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16S/18S/ITS Amplicon Sequencing-PacBio

Cov noob 16S thiab 18S rRNA, nrog rau thaj tsam Internal Transcribed Spacer (ITS), ua cov cim tseem ceeb ntawm cov ntiv tes ntiv tes vim lawv qhov sib xyaw ua ke ntawm thaj chaw ruaj khov thiab muaj qhov sib txawv, ua rau lawv cov cuab yeej muaj txiaj ntsig rau cov kab mob prokaryotic thiab eukaryotic. Amplification thiab sequencing ntawm cov cheeb tsam no muab ib qho kev cais-dawb txoj hauv kev rau kev soj ntsuam cov microbial muaj pes tsawg leeg thiab ntau haiv neeg nyob rau hauv ntau yam ecosystems. Thaum Illumina sequencing feem ntau yog lub hom phiaj luv luv hypervariable cheeb tsam xws li V3-V4 ntawm 16S thiab ITS1, nws tau pom tias zoo dua taxonomic annotation yog ua tiav los ntawm kev ua tiav qhov ntev ntawm 16S, 18S, thiab ITS. Qhov kev qhia dav dav no ua rau muaj feem pua ntawm cov kev faib tawm kom raug, ua tiav qib kev daws teeb meem uas txuas mus rau kev txheeb xyuas hom. PacBio's Single-Molecule Real-Time (SMRT) sequencing platform sawv tawm los ntawm kev muab cov ntawv nyeem ntev heev (HiFi) uas npog tag nrho-ntev amplicons, rivaling qhov tseeb ntawm Illumina sequencing. Qhov kev muaj peev xwm no tso cai rau cov kws tshawb fawb kom tau txais qhov txiaj ntsig tsis sib xws - pom qhov pom ntawm cov noob caj noob ces. Qhov kev pab cuam txuas ntxiv txhawb nqa qhov kev daws teeb meem hauv hom lus piav qhia, tshwj xeeb tshaj yog nyob rau hauv cov kab mob lossis cov kab mob fungal, ua kom nkag siab tob txog qhov tsis sib haum xeeb ntawm cov neeg microbial.

Tiv tauj peb

Cov ntsiab lus ntawm kev pabcuam

Bioinformatics

Cov txiaj ntsig Demo

Cov ntawv tshaj tawm tshwj xeeb

Kev Pabcuam Nta

● Sequencing platform: PacBio Revio

● Sequencing mode: CCS (HiFi nyeem)

● Amplification ntawm lub hom phiaj cheeb tsam ua raws li tandem txuas ntawm amplicons ua ntej HiFi SMRT tswb tsev qiv ntawv npaj

Kev Pabcuam Zoo

●Kev daws teeb meem siab dua taxonomic: Than luv-amplicon sequencing,ua kom muaj ntau dua OTU kev faib tawm ntawm cov hom tsiaj.

●Kev hu xovtooj muaj tseeb heev: PacBio CCS hom sequencing (HiFi nyeem).

●Tsis muaj kev cais tawm: Kev txheeb xyuas sai ntawm microbial muaj pes tsawg leeg hauv cov qauv ib puag ncig.

●Dav siv tau: Ntau hom kev tshawb fawb hauv zej zog microbial.

●Kev txheeb xyuas bioinformatic: Qhov tseeb QIIME2 pob (ntau qhov kev pom rau hauv microbial ecology) nrog ntau yam kev soj ntsuam ntawm cov ntaub ntawv, annotation, OTU / ASV.

●Kev txawj ntse: Nrog ntau txhiab tus amplicon sequencing tej yaam num ua txhua xyoo, BMKGENE coj ntau tshaj kaum xyoo ntawm kev paub, pab pawg neeg soj ntsuam uas txawj ntse, cov ntsiab lus dav, thiab kev txhawb nqa tom qab muag zoo heev.

Service Specifications

Lub tsev qiv ntawv	Sequence Strategy	Cov ntaub ntawv pom zoo
Amplicon	PacBio Revio: koj puas xav tau ntau tus thwjtim?	10/30/50 K tags (CCS)

Cov Qauv Xav Tau

Concentration (ng / μL)	Tag nrho cov nyiaj (µg)	Ntim (µL)
≥5	≥0.3	≥ 20

Pom zoo coj mus kuaj

Kho cov qauv hauv cov kua nitrogen rau 3-4 teev thiab khaws cia hauv cov kua nitrogen los yog -80 degree mus rau lub sijhawm ntev. Qauv shipping nrog qhuav-dej khov yog yuav tsum tau.

Kev Pabcuam Ua Haujlwm Flow

Qauv xa tuaj

Kev tsim kho tsev qiv ntawv

Sequencing

Cov ntaub ntawv tsom xam

Kev pab cuam tom qab muag

Yav dhau los: 16S/18S/ITS Amplicon Sequencing-NGS

Tom ntej: Kev ntsuam xyuas rau Transposase-Accessible Chromatin nrog High Throughput Sequencing (ATAC-seq)

Xws li cov kev txheeb xyuas hauv qab no:

● Raw cov ntaub ntawv zoo tswj

● OTU pawg/De-noise(ASV)

● OTU cov lus qhia

●Alpha diversity tsom xam: ntau qhov ntsuas, suav nrog Shannon, Simpson thiab ACE.

● Beta diversity tsom xam

●Inter-group tsom xam

● Kev txheeb xyuas kev sib txheeb: nruab nrab ntawm ib puag ncig yam thiab OUT muaj pes tsawg leeg thiab ntau haiv neeg

●16S kev ua haujlwm noob kwv yees

Histogram ntawm taxonomic faib

Zej zog faib phylogenetic tsob ntoo

Alpha diversity analysis: ACE

qhov ntsuas

Kev txheeb xyuas kev sib txawv ntawm beta: PCoA

Intergroup tsom xam: ANOVA

Tshawb nrhiav cov kev nce qib uas tau pab los ntawm BMKGene cov kev pabcuam amplicon sequencing nrog PacBio los ntawm kev sau cov ntawv tshaj tawm.

Gao, X. thiab Wang, H. (2023) 'Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with two growing stage on an Alpine Meadow', Fermentation, 9( 1), ib., p. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. et al. (2023) 'Capturing the microbial dark matters in desert av using culturomics-based metagenomics and high-resolution analysis', npj Biofilms and Microbiomes 2023 9:1, 9(1), pp. 1–14. doi: 10.1038/s41522-023-00439-8 ib.

Mu, L. et al. (2022) 'Kev cuam tshuam ntawm fatty acid ntsev ntawm fermentation yam ntxwv, kab mob ntau haiv neeg thiab aerobic stability ntawm tov silage npaj nrog alfalfa, nplej straw thiab nplej bran', Journal of the Science of Food and Agriculture, 102(4), pp. 1475– 1487 ib. doi: 10.1002/JSFA.11482.

Yang, J. et al. (2023) 'Kev sib cuam tshuam ntawm Oxidative Stress Biomarkers thiab Gut Microbiota hauv Antioxidant Effects ntawm Extracts los ntawm Sonchus brachyotus DC. hauv Oxazolone-Induced plab hnyuv Oxidative Stress in Adult Zebrafish', Antioxidants 2023, Vol. 12, Page 192, 12(1), p. 192. doi: 10.3390/ANTIOX12010192.