Alaabta

16S/18S/ITS Amplicon Sequencing-NGS

16S/18S/ITS isku xigxiga amplicon waxay ujeedadeedu tahay muujinta phylogeny, taxonomy, iyo noocyada badan ee bulshada microbial iyadoo la baarayo alaabta PCR ee calaamadaha hidda-waynta ee ka kooban qaybaha aadka loo wada sheekaysanayo iyo kuwa kala duwan.Soo bandhigida faraha molecular ee kaamil ah ee Woeses et al, (1977) waxay awood u siisaa sifaynta microbiome-ka-freeIsku xigxiga 16S (bakteeriyada), 18S (Fungi) iyo Internal transcribed spacer (ITS, fungi) waxay ogolaataa aqoonsiga noocyada badan ee noocyada kala duwan iyo sidoo kale noocyada dhifka ah iyo kuwa aan la aqoonsan.Tiknoolajiyadani waxay noqotay qalab si ballaaran loo isticmaalo oo lagu garto ka kooban microbial kala duwan ee deegaan kala duwan, sida afka aadanaha, xiidmaha, saxarada, iwm.

Platform:Illumina NovaSeq Platform

Nala soo xiriir

Faahfaahinta Adeegga

Natiijooyinka Demo

Kiis barasho

Faa'iidooyinka Adeegga

● Go'doon-la'aanta iyo aqoonsiga degdega ah ee ka kooban microbial muunadaha deegaanka

● Xallinta sare ee qaybaha hoose ee shaybaarada deegaanka

● QIIME2 u dambeeyay ee falanqaynta socodka oo leh falanqayn kala duwan marka loo eego xogta xogta, tafatirka, OTU/ASV.

● Soo saarid sare, saxnaan sare

● Lagu dabaqi karo daraasado bulsho oo kala duwan oo microbial ah

● BMK waxay leedahay waayo-aragnimo ballaaran oo leh in ka badan 100,000 muunado/sannadkii, daboolaya ciidda, biyaha, gaaska, dhoobada, saxarada, xiidmaha, maqaarka, maraq halsano, cayayaanka, dhirta, iwm.

● BMKCloud waxay fududaysay tarjumaadda xogta oo ka kooban 45 qalab oo shakhsi ah oo lafo-guro

Tilmaamaha Adeegga

Isku xigxigaPlatform	Maktabadda	Soo saarista xogta lagu taliyay	Waqtiga soo-jeedinta la qiyaasay
Illumina NovaSeq Platform	PE250	50K/100K/300K Tags	30 Maalmood

Falanqaynta bioinformatics

● Xakamaynta tayada xogta ceeriin

● Kooxda OTU/Buuq-dejinta (ASV)

● Faahfaahinta OTU

● Kala duwanaanta Alpha

● Kala duwanaanta beta

● Falanqaynta kooxaha dhexdooda

● Falanqaynta ururka ee ka soo horjeeda arrimo tijaabo ah

● Saadaasha hidda-socodka shaqada

Tusaalaha Shuruudaha iyo Bixinta

Tusaalaha Shuruudaha:

WaayoSoosaarida DNA:

Tusaalaha Nooca	Qadarka	Xoog-saarid	daahirnimo
Soosaarida DNA	＞ 30 ng	＞ 1 ng/μl	OD260/280= 1.6-2.5

Tusaalooyinka deegaanka:

Tusaalaha nooca	Habka muunad ee lagu taliyay
Ciid	Qadarka saamiga: qiyaastii.5 g;Walaxda engegan ayaa u baahan in laga saaro oogada;Shiidi qaybo waaweyn oo dhex mara filter 2 mm;Muunado Aliquot ee EP-tube ama cyrotube nadiif ah oo boos celin ah.
Saxarada	Qadarka saamiga: qiyaastii.5 g;Ku soo ururi oo ka saar shaybaarada EP-tube-ka nadiifka ah ama cryotube ee boos celinta.
Waxa ku jira mindhicirka	Tijaabooyinku waxay u baahan yihiin in lagu farsameeyo xaalad aseptic.Ku dhaq nudaha la ururiyey PBS;Centrifuge PBS oo ku soo ururi daadadka EP-tubooyinka.
dhoobo	Qadarka saamiga: qiyaastii.5 g;Ku soo ururi oo ka saar muunada dhoobada dhoobada EP-tube-ka nadiifka ah ama cryotube ee boos celinta
Biyaha	Muunad leh xaddi xaddidan oo microbial ah, sida biyaha qasabada, biyaha ceelka, iwm., Uruuri ugu yaraan 1 L oo biyo ah oo dhex mara 0.22 μm filter si loo kobciyo microbial xuubka.Ku kaydi xuubka tuubada nadiifka ah.
Maqaarka	Si taxadar leh ugu xoq dusha maqaarka suuf aan nadiif ahayn ama daab qalliin oo ku rid tuubo nadiif ah.

Tusaalaha keenista lagu taliyay

Ku qabooji shaybaarada ku jira nitrogen dareeraha ah 3-4 saacadood kuna kaydi nitrogen dareeraha ah ama -80 digrii ilaa boos sii hayn muddo dheer ah.Tusaalaha rarida baraf qallalan ayaa loo baahan yahay.

Socodka Shaqada Adeegga

Tusaalaha keenista

Dhismaha maktabadda

Isku xigxiga

Falanqaynta xogta

Adeegyada iibka kadib

Hore: Dib-u-habaynta Bakteeriyada iyo Fungal Genom Genome

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

1.Species qaybinta

2.Maabka kulaylka: Noocyada hodantinimada ururinta

3. qalooca kooxda naadir

4.NMDS falanqaynta

5.Falanqaynta bidixda

Kiiska BMK

Shakhsiyaadka Cayilka leh iyo kuwa aan lahayn Nooca 2 ee Sonkorowga waxay muujinayaan awood shaqo oo kala duwan ee mindhicirka

La daabacay:Unug Host & Microbe, 2019

Istaraatiijiyada isku xigxiga:

Cudurka macaanka ee aan caatada ahayn (n=633);Cayil aan sonkor lahayn (n=494);Nooca 2-aad ee xanuunka macaanka (n=153);
Gobolka bartilmaameedka: 16S rDNA V1-V2
Platform: Illumina Miseq (isku xigxiga amplicon ku salaysan ee NGS)
Qayb ka mid ah soosaarayaasha DNA-da ayaa lagu sammeeyay isku xigxiga metagenomic ee Illumina Hiseq

Natiijooyinka muhiimka ah

Tilmaamaha microbial ee cuduradan dheef-shiid kiimikaadka ayaa si guul leh loo kala saaray.
Marka la barbardhigo sifooyinka microbial-ka ah ee ay soo saartay isku xigxiga 16S, buurnaanta ayaa la ogaaday in ay la xiriirto isbeddelada ku yimaada halabuurka microbial, sifooyinka shakhsi ahaaneed, gaar ahaan hoos u dhac weyn oo ku yimaada Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, iwm. .

Tixraac

Thingholm, LB, iyo al."Shakhsiyaadka cayilan ee qaba iyo kuwa aan lahayn nooca 2 ee sonkorowga waxay muujinayaan awood shaqo oo kala duwan ee mindhicirka microbial."Unug Host & Microbe26.2 (2019).