Cov khoom

16S/18S/ITS Amplicon Sequencing-NGS

16S / 18S / ITS amplicon sequencing aims ntawm kev nthuav tawm phylogeny, taxonomy, thiab tsiaj abundance nyob rau hauv ib tug microbial zej zog los ntawm kev soj ntsuam PCR cov khoom ntawm tsev tu cov noob caj noob ces cov cim uas muaj ob qho tib si sib tham thiab hypervariable qhov chaw.Kev taw qhia ntawm cov ntiv tes zoo meej molecular los ntawm Woeses et al, (1977) txhawb nqa kev sib cais-dawb microbiome profiling.Sequencing ntawm 16S (cov kab mob), 18S (fungi) thiab Internal transcribed spacer (ITS, fungi) tso cai rau kev txheeb xyuas ntawm ob qho tib si ntau hom nrog rau cov hom tsis tshua muaj thiab tsis muaj npe.Cov cuab yeej siv no tau dhau los ua cov cuab yeej siv dav thiab tseem ceeb hauv kev txheeb xyuas qhov sib txawv ntawm cov kab mob microbial nyob rau hauv ntau qhov chaw, xws li tib neeg lub qhov ncauj, txoj hnyuv, quav, thiab lwm yam.

Platform:Illumina NovaSeq Platform

Tiv tauj peb

Cov ntsiab lus kev pabcuam

Cov txiaj ntsig Demo

Case Study

Kev Pabcuam Zoo

● Kev rho tawm tsis pub dawb thiab txheeb xyuas sai ntawm microbial muaj pes tsawg leeg hauv cov qauv ib puag ncig

● Kev daws teeb meem siab hauv cov khoom qis qis hauv cov qauv ib puag ncig

● Tseeb QIIME2 tsom xam ntws nrog ntau yam kev soj ntsuam ntawm cov ntaub ntawv, annotation, OTU/ASV.

● High-throughput, siab dua

● Muaj feem xyuam rau ntau hom kev tshawb fawb hauv zej zog microbial

● BMK muaj kev paub dhau los nrog ntau dua 100,000 tus qauv / xyoo, npog av, dej, roj, sludge, quav, hnyuv, tawv nqaij, fermentation broth, kab, nroj tsuag, thiab lwm yam.

● BMKCloud txhawb kev txhais cov ntaub ntawv uas muaj 45 tus kheej cov cuab yeej tshuaj xyuas

Service Specifications

SequencingPlatform	Lub tsev qiv ntawv	Pom zoo cov ntaub ntawv tawm los	Kwv yees lub sijhawm tig rov qab
Illumina NovaSeq Platform	PEB 250	50K/100K/300K Tag	30 Hnub

Bioinformatics tsom xam

● Raw cov ntaub ntawv zoo tswj

● OTU pawg/De-noise(ASV)

● OTU cov lus qhia

● Alpha ntau haiv neeg

● Beta ntau haiv neeg

● Kev tsom xam ntawm pab pawg

● Kev txheeb xyuas kev sib koom ua ke tawm tsam qhov kev sim

● Muaj nuj nqi gene kwv yees

Sample Requirements thiab Delivery

Cov Qauv Xav Tau:

RauDNA rho tawm:

Hom qauv	Tus nqi	Nco ntsoov	Purity
DNA rho tawm	30 ng	1 ng / μl	OD 260/280 = 1.6-2.5

Rau ib puag ncig cov qauv:

Hom qauv	Cov txheej txheem kuaj pom pom zoo
Av	Sampling tus nqi: approx.5 g yog ';Cov khoom uas tseem tshuav yuav tsum tau muab tshem tawm ntawm qhov chaw;Sib tsoo cov khoom loj thiab hla dhau 2 hli lim;Aliquot kuaj hauv sterile EP-tube lossis cyrotube rau kev tshwj tseg.
Cov quav	Sampling tus nqi: approx.5 g yog ';Sau thiab aliquot cov qauv hauv sterile EP-tube lossis cryotube rau kev tshwj tseg.
Cov ntsiab lus plab hnyuv	Cov qauv yuav tsum tau ua tiav nyob rau hauv aseptic mob.Ntxuav cov ntaub so ntswg nrog PBS;Centrifuge PBS thiab sau cov dej nag hauv EP-tubes.
Sludge	Sampling tus nqi: approx.5 g yog ';Sau thiab aliquot sludge qauv hauv sterile EP-tube lossis cryotube rau kev tshwj tseg
Tus dej	Rau cov qauv uas txwv tsis pub muaj microbial, xws li dej kais dej, dej zoo, thiab lwm yam, Sau tsawg kawg yog 1 L dej thiab dhau los ntawm 0.22 μm lim kom txhawb microbial ntawm daim nyias nyias.Khaws cov membrane rau hauv lub raj tsis muaj menyuam.
Daim tawv nqaij	Ua tib zoo txhuam cov tawv nqaij nrog cov paj rwb sterile swab los yog phais hniav thiab muab tso rau hauv sterile raj.

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: Cov kab mob thiab cov kab mob fungal tag nrho genome Re-Sequencing

Tom ntej: 16S/18S/ITS Amplicon Sequencing-PacBio

1. Hom kev faib tawm

2.Heat map: Hom kev nplua nuj nyob hauv pawg

3.Rare faction nkhaus

4.NMDS tsom xam

5.Lefse tsom xam

BMK Cas

Cov neeg rog rog thiab tsis muaj ntshav qab zib hom 2 qhia cov plab hnyuv microbial ua haujlwm muaj peev xwm thiab muaj pes tsawg leeg

Luam tawm:Cell Host & Microbe, 2019

Sequence Strategy:

Lean non-diabetes (n=633);Kev rog tsis muaj ntshav qab zib (n=494);Obese-Type 2 diabetes (n=153);
Lub hom phiaj cheeb tsam: 16S rDNA V1-V2
Platform: Illumina Miseq (NGS-based amplicon sequencing)
Subset ntawm DNA rho tawm tau raug rau metagenomic sequencing ntawm Illumina Hiseq

Cov txiaj ntsig tseem ceeb

Microbial profileings ntawm cov kab mob metabolic no tau ua tiav tiav.
Los ntawm kev sib piv microbial nta tsim los ntawm 16S sequencing, rog tau pom muaj feem xyuam nrog cov kev hloov hauv microbial muaj pes tsawg leeg, cov yam ntxwv ntawm tus kheej, tshwj xeeb tshaj yog txo qis hauv Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, thiab lwm yam. Ntxiv rau, T2D tau pom muaj cuam tshuam nrog nce hauv Escherichia / shigella. .

Kev siv

Thingholm, LB, et al."Cov neeg rog rog uas muaj thiab tsis muaj ntshav qab zib hom 2 qhia qhov sib txawv ntawm plab hnyuv microbial muaj peev xwm ua haujlwm thiab muaj peev xwm."Cell Host & Microbe26.2 (2019).