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16S/18S/ITS Amplicon Sequencing-PacBio

Iijini ze-rRNA ze-16S kunye ne-18S, kunye nengingqi ye-Internal Transcribed Spacer (ITS), isebenza njengeyona nto ibalulekileyo yokuphawula iminwe yemolekyuli ngenxa yokudityaniswa kwemimandla egcinwe kakhulu kunye ne-hyper-variable, izenza izixhobo ezixabiseke kakhulu zokubonisa iimpawu zeprokaryotic kunye ne-eukaryotic. Ukwandiswa kunye nolandelelwano lwale mimandla lubonelela ngendlela engenakwahlulwa yokuphanda ubume bemicrobial kunye neyantlukwano kuzo zonke ii-ecosystems. Ngelixa ulandelelwano lwe-Illumina ngokuqhelekileyo lujolise kwimimandla emifutshane ye-hypervariable efana ne-V3-V4 ye-16S kunye ne-ITS1, kubonakaliswe ukuba i-taxonomic annotation ephezulu ifikeleleke ngokulandelelana ubude obupheleleyo be-16S, 18S, kunye ne-ITS. Le ndlela ibanzi iphumela kwiipesenti eziphezulu zolandelelwano oluhlelwe ngokuchanekileyo, ukufikelela kwinqanaba lesisombululo esinabela ekuchongweni kweentlobo. I-PacBio ye-Single-Molecule ye-Real-Time (SMRT) iqonga lokulandelelanisa iqonga livelele ngokubonelela ngofundo oluchanekileyo olude (i-HiFi) olugubungela i-amplicon yobude obupheleleyo, ikhuphisana nokuchaneka kokulandelelana kwe-Illumina. Esi sikhundla sivumela abaphandi ukuba bafumane inzuzo engenakuthelekiswa - imbono yepanoramic ye-genetic landscape. Ugutyulo olwandisiweyo lusiphakamisa kakhulu isisombululo kwingcaciso yeentlobo, ngakumbi phakathi kweebhaktiriya okanye kuluntu lomngundo, okwenza ukuba kuqondwe nzulu ukuntsonkotha kobuninzi bentsholongwane.

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Bioinformatics

Iziphumo zeDemo

Iimpapasho ezikhoyo

Iimpawu zeNkonzo

● Iqonga lokulandelelanisa: I-PacBio Revio

● Imo yolandelelwano: CCS (i-HiFi ifundeka)

● Ukwandiswa kwendawo ekujoliswe kuyo kulandelwa ludibaniso oludibeneyo lweeamplicon phambi kolungiselelo lwethala leencwadi lentsimbi ye-HiFi SMRT.

Iinkonzo eziluncedo

●Isisombululo esiphezulu se-taxonomic: Than short-amplicon ulandelelwano,ivumela amazinga aphezulu okuhlelwa kwe-OTU kwinqanaba leentlobo.

●Ubizo olusisiseko oluchanekileyo: Ulandelelwano lwemodi ye-PacBio CCS (i-HiFi ifundeka).

●Ukwahlukaniswa-simahla: Ukuchongwa okukhawulezayo kokubunjwa kweentsholongwane kwiisampulu zokusingqongileyo.

●Isebenza ngokubanzi: Izifundo zoluntu ezahlukeneyo ze-microbial.

●Uhlalutyo olubanzi lwe-bioinformatic: Iphakheji ye-QIIME2 yamva nje (ingqiqo yobungakanani kwi-microbial ecology) enohlalutyo olwahlukeneyo ngokwesiseko sedatha, inkcazo, i-OTU/ASV.

●UbuGcisa obuBanzi: Ngamawaka eeprojekthi zokulandelelana kwe-amplicon eziqhutywa minyaka le, i-BMKGENE izisa ngaphezulu kweminyaka elishumi yamava, iqela lokuhlalutya abanobuchule obuphezulu, umxholo obanzi, kunye nenkxaso ebalaseleyo emva kokuthengisa.

IiNgcaciso zeNkonzo

Ithala leencwadi	Isicwangciso solandelelwano	Idatha iyacetyiswa
Amplicon	I-PacBio Revio	10/30/50 K iithegi (CCS)

IiMfuno zeSampulu

Ugxininiso (ng/µL)	Imali iyonke (µg)	Umthamo (µL)
≥5	≥0.3	≥20

Unikezelo lweSampuli eCetyisiweyo

Umkhenkce iisampulu kulwelo nitrogen iiyure 3-4 kwaye ugcine kulwelo nitrogen okanye -80 degree ukuya ugcino ixesha elide. Isampulu yokuthumela ngenqanawe eyomileyo iyafuneka.

Ukuhamba komsebenzi weNkonzo

Ukuhanjiswa kwesampuli

Ukwakhiwa kwethala leencwadi

Ukulandelelanisa

Uhlalutyo lwedatha

Iinkonzo emva kokuthengisa

Ngaphambili: 16S/18S/ITS Amplicon Sequencing-NGS

Okulandelayo: I-Assay ye-Chromatin eFikelelekayo yeTransposase kunye noSetyenziso lwe-High throughput (ATAC-seq)

Ibandakanya uhlalutyo olulandelayo:

●Ulawulo lomgangatho wedatha ekrwada

● Ukudibanisa kwe-OTU/Ukukhupha ingxolo(ASV)

● Inkcazo ye-OTU

● Uhlalutyo lokwahluka kwe-Alpha: izalathisi ezininzi, kuquka i-Shannon, i-Simpson kunye ne-ACE.

● Uhlalutyo lokwahluka kweBeta

● Uhlalutyo lwamaqela

● Uhlalutyo lonxulumano: phakathi kwemiba yokusingqongileyo kunye nokwakheka kwe-OUT kunye neyantlukwano

● I-16S esebenzayo yokuqikelela imfuza

I-Histogram yokusasazwa kwe-taxonomic

Ukusasazwa koluntu umthi wephylogenetic

Uhlalutyo lokwahluka kwe-Alpha: ACE

isalathisi

Uhlalutyo lokwahluka kweBeta: PCoA

Uhlalutyo lwamaqela: ANOVA

Phonononga inkqubela phambili eququzelelwa yi-BMKGene yeenkonzo zolandelelwano lweamplicon kunye ne-PacBio ngengqokelela egciniweyo yopapasho.

I-Gao, X. kunye noWang, H. (2023) 'Uhlalutyo oluthelekisayo lweeProfayili zeBhaktheriya ze-Rumen kunye neMisebenzi ngexesha loKuqhelanisa nePhenology ehlukeneyo (iRegreen vs. Grassy) kwi-Alpine Merino yeegusha ezineNqanaba ezimbini zokuKhula kwi-Alpine Meadow', Ukuvutshelwa, i-9( 1), iphe. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. et al. (2023) 'Ukubamba i-microbial dark matter kwimihlaba yentlango usebenzisa i-culturomics-based metagenomics kunye nohlalutyo oluphezulu', npj Biofilms kunye neMicrobiomes 2023 9: 1, 9 (1), iphe. 1-14. doi: 10.1038/s41522-023-00439-8.

Mu, L. et al. (2022) 'Iziphumo zeetyuwa ze-asidi ezinamafutha kwiimpawu zokubiliswa, ukuhlukahluka kwebhaktheriya kunye nokuzinza kwe-aerobic ye-silage exutywe ne-alfalfa, i-rice straw kunye nengqolowa yengqolowa', Ijenali yeNzululwazi yoKutya kunye nezoLimo, i-102 (4), iphe. 1475– 1487. doi: 10.1002/JSFA.11482.

Yang, J. et al. (2023) 'Intsebenziswano phakathi kwe-Oxidative Stress Biomarkers kunye ne-Gut Microbiota kwi-Antioxidant Effects of Extracts esuka kwi-Sonchus brachyotus DC. kwi-Oxazolone-Induced Intestinal Oxidative Stress in Adult Zebrafish ', Antioxidants 2023, Vol. 12, Iphepha 192, 12(1), iphe. 192. doi: 10.3390/ANTIOX12010192.