Iimveliso

16S/18S/ITS Amplicon Sequencing-PacBio

Iyunithi ekwi-16S kunye ne-18S rRNA equlathe yomibini imimandla egcinwe kakhulu kunye ne-hyper-variable printing yeminwe egqibeleleyo yemolekyuli yokuchongwa kweprokaryotic kunye ne-eukaryotic organisms.Ukuthatha ithuba lolandelelwano, ezi amplicon zinokujoliswa ngokusekwe kumacandelo agciniweyo kwaye imimandla eguquguqukayo ephezulu inokubonakaliswa ngokupheleleyo ukuchongwa kwemicrobial enegalelo kwizifundo ezibandakanya uhlalutyo lwentlupheko yemicrobial, taxonomy, phylogeny, njl. ) ulandelelwano lweqonga lePacBio lwenza ukuba kufumaneke ufundo olude oluchaneke kakhulu, olunokugubungela i-amplicon yobude obupheleleyo (malunga ne-1.5 KB).Imbono ebanzi yendawo yemfuza yasiphucula kakhulu isisombululo kwingcaciso yeentlobo kwiibhaktiriya okanye kuluntu lomngundo.

Iqonga:I-PacBio Sequel II

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Iziphumo zeDemo

Into yokuphonononga

Iinkonzo eziluncedo

● Ufundo olude luveza ulandelelwano olupheleleyo lwe-16S/18S/ITS

● Isiseko esichanekileyo sokufowuna kunye nolandelelwano lwemodi ye-PacBio CCS

● Isisombululo senqanaba leentlobo kwinkcazo ye-OTU/ASV

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

● 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	Idatha ecetyiswayo	Ixesha lokuguqula
I-PacBio Sequel II	SMRT-intsimbi	5K/10K/20K iithegi	44 Iintsuku zokusebenza

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	＞ 1 μg	＞ 20 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: 16S/18S/ITS Amplicon Sequencing-NGS

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

I-1.Izinga le-Annotation ye-V3 + V4 (Illumina)-based microbial community profiling vs. Full-length (PacBio)-based profiling.
(Idatha yeeprojekthi ezingama-30 ezichongwe ngokungacwangciswanga ziye zafakwa kwizibalo)

2.Ireyithi yenkcazo yolandelelwano lweamplicon yobude obupheleleyo kwinqanaba leentlobo ezahlukeneyo zesampulu

3.Ukuhanjiswa kweentlobo

4.Umthi wePhylogenetic

Ityala le-BMK

Ukuvezwa kwe-arsenic kubangela umonakalo othintela amathumbu kunye nokulandelwa kokusebenza kwe-gut-liver axis ekhokelela ekudumbeni kunye ne-pyroptosis yesibindi kumadada.

Ipapashiwe:Inzululwazi yeNdawo yonke,2021

Iqhinga lokulandelelanisa:

Iisampulu: Ukulawula vs 8 mg / kg ATO evezwe iqela
Ukulandelelanisa isivuno sedatha: 102,583 ulandelelwano lwe-CCS ekrwada iyonke
Ulawulo: 54,518 ± 747 CCS esebenzayo
I-ATO-eveziweyo : 45,050 ± 1675 i-CCS esebenzayo

Iziphumo eziphambili

Ukwahluka kweAlpha:Ukuvezwa kwe-ATO kutshintshe kakhulu ubutyebi be-intestinal microbial kunye nokwahluka kwamadada.

Uhlalutyo lwe-Metastats:
Kwinqanaba le-phylum: I-2 ibhaktheriya phyla ifunyenwe kuphela kumaqela olawulo
Kwinqanaba le-genus: I-6 genera ifunyenwe yahluke kakhulu kubuninzi obunxulumene
Kwinqanaba leentlobo: Iindidi ezingama-36 zachongwa zizonke, kunye ne-6 yazo yahluke kakhulu kubuninzi be-relavive

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).