Metagenomic Sequencing-Nanopore
Zvakanakira Basa
ØKuungana kwepamusoro-Kuwedzera kurongeka kwekuzivikanwa kwezvipenyu uye kufanotaura kwemajini funcational
ØYakavharwa bhakitiriya genome isolation
ØYakawedzera simba uye yakavimbika application munzvimbo dzakasiyana siyana, semuenzaniso kucherechedzwa kwemapathogenic microorganisms kana maantibiotic resistance ane chekuita nemajini.
ØKuenzanisa metagenome kuongorora
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 uye kuendesa
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.Heatmap: Species hupfumi kuunganidza
2.Magenes anoshanda anotsanangurwa kuKEGG nzira dzemagetsi
3.Species correlation network
4.Circos yeCARD antibiotic resistance genes
Nyaya yeBMK
Nanopore metagenomics inogonesa kukurumidza kuongororwa kwekiriniki yehutachiona hwehutachiona hwepazasi
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 kusanzwa zvakanaka, kutendeuka-nguva-kutenderera-nguva uye kushomeka kwekutungamira mukutanga kwemishonga inorwisa mabhakitiriya.Kukurumidza uye kwakarurama kuongororwa kwehutachiona hwagara huri chido chekukurumidzira.Dr. Justin kubva 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 anogona kupera mumaawa matanhatu.
Reference
Charalampous, T. , Kay, GL , Richardson, H. , Aydin, A. , & O'Grady, J. .(2019).Nanopore metagenomics inogonesa kukurumidza kuongororwa kwekiriniki yehutachiona hwehutachiona hwepazasi.Nature Biotechnology, 37(7), 1.
