Metagenomic Sequencing -NGS
Tulaga Auaunaga
● Tu'u'ese ma fa'ato'aga e leai se totogi mo fa'amatalaga fa'alaua'itele a microbial
● Maualuluga maualuga i le suʻeina o ituaiga e maualalo le tele i faʻataʻitaʻiga o le siosiomaga
● O le manatu o le "meta-" o loʻo tuʻufaʻatasia uma uiga faʻaola i le tulaga galue, tulaga o ituaiga ma le gene, lea e atagia mai ai se vaaiga malosi e latalata i le mea moni.
● BMK faʻaputuina le poto masani i ituaiga faʻataʻitaʻiga eseese ma sili atu i le 10,000 faʻataʻitaʻiga faʻatautaia.
Fa'amatalaga Auaunaga
| Fa'avae | Fa'asologa | Fa'amatalaga fautuaina | Taimi liliu |
| Illumina NovaSeq Platform | PE150 | 6 G/10 G/20 G | 45 Aso faigaluega |
Bioinformatics su'esu'ega
● Pulea lelei o fa'amaumauga mata'utia
● Fonotaga Metagenome
● Seti o kenera e le toe faia ma fa'amatalaga
● Su'esu'ega o ituaiga eseese
● Su'esu'ega o galuega fa'atupu kenera
● Su'esu'ega va'aiga
● Su'esu'ega fa'atasi e fa'atatau i mea fa'ata'ita'i
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, e pei o le paipa vai, vai vai, ma isi, Aʻoaʻo mai le itiiti ifo i le 1 L vai ma pasi ile 0.22 μm faamama e faʻatamaoaigaina ai microbial i luga ole 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.Fa'ata'ita'iga felauaiga fa'atasi ai ma le aisa mago e mana'omia.
Auala o Galuega Au'aunaga
Fa'ata'ita'iga tu'uina atu
Fausia faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
1.Histogram: Tufatufaina o ituaiga
2. Functional genes annotated to KEGG metabolic pathways
3. Heat fa'afanua: 'ese'ese galuega fa'avae e fa'atatau i le tele o gafa
4.Circos of CARD antibiotic resistance genes
BMK mataupu
Fa'ateleina o kenera fa'ama'i fa'ama'i fa'ama'i ma fa'ama'i fa'ama'i i luga ole eleele-togatogo fa'aauau
Lolomiina:Tusitala o Mea Mata'utia, 2021
Fuafuaga fa'asologa:
Meafaitino: DNA fa'asoa mai vaega e fa o a'a togatogo fa'ata'ita'iga e feso'ota'i ma fa'ata'ita'iga: palapala e le'i totōina, rhizosphere, episphere ma vaega endosphere.
Fa'avae: Illumina HiSeq 2500
Sini: Metagenome
16S rRNA gene V3-V4 itulagi
I'uga autu
Metagenomic sequencing ma metabarcoding profiling i luga o le eleele-a'a fa'aauau o togatogo sa fa'agasolo ina ia mafai ai ona su'esu'eina le fa'asalalauina o kenera fa'ama'i fa'ama'i (ARGs) mai le palapala i totonu o la'au.Fa'amatalaga Metagenomic na fa'aalia ai e 91.4% o kenera e tetee atu ai fa'ama'i fa'ama'i e masani ona iloa i vaega uma e fa o le palapala o lo'o ta'ua i luga, lea na fa'aalia ai le fa'aauau pea.16S rRNA amplicon sequencing gaosia 29,285 sequencing, fai ma sui o 346 ituaiga.O le tu'ufa'atasia ma le fa'avasegaina o ituaiga e ala i le fa'asologa o le amplicon, o lenei fa'asalalauga na maua e tuto'atasi mai le microbiota e feso'otai i a'a, peita'i, e mafai ona fa'afaigofieina e le telefoni feavea'i o elemene kenera.O lenei su'esu'ega na iloa ai le tafe mai o ARGs ma pathogens mai le palapala i totonu o la'au e ala i feso'ota'iga o le eleele-a'a.
Fa'asinomaga
Wang, C. , Hu, R. , Malosi, PJ , Zhuang, W. , & Shu, L. .(2020).Fa'ateleina o kenera fa'ama'i fa'ama'i ma fa'ama'i fa'ama'i i luga ole eleele-fa'aauau a'a togatogo.Tusitala o Mea Mata'utia, 408, 124985.
