16S/18S/ITS Amplicon Sequencing-PacBio
Basa Rinobatsira
● Kuverenga kwenguva refu kunoratidza zvizere-kureba kutevedzana kwe16S/18S/ITS
● Yakanyanya kurongeka base kufona nePacBio CCS mode sequencing
● Kugadziriswa kwemhando yemhando muOTU/ASV chirevo
● Yazvino QIIME2 inoongorora kuyerera ine ongororo dzakasiyana maererano nedatabase, zvirevo, OTU/ASV.
● 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 | Yakakurudzirwa data | Nguva yekuchinja |
| PacBio Sequel II | SMRT-bhero | 5K/10K/20K Tags | 44 Mazuva ekushanda |
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 | ＞ 1 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.Annotation rate yeV3 + V4 (Illumina)-based microbial community profiling vs. Full-length (PacBio)-based profiling.
(Data remapurojekiti makumi matatu akatorwa zvisina tsarukano akaiswa pahuwandu)
2.Annotation mwero weakazara-kureba amplicon sequencing pamhando-nhanho mumhando dzakasiyana dzemuenzaniso
3.Kugovera marudzi
4.Phylogenetic muti
Nyaya yeBMK
Arsenic exposure inoita kuti intestinal barriers ikuvadze uye zvinokonzeresa activation yegut-chiropa axis inotungamira mukuzvimba uye pyroptosis yechiropa mumadhadha.
Rakatsikiswa:Sayenzi Yenzvimbo Yese,2021
Sequencing strategy:
Samples: Kudzora vs 8 mg / kg ATO yakafumurwa boka
Sequencing data goho: 102,583 mbishi CCS sequences muhuwandu
Kudzora: 54,518 ± 747 inoshanda CCS
ATO-yakafumurwa : 45,050 ± 1675 inoshanda CCS
Mibairo yakakosha
Alpha kusiyana:Kuratidzwa kweATO kwakachinja zvakanyanya hupfumi hwemukati memukati uye kusiyana mumadhadha.
Metastats kuongororwa:
Mune phylum level: 2 bhakitiriya phyla inongowanikwa mumapoka ekutonga
Mune genus level: 6 genera yakawanikwa yakasiyana zvakanyanya muhuwandu hwakawanda
Muchikamu chezvipenyu: 36 marudzi akaonekwa akazara, uye 6 azvo akasiyana zvakanyanya mukuwanda kwerelavive.
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).
