Alaabta

16S/18S/ITS Amplicon Sequencing-PacBio

Qaybta hoose ee 16S iyo 18S rRNA oo ka kooban labada gobol ee aadka loo ilaaliyo iyo kuwa isbedbeddela leh ayaa ah faraha molikula ee ugu fiican ee aqoonsiga noolaha prokaryotic iyo eukaryotic.Ka faa'iidaysiga isku xigxiga, amplicons-yadan waxaa lagu bartilmaameedsan karaa iyadoo lagu salaynayo qaybaha la xafiday iyo gobollada kala duwanaanshiyaha ayaa si buuxda loogu sifeyn karaa aqoonsiga microbial ee gacan ka geysanaya daraasadaha daboolaya falanqaynta kala duwanaanta microbial, taxonomy, phylogeny, iwm. Hal-molecule-waqtiga dhabta ah (SMRT) Isku xigxiga madal PacBio waxa ay sahlaysa in la helo akhrin dheer oo sax ah, kaas oo dabooli kara amplicons dhererkiisu dhan yahay (qiyaastii 1.5 Kb).Aragtida ballaaran ee beerta hidde-sidaha ayaa si weyn u xoojisay xallinta sharraxaadda noocyada bakteeriyada ama fangaska bulshada.

Platform:Talaabada PacBio II

Nala soo xiriir

Faahfaahinta Adeegga

Natiijooyinka Demo

Kiis barasho

Faa'iidooyinka Adeegga

● Akhris dheer oo muujinaya taxanaha dhererka buuxa ee 16S/18S/ITS

● Wicitaan sal leh oo sax ah oo leh habka PacBio CCS ee isku xigxiga

● Xallinta heerka noocyada ee sharraxaadda OTU/ASV

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

● 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	Xogta lagu taliyay	Wakhtiga Wareega
Talaabada PacBio II	SMRT-gambaleelka	5K/10K/20K Tags	44 Maalmo shaqo

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

Xiga: Qiimaynta chromatin-ka-wareejin-ka-hortagga leh ee la heli karo

1.Heerka sharraxaadda ee V3+V4(Illumina) -ku salaysan xog-ururinta beesha microbial vs. dhererka buuxa (PacBio) -ku-salaysan sifada.
(Xogta 30 mashruuc oo si aan kala sooc lahayn loo soo xulay ayaa lagu dabaqay tirakoob)

2.Qoritaanka qiyaasta dhererka dhererka buuxa ee isku xigxiga heerka noocyada noocyada muunada ee kala duwan

3.Species qaybinta

4.Geedka phylogenetic

Kiiska BMK

Soo-gaadhista arsenic waxay keentaa dhaawaca xannibaadda mindhicirka iyo ka-shaqaynta dhidibka beerka-beeraha taasoo horseedaysa barar iyo pyroptosis ee beerka shimbiraha.

La daabacay:Sayniska Guud ee Deegaanka,2021

Istaraatiijiyada isku xigxiga:

Tusaalooyinka: Xakamaynta vs 8 mg/kg ATO kooxda daaha ka qaaday
Soo saarista xogta isku xigta: 102,583 cayriin CCS isku xigxiga wadarta guud
Xakamaynta: 54,518 ± 747 wax ku ool ah CCS
ATO-soo bandhigay: 45,050 ± 1675 CCS waxtar leh

Natiijooyinka muhiimka ah

Kala duwanaanta Alpha:Soo-gaadhista ATO waxay si weyn u beddeshay hodantinimada microbial mindhicirka iyo kala duwanaanshaha shinbiraha.

Falanqaynta Metastats:
Heerka phylum: 2 phyla bakteeriyada kaliya ayaa laga helay kooxaha kantaroolka
Heerka hiddaha: 6 generas ayaa la helay si aad ah uga duwan marka loo eego tirada badan
Heerka noocyada: 36 nooc ayaa la aqoonsaday guud ahaan, iyadoo 6 ka mid ah ay aad uga duwan yihiin tirada badan

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).