16S/18S/ITS Amplicon Sequencing-NGS
Basa Rinobatsira
● Kuzviparadzanisa-kusina uye kukurumidza kuzivikanwa kwehutachiona hwehutachiona mumasampuli ezvakatipoteredza
● Kugadziriswa kwepamusoro muzvikamu zvakaderera-zvizhinji mumasampuli ezvakatipoteredza
● Yazvino QIIME2 inoongorora kuyerera ine ongororo dzakasiyana maererano nedatabase, zvirevo, OTU/ASV.
● High-throughput, yakakwirira kururama
● Inoshanda kuzvidzidzo zvakasiyana-siyana zvenharaunda
● BMK ine ruzivo rwakakura neinopfuura 100,000 samples / gore, inovhara ivhu, mvura, gasi, sludge, tsvina, ura, ganda, kuvirisa muto, tumbuyu, zvirimwa, nezvimwe.
● BMKCloud yakagonesa kududzira dhata ine makumi mana neshanu emunhu ekuongorora maturusi
Sevhisi Zvinotsanangurwa
| SequencingPlatform | Library | Inokurudzirwa kuburitsa data | Inofungidzirwa kutendeuka-nguva |
| Illumina NovaSeq Platform | PE250 | 50K/100K/300K Tags | 30 Mazuva |
Bioinformatics inoongorora
● Raw data quality control
● OTU clustering/De-noise(ASV)
● OTU chirevo
● Alpha kusiyana-siyana
● Beta siyana
● Kuongorora kweboka
● Kuongororwa kwesangano kunopesana nekuedza zvinhu
● Basa rekufanotaura kwemajini
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;Geda zvidimbu zvakakura uye upfuure ne2 mm sefa;Aliquot samples mune sterile EP-chubhu kana cyrotube yekuchengetera. |
| Feces | Sampling huwandu: approx.5 g;Unganidza uye aliquot samples mune isinga bereki EP-tube kana cryotube yekuchengetera. |
| Intestinal contents | Samples dzinofanirwa kugadziriswa pasi peaseptic mamiriro.Geza matishu akaunganidzwa nePBS;Centrifuge iyo PBS uye tora iyo precipitant mu EP-machubhu. |
| Sludge | Sampling huwandu: approx.5 g;Unganidza uye aliquot sludge sampuli mune isina utachiona EP-tube kana cryotube yekuchengetera |
| Mvura | Kune sampuli ine hushoma huwandu hwehutachiona, hwakadai semvura yemupombi, mvura yemugodhi, nezvimwewo, Unganidza ingangoita 1 L mvura uye upfuure nepakati pe0.22 μm sefa yekupfumisa hutachiona pa membrane.Chengetedza membrane muchubhu isina utachiona. |
| Ganda | Nyatsokweva pamusoro peganda nemucheka wedonje usina 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 kurebesa kwenguva refu.Sample shipping with dry-ice inodiwa.
Kuyerera Kwebasa Rebasa
Muenzaniso wekutumira
Kuvakwa kweraibhurari
Sequencing
Data analysis
Mushure mekutengesa masevhisi
1.Kugovera marudzi
2.Kupisa mepu: Zvisikwa zvehupfumi kuunganidza
3.Rare faction curve
4.NMDS kuongorora
5.Lefse kuongorora
Nyaya yeBMK
Vakafutisa Vanhu vane uye vasina Type 2 Diabetes vanoratidza akasiyana gut microbial inoshanda kugona uye kuumbwa
Rakatsikiswa:Cell Host & Microbe, 2019
Sequencing strategy:
Kuonda kusina chirwere cheshuga (n=633);Kufutisa asiri chirwere cheshuga (n=494);Kufutisa-Type 2 chirwere cheshuga (n=153);
Nzvimbo yakatarisa: 16S rDNA V1-V2
Platform: Illumina Miseq (NGS-based amplicon sequencing)
Subset yezvinyorwa zveDNA zvakaiswa pasi pemetagenomic sequencing paIllumina Hiseq
Mibairo yakakosha
Microbial profilings yezvirwere izvi zvemetabolism zvakabudirira kusiyaniswa.
Nokuenzanisa maitiro ehutachiona anokonzerwa ne16S sequencing, kufutisa kwakawanikwa kunobatanidza nekuchinja kwehutachiona hwehutachiona, humwe humwe humwe, kunyanya kuderera kukuru muAkkermansia, Faecalibacterium, Oscillibacter, Alistipes, nezvimwewo. Mukuwedzera, T2D yakawanikwa yakabatana nekuwedzera kweEscherichia / shigella. .
Reference
Thingholm, LB, et al."Vanhu Vakafutisa vane uye vasina Type 2 Diabetes Vanoratidza Yakasiyana Gut Microbial Functional Capacity and Composition."Cell Host & Microbe26.2(2019).
