16S/18S/ITS Amplicon Sequencing-PacBio

O le vaega pito i luga ole 16S ma le 18S rRNA o lo'o i ai vaega e lua e sili ona fa'asao ma hyper-feuia'i itulagi ose mole mole tamatamai lima mole prokaryotic ma eukaryotic organisms faailoagofieina.I le faʻaaogaina o le faʻasologa, o nei amplicons e mafai ona faʻatatau i luga o vaega faʻasao ma o le hyper-variable regions e mafai ona faʻamalamalamaina atoatoa mo microbial identification e fesoasoani i suʻesuʻega e aofia ai microbial diversity analysis, taxonomy, phylogeny, ma isi. ) fa'asologa o PacBio platform e mafai ai ona maua le sa'o sa'o umi o faitau, lea e mafai ona aofia ai amplicons atoa (pe. 1.5 Kb).O le fa'alauteleina o le va'aiga o fa'atupu kenera na matua fa'aleleia atili ai le fa'ai'uga i fa'amatalaga o ituaiga i siama po'o sigi nu'u.

Fa'avae:PacBio Sequel II

Faafesootai matou

Au'aunaga Fa'amatalaga

I'uga Fa'aaliga

Tulaga Suesuega

Tulaga Auaunaga

● Faitau umi e fa'aalia ai le fa'asologa atoa o le 16S/18S/ITS

● Sa'o maualuga vala'au fa'atasi ma PacBio CCS mode fa'asologa

● Fa'ai'uga tulaga o ituaiga ile fa'amatalaga OTU/ASV

● Su'esu'ega QIIME2 lata mai fa'atasi ma su'esu'ega 'ese'ese i tulaga o fa'amaumauga, fa'amatalaga, OTU/ASV.

● Fa'aoga i su'esu'ega tu'ufa'atasiga o microbial community

● E iai le poto masani a le BMK i le silia ma le 100,000 faʻataʻitaʻiga / tausaga, ufiufi palapala, vai, kesi, sludge, feces, intestinas, paʻu, faʻafefete susu, iniseti, laau, ma isi.

● BMKCloud faʻafaigofieina faʻamatalaga faʻamatalaga o loʻo i ai 45 meafaigaluega suʻesuʻe faʻapitoa

Fa'amatalaga Auaunaga

Fa'asologaFa'avae	Faletusi	Fa'amatalaga fautuaina	Taimi Fa'aliliu
PacBio Sequel II	SMRT- logo	5K/10K/20K fa'ailoga	44 Aso faigaluega

Bioinformatics su'esu'ega

● Pulea lelei o fa'amaumauga mata'utia

● OTU fa'apipi'i/Ta'ese-leo(ASV)

● OTU faʻamatalaga

● Alfa eseese

● Beta eseese

● Su'esu'ega va'aiga

● Su'esu'ega fa'atasi e fa'atatau i mea fa'ata'ita'i

● Fuafuaga gafa galue

Fa'ata'ita'iga Mana'oga ma le Tu'uina atu

Fa'ata'ita'iga Mana'oga:

MoDNA extracts:

Ituaiga Fa'ata'ita'i	Aofaiga	Fa'atotonuga	Mama
DNA extracts	1 μg	+ 20 ng/μl	OD260/280= 1.6-2.5

Mo fa'ata'ita'iga tau si'osi'omaga:

Fa'ata'ita'iga ituaiga	Fautuaina faiga faataitai
Eleele	Aofa'i fa'ata'ita'iga: tusa.5 g;O mea maguma totoe e tatau ona aveese mai luga;Oli fasipepa lapopoa ma pasi i totonu ole faamama 2 mm;Aliquot faʻataʻitaʻiga i le sterile EP-tube poʻo le cyrotube mo le faʻaagaga.
Feces	Aofa'i fa'ata'ita'iga: tusa.5 g;Aoina ma fa'ata'ita'i fa'ata'ita'iga ile EP-tube po'o le cryotube mo le fa'aagaga.
I totonu o le manava	E mana'omia le fa'agaioiina o fa'ata'ita'iga i lalo ole tulaga aseptic.Fufulu aano o lo'o aoina ile PBS;Centrifuge le PBS ma fa'aputu le mea fa'afefe ile EP-tubes.
Sludge	Aofa'i fa'ata'ita'iga: tusa.5 g;Aoina ma fa'ata'ita'i fa'ata'ita'i palapala ile EP-tube po'o le cryotube mo le fa'aagaga
Vaivai	Mo faʻataʻitaʻiga e faʻatapulaʻa le aofaʻi o microbial, pei ole paipa vai, vai lelei, ma isi, Aʻoaʻo mai le itiiti ifo ma le 1 L vai ma pasi ile 0.22 μm faamama e faʻatamaoaigaina ai microbial ile membrane.Teu le pa'u i totonu o le faagaau mama.
Pa'u	'A'e ma le fa'aeteete le pa'u i le su'e fugala'au po'o le lau tipitipi ma tu'u i totonu ole faagaau mama.

Fautuaina Fa'ata'ita'iga Tu'uina atu

Fa'a'aisa fa'ata'ita'iga ile sua vai mo le 3-4 itula ma teu ile fa'asusu vai po'o le -80 tikeri ile fa'aagaga umi.E manaʻomia le faʻataʻitaʻiga o felauaiga ma le aisa mago.

Auala o Galuega Au'aunaga

Fa'ata'ita'iga tu'uina atu

Fauga faletusi

Fa'asologa

Iloiloga fa'amaumauga

Au'aunaga pe a uma ona fa'atau

Muamua: 16S/18S/ITS Amplicon Sequencing-NGS

Sosoo ai: Su'esu'ega mo le Transposase-Accessible Chromatin fa'atasi ai ma le fa'asologa maualuga (ATAC-seq)

1. Fa'amatalaga fua faatatau o le V3 + V4(Illumina)-fa'avae microbial fa'alaua'itele fa'alaua'itele vs. Full-umi (PacBio)-fa'avae profiling.
(Fa'amaumauga o poloketi e 30 na filifilia fa'afuase'i na fa'aaogaina mo fa'amaumauga)

2. Fa'amatalaga fa'amatalaga o le fa'asologa o le amplicon umi atoa i ituaiga-tulaga i ituaiga fa'ata'ita'iga eseese

3. Tufatufaina o ituaiga

4. Laau Phylogenetic

BMK mataupu

O le faʻaalia o le arsenic e aʻafia ai le faʻaleagaina o pa puipui o le intestinal ma faʻaosoina ai le gut-liver axis e mafua ai le mumū ma le pyroptosis o le ate i pato.

Lolomiina:Saienisi o le Siosiomaga Aofa'i,2021

Fuafuaga fa'asologa:

Fa'ata'ita'iga: Pulea vs 8 mg/kg ATO vaega fa'aalia
Fa'asologa o fa'amaumauga maua mai: 102,583 fa'asologa mata'utia CCS i le aofa'i
Puleaina: 54,518 ± 747 CCS aoga
ATO-fa'aalia : 45,050 ± 1675 CCS aoga

I'uga autu

Alfa eseese:O le fa'aalia o le ATO na matua'i suia ai le tamaoaiga o le intestinal microbial ma le eseesega o pato.

Iloiloga metastats:
I le phylum level: 2 siama siama e na'o vaega e pulea
I le tulaga o ituaiga: 6 genera na maua e matua'i ese'ese i le tele o mea
I le tulaga o ituaiga: 36 ituaiga na iloa i le aofaiga atoa, ma le 6 o i latou e matua'i ese'ese i le toe fa'afouina.

Fa'asinomaga

Thingholm, LB, ma isi.“O tagata lapopoa e iai ma e aunoa ma le ma'isuka ituaiga 2 e fa'aalia ai le ese'esega o le gaogao ma le fa'aogaina ole gut microbial.Cell Host & Microbe26.2(2019).