda

Falanqaynta kale ee plandeyadenynyelylation (APA)

Qoraalka Shaqada ee Shaqada ee Qoraalka cusub

Baadh horumarka ay fududeeyeen adeegsiga Bmkgene Nanopore oo ah adeegyo isku xigxiga M Mrna oo ah qiimeyntaan ku jira daabacaadan.

MA, y. et al. (2023) Falanqaynta Isbarbardhiga Pacio iyo Hababka Isku-darka ee Jnacyada ee loo yaqaan 'Nebopilama noterai', Genomas, 115 (6), p. 110709. DOI: 10.1016 / J.ygego.2023.110709.

Chao, Q. Et al. (2019) 'Dhaqdhaqaaqa koritaanka ee populus stem transcriptome', joornaalka bayootarka, 17 (1), pp. 206-219. doi: 10.1111 / PBI.12958.

Deng, H. et al. (2022) 'isbeddelada firfircoon ee asiid asific-ka inta lagu jiro horumarka miraha iyo bislaada Ficifidis Latifolia (dalag miro hodanka ah oo loo yaqaan' ancorbate 'iyo qaababka miraha-molinicaular', joornaalka caalamiga ah ee cilmiga molikalar, 23 (10), p. 5808. Do: 10.3390 / Ijmss23105808 / S1.

Hua, x. et al. (2022) 'saadaalin wax ku ool ah oo jidka loo yaqaan' biosynthetic wadooyinka 'hiddo-wadaha' doi: 10.1038 / s42003-022-03000-z.

Liu, M. et al. (2023) 'Isku-darka Pacbio ISO-SEQ iyo CLEQ RANCE-Seq ee The TATA-Seq Actoluta (Meyyrick) Transcritome iyo Cytochrome P450 hiddo-wadaha' ', cayayaanka, 14 (4), p. 363. Do: 10.3390 / cayayaanka14040363 / S1.

Wang, lijun et al. (2019) Sahaminta kakanaanta qoraalka ku-dhafka ah iyadoo la adeegsanayo falanqaynta waqtiga-dhabta ah ee pacbio-menynthisis-ka ee loo yaqaan 'Iluenic RNENTESIS' ,, 1 (1), PP. 1-17. doi: 10.1186 / s1286-019-5832-9.