Iimveliso

16S/18S/ITS Amplicon Sequencing-NGS

I-16S/18S/ITS i-amplicon sequencing ijolise ekuvezeni i-phylogeny, taxonomy, kunye nobuninzi beentlobo kwi-microbial community ngokuphanda iimveliso ze-PCR zeempawu zofuzo zokugcina izindlu eziqulethe zombini iindawo ezixoxwa kakhulu kunye ne-hypervariable.Ukwaziswa kwezi minwe zigqibeleleyo zemolekyuli ngu-Woeses et al, (1977) ixhobisa iprofayili ye-microbiome ezimeleyo.Ulandelelwano lwe-16S (iibhaktheriya), i-18S (umngundo) kunye ne-Internal transcribed spacer(ITS, umngundo) ivumela ukuchongwa kweentlobo ezininzi kunye neentlobo ezinqabileyo nezingaziwa.Le teknoloji iye yaba sisixhobo esisetyenziswa ngokubanzi kunye nesiphambili ekuchongeni ukubunjwa kwe-microbial eyahlukileyo kwiindawo ezahlukeneyo, ezinjengomlomo womntu, amathumbu, ilindle, njl.

Iqonga:I-Illumina NovaSeq Platform

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Iziphumo zeDemo

Into yokuphonononga

Iinkonzo eziluncedo

● Ukuchongwa okungekho bodwa kunye nokuchongwa ngokukhawuleza kokwakheka kweentsholongwane kwiisampulu zokusingqongileyo

● Isisombululo esiphezulu kumacandelo aphantsi kakhulu kwiisampuli zokusingqongileyo

● I-QIIME2 yamva nje yokuhlalutya ukuhamba nohlalutyo olwahlukeneyo ngokwemigaqo yogcino-lwazi, inkcazo, i-OTU/ASV.

● Ukuphumelela okuphezulu, ukuchaneka okuphezulu

● Isetyenzwa kwizifundo zoluntu ezahlukeneyo zemicrobial

● I-BMK inamava abanzi ngeesampulu ezingaphezulu kwe-100,000 ngonyaka, ezigquma umhlaba, amanzi, igesi, udaka, ilindle, amathumbu, ulusu, umhluzi wokubila, izinambuzane, izityalo, njl.

● I-BMKCloud iququzelele ukutolikwa kwedatha equlethe i-45 yezixhobo zokuhlalutya zomntu

IiNgcaciso zeNkonzo

UkulandelelanisaIqonga	Ithala leencwadi	Imveliso yedatha ecetyiswayo	Ixesha eliqikelelweyo lokujika
I-Illumina NovaSeq Platform	PE250	50K/100K/300K iithegi	Iintsuku ezingama-30

Uhlalutyo lwe-Bioinformatics

● Ulawulo lomgangatho wedatha ekrwada

● I-OTU clustering/De-noise(ASV)

● Inkcazo ye-OTU

● Ukwahluka kweAlpha

● Ukwahluka kweBeta

● Uhlalutyo lwamaqela ngamaqela

● Uhlalutyo lombutho ngokuchasene nemiba yovavanyo

● Uqikelelo lwemfuza yokusebenza

IiMfuno zeSampulu kunye nokuhanjiswa

IiMfuno zeSampulu:

KubaIzicatshulwa zeDNA:

Uhlobo lweSampuli	Isixa	Ukugxininisa	Ubunyulu
Izicatshulwa zeDNA	＞ 30 ng	＞ 1 ng/μl	OD260/280= 1.6-2.5

Ngeesampuli zokusingqongileyo:

Uhlobo lwesampuli	Inkqubo yesampulu ecetyiswayo
Umhlaba	Isixa sesampulu: malunga.5 g;Into eseleyo ebunayo kufuneka isuswe kumphezulu;Gaya iinqununu ezinkulu kwaye udlule kwi-2 mm filter;Iisampulu ze-Aliquot kwi-EP-tube engcolileyo okanye i-cyrotube yogcino.
Ilindle	Isixa sesampulu: malunga.5 g;Qokelela kwaye iisampulu ze-aliquot kwi-EP-tube engcolileyo okanye i-cryotube yokubhukisha.
Iziqulatho zamathumbu	Iisampuli kufuneka ziqwalaselwe phantsi kwemeko ye-aseptic.Hlamba izicubu eziqokelelweyo nge-PBS;I-Centrifuge i-PBS kwaye uqokelele i-precipitant kwi-EP-tubes.
Udaka	Isixa sesampulu: malunga.5 g;Qokelela kwaye isampuli ye-aliquot ye-sludge kwi-EP-tube engcolileyo okanye i-cryotube yokugcinelwa
Amanzi	Kwisampulu enesixa esilinganiselweyo se-microbial, njengamanzi epompo, amanzi omthombo, njl., Qokelela ubuncinci be-1 L yamanzi kwaye udlule kwi-0.22 μm yokucoca ukutyebisa i-microbial kwi-membrane.Gcina inwebu kwityhubhu enyumba.
Ulusu	Khulula ngononophelo umphezulu wolusu ngeswab yomqhaphu engcolileyo okanye iblade yotyando kwaye uyibeke kwityhubhu engenanto.

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: IBacterial kunye neFungal Whole Genome Re-Sequencing

Okulandelayo: 16S/18S/ITS Amplicon Sequencing-PacBio

1.Ukuhanjiswa kweentlobo

2.Imephu yobushushu: Ubuncwane beentlobo zeentlobo

3.Ijika elinqabileyo leqela

Uhlalutyo lwe-4.NMDS

5.Uhlalutyo lweLefse

Ityala le-BMK

Abantu abatyebileyo abaneSifo seswekile esi-2 kunye nangaphandle babonisa amandla okusebenza amathumbu amancinci kunye nokwakheka

Ipapashiwe:Umamkeli weSeli kunye neMicrobe, ngo-2019

Iqhinga lokulandelelanisa:

I-Lean non-diabetes (n=633);Ukutyeba ngokugqithisileyo okungenasifo seswekile (n=494);Ukutyeba-Uhlobo lwe-2 yeswekile (n=153);
Ummandla ekujoliswe kuwo: 16S rDNA V1-V2
Iqonga: Illumina Miseq (NGS-based amplicon sequencing)
I-Subset ye-extracts ye-DNA iphantsi kolandelelwano lwe-metagenomic kwi-Illumina Hiseq

Iziphumo eziphambili

Iiprofayili zeMicrobial zezi zifo zemetabolism zahlulwa ngempumelelo.
Ngokuthelekisa iimpawu ze-microbial eziveliswa ngokulandelelana kwe-16S, ukutyeba kwafunyaniswa kuhambelana nokutshintsha kwe-microbial composition, iimpawu zomntu ngamnye, ngokukodwa ukuncipha okukhulu kwe-Akkermansia, i-Faecalibacterium, i-Oscillibacter, i-Alistipes, njl. Ukongeza, i-T2D yafunyanwa idibene nokunyuka kwe-Escherichia / shigella .

Isalathiso

Thingholm, LB , et al."Abantu abatyebileyo abanesifo seswekile kunye nabangenaso iSifo seswekile sesi-2 babonisa isakhono esiSebenzayo seThumbu leMicrobial."Umamkeli weSeli kunye neMicrobe26.2(2019).