Metagenomic Sequencing-Nanopore
Basa Rinobatsira
● Kuungana kwepamusoro-Kuwedzera kurongeka kwekuzivikanwa kwemarudzi uye kufanotaura kwemajini funcational
● Yakavharwa bhakitiriya genome isolation
● Kushandisa kwakasimba uye kwakavimbika munzvimbo dzakasiyana siyana, semuenzaniso kuona utachiona hwepathogenic kana majini ane chekuita nemishonga inorwisa mabhakitiriya.
● Kuenzanisa metagenome kuongorora
Sevhisi Zvinotsanangurwa
| Platform | Sequencing | Yakakurudzirwa data | Nguva yekuchinja |
| Nanopore | ONT | 6 G/10 G | 65 Mazuva ekushanda |
Bioinformatics inoongorora
● Raw data quality control
● Metagenome musangano
● Non-redundant gene set uye annotation
● Kuongororwa kwemhando dzakasiyana-siyana
● Genetic function diversity analysis
● Kuongororwa kweboka
● Kuongororwa kwesangano kunopesana nekuedza zvinhu
Sample Zvinodiwa uye Delivery
Sample zvinodiwa uye kuendesa
Sample Zvinodiwa:
ForDNA zvinyorwa:
| Muenzaniso Type | Mari | Concentration | Kuchena |
| DNA zvinyorwa | 1-1.5 μg | ＞ 20 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.Heatmap: Species hupfumi kuunganidza
2.Magetsi anoshanda anotsanangurwa kuKEGG metabolic pathways
3.Species correlation network
4.Circos yeCARD antibiotic resistance genes
Nyaya yeBMK
Nanopore metagenomics inogonesa kukurumidza kuongororwa kwekiriniki yehutachiona hwehutachiona hwepasi pekufema
Rakatsikiswa:Nature Biotechnology, 2019
Zvinokosha Zveunyanzvi
Sequencing: Nanopore MinION
Clinical metagenomics bioinformatics: Host DNA kupera, WIMP uye ARMA kuongorora.
Kukurumidza kuona: maawa matanhatu
Kunzwa kwakanyanya: 96.6%
Mibairo yakakosha
Muna 2006, chirwere chepasi chekufema (LR) chakakonzera kufa kwevanhu mamiriyoni matatu pasi rose.Iyo yakajairwa nzira yeLR1 pathogen yekuona kurima, iyo ine kutadza kunzwa, kureba-kutenderera-nguva uye kushomeka kwekutungamira mukutanga kwemishonga inorwisa mabhakitiriya.Kukurumidza uye kwakarurama kuongororwa utachiona hwagara huri chinhu chinodiwa nekukurumidzira.Dr. Justin anobva kuYunivhesiti yeEast Anglia nevamwe vake vakabudirira kugadzira nzira yeNanopore-based metagenomic yekutsvaga utachiona.Zvinoenderana nekufamba kwavo, 99.99% yevaenzi DNA inogona kudzikiswa.Kuonekwa muzvirwere uye majini asingachinjiki nemishonga inorwisa mabhakitiriya kunogona kupera mumaawa matanhatu.
Reference
Charalampous, T. , Kay, GL , Richardson, H. , Aydin, A. , & O'Grady, J. .(2019).Nanopore metagenomics inogonesa kukurumidza kuongororwa kwekiriniki yehutachiona hwehutachiona hwepasi pekufema.Nature Biotechnology, 37(7), 1.
