Lihlahisoa

16S/18S/ITS Amplicon Sequencing-NGS

Tatelano ea amplicon ea 16S/18S/ITS e ikemiselitse ho senola phylogeny, taxonomy, le mefuta e mengata sechabeng sa likokoana-hloko ka ho batlisisa lihlahisoa tsa PCR tsa liphatsa tsa lefutso tsa tlhokomelo ea ntlo tse nang le likarolo tse peli tse buuoang haholo le tse sa fetoheng.Kenyelletso ea likhatiso tsena tsa menoana ea limolek'hule tse phethahetseng ka Woeses et al, (1977) e matlafatsa profiling ea microbiome e se nang ho itšehla thajana.Tatellano ea 16S (libaktheria), 18S (fungus) le Internal transcribed spacer(ITS, fungus) e lumella ho tsebahatsa mefuta e mengata e mengata hammoho le mefuta e sa tloaelehang le e sa tsejoeng.Theknoloji ena e se e le sesebelisoa se sebelisoang haholo le se seholo ho khetholla sebopeho sa likokoana-hloko tse fapaneng libakeng tse fapaneng, joalo ka molomo oa motho, mala, mantle, joalo-joalo.

Sethala:Illumina NovaSeq Platform

Iteanye le rona

Lintlha tsa Tšebeletso

Demo Results

Thuto-pale

Melemo ea Tšebeletso

● Ho tsebahatsa ntle le ho itšehla thajana le ho tseba ka potlako sebopeho sa likokoana-hloko lisampoleng tsa tikoloho

● Boemo bo phahameng ka likarolo tse fokolang haholo lisampoleng tsa tikoloho

● QIIME2 ea morao-rao e hlahlobang phallo e nang le litlhahlobo tse fapaneng ho latela bobolokelo, litlhaloso, OTU/ASV.

● Ho sebetsa ka mokhoa o phahameng, ho nepahala ho phahameng

● E sebetsa lithutong tse fapaneng tsa sechaba ka likokoana-hloko

● BMK e na le boiphihlelo bo batsi ka disampole tse fetang 100,000/selemo, e kwahelang mobu, metsi, kgase, seretse, mantle, mala, letlalo, moro o bedisang, dikokonyana, dimela, jj.

● BMKCloud e nolofalitse tlhaloso ea data e nang le lisebelisoa tse 45 tsa ho hlahloba motho ka mong

Litlhaloso tsa Tšebeletso

TatelanoSethala	Laebrari	Tlhahiso ea data e khothalelitsoeng	Khakanyo ea nako ea ho fetoha
Illumina NovaSeq Platform	PE250	50K/100K/300K Li-tag	Matsatsi a 30

Litlhahlobo tsa Bioinformatics

● Taolo ea boleng ba data e tala

● OTU clustering/De-noise(ASV)

● Tlhaloso ea OTU

● Mefuta e sa tšoaneng ea alpha

● Ho fapana ha Beta

● Tlhahlobo ea lihlopha

● Tlhahlobo ea mokhatlo khahlanong le lintlha tsa liteko

● Ho bolela esale pele ka liphatsa tsa lefutso

Litlhoko tsa Mohlala le Phano

Litlhoko tsa Mohlala:

Bakeng saLintlha tsa DNA:

Mofuta oa Mohlala	Chelete	Ho tsepamisa maikutlo	Bohloeki
Lintlha tsa DNA	＞ 30 ng	＞ 1 ng/μl	OD260/280= 1.6-2.5

Bakeng sa mehlala ea tikoloho:

Mofuta oa mohlala	Mokhoa o khothaletsoang oa sampole
Mobu	Chelete ea mohlala: hoo e ka bang.5 g;Lintho tse setseng tse omeletseng li hloka ho tlosoa holimo;Sila likotoana tse kholo 'me u fete ka har'a filthara ea 2 mm;Lisampole tsa Aliquot ka har'a EP-tube kapa cyrotube bakeng sa ho behelloa.
Mantle	Chelete ea mohlala: hoo e ka bang.5 g;Bokella le lisampole tsa aliquot ho EP-tube kapa cryotube e sa sebetseng bakeng sa ho behelloa.
Likahare tsa mala	Mehlala e hloka ho sebetsoa tlas'a boemo ba aseptic.Hlatsoa lithane tse bokeletsoeng ka PBS;Hlakola PBS 'me u bokelle precipitant ka EP-tubes.
Sludge	Chelete ea mohlala: hoo e ka bang.5 g;Bokella le aliquot sludge sampole ho EP-tube e hloekisitsoeng kapa cryotube bakeng sa ho behelloa
Metsi	Bakeng sa sampole e nang le palo e lekanyelitsoeng ea likokoana-hloko, joalo ka metsi a pompo, metsi a seliba, joalo-joalo, Bokella bonyane metsi a 1 L 'me u fete ka 0.22 μm filthara ho ntlafatsa likokoana-hloko tse holim'a lera.Boloka lera ka har'a tube e hloekisitsoeng.
Letlalo	Hlakola bokaholimo ba letlalo ka hloko ka swab e hloekisitsoeng ea k'hothone kapa lehare la ho buoa 'me u le kenye ka har'a tube e sa hloekang.

Tlhahiso ea Mehlala e khothalelitsoeng

Emisa lisampole ka har'a naetrojene e metsi bakeng sa lihora tse 3-4 'me u li boloke ka har'a metsi a naetrojene kapa -80 degree ho boloka nako e telele.Ho hlokahala ho romelloa ka sekepe se nang le leqhoa le omileng.

Phallo ea Mosebetsi oa Ts'ebeletso

Mohlala oa thomello

Kaho ea laebrari

Tatelano

Tlhahlobo ea lintlha

Litšebeletso tsa ka mor'a thekiso

E fetileng: Libaktheria le Fungus Whole Genome Re-Sequencing

E 'ngoe: 16S/18S/ITS Amplicon Sequencing-PacBio

1.Kabo ea mefuta

2.'Mapa oa mocheso: Ho kopanya mefuta e mengata ea mefuta

3.Mokokotlo o sa tloaelehang oa lihlopha

Tlhahlobo ea 4.NMDS

5.Lefse analysis

Taba ea BMK

Batho ba batenya ba nang le lefu la tsoekere la Mofuta oa 2 le ba se nang bona ba bonts'a bokhoni bo fapaneng ba ts'ebetso ea likokoana-hloko le sebopeho

E hatisitsoe:Cell Host & Microbe, 2019

Leano la tatellano:

Lean non-diabetes (n=633);Batho ba nonneng ba nang le lefu la tsoekere (n=494);Lefu la tsoekere la Mofuta oa 2 oa Botenya (n=153);
Sebaka se reriloeng: 16S rDNA V1-V2
Sethala: Illumina Miseq (Sequencing ea amplicon e thehiloeng ho NGS)
Karolo e nyane ea likhechana tsa DNA li ile tsa hlophisoa ka tatellano ea metagenomic ho Illumina Hiseq

Liphetho tsa bohlokoa

Litlhaloso tsa likokoana-hloko tsa mafu ana a metabolism li ile tsa aroloa ka katleho.
Ka ho bapisa likarolo tsa likokoana-hloko tse hlahisoang ke tatellano ea 16S, botenya bo fumanoe bo amahanngoa le liphetoho tse entsoeng ka likokoana-hloko, likarolo tsa motho ka mong, haholo-holo ho fokotseha ho hoholo ho Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, joalo-joalo. Ho phaella moo, T2D e fumanoe e amahanngoa le ho eketseha ha Escherichia / shigella. .

Referense

Thingholm, LB , et al."Batho ba Batenya ba nang le Lefu la tsoekere la Mofuta oa 2 le ba se nang bona ba Bontša Matla le Sebopeho se Fapaneng sa Maleng a Microbial."Cell Host & Microbe26.2(2019).