16S/18S/ITS Amplicon Sequencing-NGS
Tulaga Auaunaga
● E leai se fa'aesea ma vave fa'ailoaina ole tu'ufa'atasiga ole microbial ile fa'ata'ita'iga ole si'osi'omaga
● Faʻasalaga maualuga i vaega maualalo-tele i faʻataʻitaʻiga o le siosiomaga
● Su'esu'ega QIIME2 lata mai fa'atasi ma su'esu'ega 'ese'ese i tulaga o fa'amaumauga, fa'amatalaga, OTU/ASV.
● maualuga-faʻaogaina, maualuga le saʻo
● 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 | Fautuaina fa'amaumauga maua mai | Fa'atatau le taimi e liliu ai |
| Illumina NovaSeq Platform | PE250 | 50K/100K/300K fa'ailoga | 30 Aso |
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 | ＞ 30 ng | ＞ 1 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
1. Tufatufaina o ituaiga
2. Fa'afanua vevela: Fa'aputuina le tamaoaiga o ituaiga
3. Faction curve seasea
4.NMDS auiliiliga
5. Lefse su'esu'ega
BMK mataupu
O tagata lapopoa o lo'o i ai ma e leai se ma'isuka Ituaiga 2 o lo'o fa'aalia ai le 'ese'ese o le gaioiina ole gaioiina ole manava
Lolomiina:Cell Host & Microbe, 2019
Fuafuaga fa'asologa:
Pa'u ma'isuka (n=633);Oona e le o le ma'i suka (n=494);Obese-Type 2 suka (n=153);
Itulagi fa'amoemoe: 16S rDNA V1-V2
Fa'avae: Illumina Miseq (fa'asologa o le amplicon fa'avae NGS)
O vaega ole vaega ole DNA na maua ile fa'asologa o metagenomic ile Illumina Hiseq
I'uga autu
O fa'ata'ita'iga fa'ama'i fa'ama'i o nei fa'ama'i fa'ama'i na fa'amanino lelei.
E ala i le faʻatusatusaina o uiga microbial faʻatupuina e le 16S sequencing, obesity na maua e fesoʻotaʻi ma suiga i le microbial composition, uiga taʻitoʻatasi, aemaise lava le faʻaitiitia o le Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, ma isi. .
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).
