Metagenomic Sequencing (NGS)
Zvakanakira Basa
ØKuzviparadzanisa nevamwe uye kurima-yemahara kune microbial nharaunda profiling
ØKugadziriswa kwepamusoro mukuona mhando dzemhando dzakaderera mumasampuli ezvakatipoteredza
ØPfungwa ye "meta-" inobatanidza zvinhu zvose zvehupenyu pahutano hwekushanda, rudzi rwemhando uye gene level, iyo inoratidza maonero ane simba ari pedyo nechokwadi.
ØBMK inounganidza ruzivo rwakakura mumhando dzakasiyana dzemasampuli ane anopfuura zviuru gumi masampuli akagadziriswa.
Sevhisi Zvinotsanangurwa
| SequencingPlatform | Library | Inokurudzirwa kuburitsa data | Inofungidzirwa kutendeuka-nguva |
| Illumina NovaSeq 6000 | PE250 | 50K/100K/300K Tags | 30 Mazuva |
Bioinformatics inoongorora
üRaw data quality control
üMetagenome musangano
üNon-redundant gene set uye annotation
üKuongororwa kwemhando dzakasiyana-siyana
üGenetic function diversity analysis
üInter-group analysis
üKuongororwa kwemubatanidzwa kunopesana nekuedza zvinhu
Sample Zvinodiwa uye Delivery
Sample Zvinodiwa:
ForDNA zvinyorwa:
| Muenzaniso Type | Mari | Concentration | Kuchena |
| DNA zvinyorwa | ＞ 30 ng | ＞ 1 ng/μl | OD260/280= 1.6-2.5 |
Zvezvakatipoteredza samples:
| Muenzaniso mhando | Inokurudzirwa sampling maitiro |
| Ivhu | Sampling huwandu: approx.5 g;Chinhu chakasara chakasvava chinoda kubviswa pamusoro;Geya zvidimbu zvakakura uye upfuure ne2 mm sefa;Aliquot samples mune isingabereki EP-chubhu kana cyrotube yekuchengetera. |
| Feces | Sampling huwandu: approx.5 g;Unganidza uye aliquot samples mune isina utachiona EP-tube kana cryotube yekuchengetera. |
| Zviri mukati mematumbo | Samples dzinofanirwa kugadziriswa pasi peaseptic mamiriro.Geza matishu akaunganidzwa nePBS;Centrifuge iyo PBS uye tora iyo precipitant muEP-machubhu. |
| Sludge | Sampling huwandu: approx.5 g;Unganidza uye aliquot sludge sampuli mune isina utachiona EP-tube kana cryotube yekuchengetera |
| Mvura | Yemuenzaniso ine hushoma hushoma hwehutachiona, senge mvura yemupombi, mvura yemugodhi, nezvimwewo, Unganidza ingangoita 1 L mvura uye upfuure nepakati pe 0.22 μm sefa yekupfumisa hutachiona pa membrane.Chengetedza membrane muchubhu isina utachiona. |
| Ganda | Nyatsokwenya ganda nedonje swab isina utachiona kana blade yekuvhiya woiisa muchubhu isina utachiona. |
Yakakurudzirwa Sample Delivery
Svitsa masampula munitrogen yemvura kwemaawa 3-4 uye chengetedza mune yemvura nitrogen kana -80 dhigirii kusvika kureba kureba.Sample shipping with dry-ice inodiwa.
Kuyerera Kwebasa Rebasa
Muenzaniso wekutumira
Kuvakwa kweraibhurari
Sequencing
Data analysis
Mushure mekutengesa masevhisi
1.Histogram: Kugoverwa kwemarudzi
2.Magenes anoshanda anotsanangurwa kuKEGG nzira dzemagetsi
3.Heat mepu: Mabasa akasiyana zvichienderana nehuwandu hwemajini
4.Circos yeCARD antibiotic resistance genes
Nyaya yeBMK
Kuwanda kwemajini ekudzivirira kwemishonga inorwisa mabhakitiriya uye hutachiona hwehutachiona pamwe nevhu-mangrove midzi inoenderera.
Rakatsikiswa:Chinyorwa cheHazardous Materials, 2021
Sequencing strategy:
Zvishandiso: Zvikamu zveDNA zvezvikamu zvina zvemangrove midzi inosanganiswa masamples: ivhu risina kusimwa, rhizosphere, episphere uye endosphere compartments.
Platform: Illumina HiSeq 2500
Zvinangwa: Metagenome
16S rRNA gene V3-V4 dunhu
Mibairo yakakosha
Metagenomic sequencing uye metabarcoding profiling paivhu-mudzi kuenderera mberi kwemangrove saplings yakagadziriswa kuti iongorore kuparadzirwa kwemishonga inorwisa mabhakitiriya (ARGs) kubva muvhu kuita zvirimwa.Metagenomic data yakaratidza kuti 91.4% yemishonga inorwisa mabhakitiriya yaiwanzozivikanwa muzvikamu zvina zvevhu zvataurwa pamusoro apa, izvo zvakaratidza fashoni inopfuurira.16S rRNA amplicon sequencing yakagadzira 29,285 sequences, inomiririra 346 marudzi.Kusanganiswa nemhando dzemhando dzemhando neamplicon sequencing, kuparadzirwa uku kwakawanikwa kwakasununguka kubva kune midzi-inosanganiswa microbiota, zvisinei, inogona kufambiswa nenharembozha yemajini.Chidzidzo ichi chakaratidza kuyerera kweArGs neutachiona kubva muvhu kupinda muzvirimwa kuburikidza nekubatana kwevhu-midzi inoenderera.
Reference
Wang, C. , Hu, R. , Strong, PJ , Zhuang, W. , & Shu, L. .(2020).Kuwanda kwemajini ekudzivirira kwemishonga inorwisa mabhakitiriya uye utachiona hwehutachiona padyo nevhu-mangrove midzi inoenderera.Nyaya yeHazardous Materials, 408, 124985.
