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16S/18S/ITS Amplicon Sequencing-PacBio

Hidde-sidayaasha 16S iyo 18S rRNA, oo ay weheliyaan gudaha gudaha Transcribed Spacer (ITS), waxay u adeegaan calaamadaynta faraha molecular ee muhiimka ah sababtoo ah isku darka gobollada aadka loo ilaaliyo iyo kuwa kala duwan, iyaga oo ka dhigaya qalab qiimo leh oo lagu garto noolaha prokaryotic iyo eukaryotic. Kordhinta iyo isku xigxiga gobolladan waxay bixiyaan hab gooni-gooni ah oo ka xor ah baaritaanka ka kooban microbial iyo kala duwanaanshiyaha dhammaan nidaamyada deegaanka ee kala duwan. Iyadoo isku xigxiga Illumina ay caadi ahaan bartilmaameedsato gobollada gaagaaban ee kala duwanaansho leh sida V3-V4 ee 16S iyo ITS1, waxaa la muujiyay in taxonomic taxonomic sare ah lagu gaari karo iyadoo la raacayo dhererka buuxa ee 16S, 18S, iyo ITS. Habkan dhammaystiran waxa uu keenayaa boqolley sare oo taxane ah oo si sax ah loo kala saaray, iyada oo la gaadhayo heer xal ah oo ku fidsan aqoonsiga noocyada. PacBio's Single-Molecule Real-Time Real-Time (SMRT) isku xigxiga madal waxay u taagan tahay siinta akhrin aad u dheer oo sax ah (HiFi) kaas oo daboolaya amplicon-yada dhererka buuxa, iyadoo la tartameysa saxnaanta isku xigxiga Illumina. Awooddani waxay u oggolaanaysaa cilmi-baarayaasha inay helaan faa'iido aan la isku mid ahayn - aragtida muuqaalka muuqaalka hiddaha. Daboolista la dheereeyey ayaa si weyn kor ugu qaadeysa xallinta sharraxaadda noocyada, gaar ahaan gudaha bulshooyinka bakteeriyada ama fangaska, taas oo awood u siineysa faham qoto dheer oo ku saabsan qallafsanaanta dadka microbial-ka ah.

Nala Soo Xidhiidh

Faahfaahinta Adeegga

Bioinformatics

Natiijooyinka Demo

Daabacaada sifada

Tilmaamaha Adeegga

● Madal isku xigxiga: PacBio Revio

● Habka isku xigxiga: CCS (HiFi wax akhriya)

● Kordhinta gobolka bartilmaameedka ah oo ay ku xigto isku xirka amplicons ka hor diyaarinta maktabadda gambaleelka HiFi SMRT

Faa'iidooyinka Adeegga

●Xallinta cashuurta sare: Than gaaban-amplicon isku xigxiga,awood sare u qaadista heerarka OTU ee heerka noocyada.

●Wicitaan sal leh oo aad u saxan: Habka isku xigxiga ee PacBio CCS (HiFi wuu akhriyaa).

●Go'doon-free: Aqoonsiga degdega ah ee ka kooban microbial muunadaha deegaanka.

●Si ballaaran loo dabaqi karoDaraasado bulsho oo kala duwan oo microbial ah.

●Falanqaynta bioinformatic oo dhamaystiranXirmada QIIME2 ee ugu dambeysay (aragti tirooyin ah oo ku saabsan ecology microbial) oo leh falanqayn kala duwan marka loo eego xogta xogta, sharraxaadda, OTU/ASV.

●Khibrad Balaadhan: Iyada oo kumanaan mashruucyo isku xigxig ah oo amplicon ah la qabto sanadkiiba, BMKGENE waxay keentaa in ka badan toban sano oo waayo-aragnimo ah, koox falanqaynta xirfad sare leh, nuxur dhamaystiran, iyo taageero iib ah oo heer sare ah.

Tilmaamaha Adeegga

Maktabadda	Istaraatiijiyada Taxanaha	Xogta lagu taliyay
Amplicon	PacBio Revio	10/30/50 K tags (CCS)

Shuruudaha Tusaalaha

Xooga saarida (ng/µL)	Tirada guud (µg)	Mugga (µL)
≥5	≥0.3	≥20

Tusaalaha keenista lagu taliyay

Ku qabooji shaybaarada ku jira nitrogen dareeraha ah 3-4 saacadood oo ku kaydi nitrogen dareeraha ah ama -80 digrii ilaa boos sii hayn muddo dheer ah. Tusaalaha rarida baraf engegan ayaa loo baahan yahay.

Socodka Shaqada Adeegga

Tusaalaha keenista

Dhismaha maktabadda

Isku xigxiga

Falanqaynta xogta

Adeegyada iibka kadib

Kii hore: 16S/18S/ITS Amplicon Sequencing-NGS

Xiga: Qiimaynta chromatin-ka-wareejin-ka-hortagga leh ee la heli karo

Waxa ku jira falanqaynta soo socota:

●Kontoroolka tayada xogta ceeriin

●Kooxaha OTU/Buuq-bax (ASV)

●Sharaxaad OTU

● Falanqaynta kala duwanaanta Alpha: tusmooyin badan, oo ay ku jiraan Shannon, Simpson iyo ACE.

●Falanqaynta kala duwanaanta beta

●Falanqaynta kooxaha dhexdooda

●Falanqaynta isku xidhka: ee udhaxeeya arrimaha deegaanka iyo ka koobanaanta OUT iyo kala duwanaanta

●16S odoroska hidda-socodka shaqada

Histogram-ka qaybinta taxonomic

Qaybinta bulshada geed phylogenetic

Falanqaynta kala duwanaanta Alpha: ACE

index

Falanqaynta kala duwanaanta beta: PCoA

Falanqaynta kooxaha: ANOVA

Sahami horumarka ay fududaysay BMKGene adeegyada isku xigxiga amplicon ee PacBio iyada oo loo marayo ururinta daabacaadyada.

Gao, X. iyo Wang, H. (2023) 'Falanqaynta isbarbardhigga ee Rumen Profiles Bacterial Profiles and Functions inta lagu guda jiro laqabsiga Phenology kala duwan (Regreen vs. Grassy) ee Alpine Merino Sheep oo leh laba marxaladood oo koraya on cawska Alpine', halsano, 9 ( 1), b. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. iyo al. (2023) ' Qabashada maadada mugdiga ah ee microbial ee ciidda saxaraha ah iyadoo la adeegsanayo metagenomics-ku-saleysan culturomics iyo falanqaynta xallinta sare', npj Biofilms iyo Microbiomes 2023 9:1, 9(1), pp. 1-14. doi: 10.1038/s41522-023-00439-8.

Mu, L. iyo al. (2022) 'Saamaynta milixyada dufanka leh ee sifooyinka halsano, kala duwanaanta bakteeriyada iyo xasilloonida aerobic ee silage isku dhafan oo lagu diyaariyey alfalfa, caws bariis iyo qamadi', Journal of Science of Food and Agriculture, 102(4), bogga 1475- 1487. doi: 10.1002/JSFA.11482.

Yang, J. iyo al. (2023) 'Isdhexgalka ka dhexeeya Oxidative Stress Biomarkers iyo Gut Microbiota ee Saamaynta Antioxidant ee Soosaarka Sonchus brachyotus DC. ku jirta Cadaadis Oxidative Mindhicirka Oxazolone-ku-abuuray ee Zebrafish dadka waaweyn', Antioxidants 2023, Vol. 12, Bogga 192, 12(1), b. 192. doi: 10.3390/ANTIOX12010192.