Ukulandelana kwe-Metagenomic -NGS
Izinzuzo Zesevisi
● Ukuzihlukanisa nokutshalwa ngaphandle kokulinywa ukuze kufakwe iphrofayili yomphakathi yezilwanyana ezincane
● Ukulungiswa okuphezulu ekutholeni izinhlobo ezinenala ephansi kumasampula emvelo
● Umbono we-“meta-” uhlanganisa zonke izici zebhayoloji ezingeni lokusebenza, izinga lezinhlobo zezilwane kanye nezinga lofuzo, okubonisa umbono oguquguqukayo oseduze neqiniso.
● I-BMK iqongelela ulwazi olukhulu ngezinhlobo zamasampuli ahlukahlukene anamasampuli angaphezu kuka-10,000 acutshunguliwe.
Imininingwane Yesevisi
| Inkundla | Ukulandelanisa | Idatha enconyiwe | Isikhathi sokushintsha |
| Illumina NovaSeq 6000 | I-PE150 | 6 G/10 G/20 G | 45 Izinsuku zokusebenza |
I-Bioinformatics ihlaziya
● Ukulawulwa kwekhwalithi yedatha eluhlaza
● Ukuhlanganiswa kwe-Metagenome
● Isethi yofuzo engadideki nesichasiselo
● Ukuhlaziya ukuhlukahluka kwezinhlobo zezilwane
● Ukuhlaziywa kokuhlukahluka kokusebenza kofuzo
● Ukuhlaziya kwamaqembu
● Ukuhlaziywa kwenhlangano ngokumelene nezici zokuhlola
Izidingo Zesampula Nokulethwa
Izimfuneko zesampula:
NgobaIzingcaphuno ze-DNA:
| Uhlobo lwesampula | Inani | Ukugxila | Ubumsulwa |
| Izingcaphuno ze-DNA | ＞ 100ng | ＞ 1 ng/μl | OD260/280= 1.6-2.5 |
Ngamasampuli emvelo:
| Uhlobo lwesampula | Inqubo yokusampula enconyiwe |
| Umhlabathi | Inani lesampula: cishe.5 g;Izinto ezisele ezibunile zidinga ukususwa phezulu;Gaya izingcezu ezinkulu futhi udlule ngesihlungi esingu-2 mm;Amasampuli e-Aliquot ku-EP-tube eyinyumba noma i-cyrotube ukuze abhukhe. |
| Indle | Inani lesampula: cishe.5 g;Qoqa futhi amasampula e-aliquot ku-EP-tube eyinyumba noma i-cryotube ukuze ubhukhe. |
| Okuqukethwe kwamathumbu | Amasampula adinga ukucutshungulwa ngaphansi kwesimo se-aseptic.Geza izicubu eziqoqiwe nge-PBS;I-Centrifuge i-PBS futhi uqoqe i-precipitant kumashubhu e-EP. |
| Udaka | Inani lesampula: cishe.5 g;Qoqa futhi isampula ye-aliquot sludge ku-EP-tube eyinyumba noma i-cryotube ukuze ubhukhe |
| Amanzi | Ukuze uthole isampula enenani elilinganiselwe le-microbial, njengamanzi kampompi, amanzi omthombo, njll., Qoqa okungenani amanzi angu-1 L futhi udlule kusihlungi esingu-0.22 μm ukuze unothise i-microbial kulwelwesi.Gcina ulwelwesi kushubhu oyinyumba. |
| Isikhumba | Hlanza ngokucophelela indawo yesikhumba ngeswabhu kakotini oyinyumba noma i-blade yokuhlinza bese uyibeka eshubhuni eliyinyumba. |
Ukulethwa Kwesampula Okunconyiwe
Friza amasampula ku-nitrogen ewuketshezi amahora angu-3-4 futhi uwagcine ku-nitrogen ewuketshezi noma -80 degree ekubhukeni isikhathi eside.Kudingeka isampula yokuthunyelwa ngeqhwa elomile.
Ukugeleza Komsebenzi Wesevisi
Ukulethwa kwesampula
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
1.I-Histogram: Ukusatshalaliswa kwezinhlobo zezilwane
2.Izakhi zofuzo ezisebenzayo ezichazelwe ezindleleni ze-KEGG ze-metabolic
3.Imephu yokushisa: Imisebenzi ehlukile esekelwe kubuningi bofuzo oluhlobene
4.Ama-Circos we-CARD antibiotic resistance genes
Icala le-BMK
Ukusabalala kwezakhi zofuzo zokumelana nama-antibiotic kanye namagciwane amagciwane eduze kwezimpande zenhlabathi-umhlume
Kushicilelwe:Ijenali Yezinto Eziyingozi, 2021
Isu lokulandelanisa:
Izinto zokwakha:Izicucu ze-DNA zezingcezu ezine zezimpande zomhlume ezihambisana namasampula: inhlabathi engatshaliwe, i-rhizosphere, i-episphere kanye ne-endosphere compartments
Ipulatifomu: Illumina HiSeq 2500
Okuhlosiwe: Metagenome
16S rRNA isifunda V3-V4
Imiphumela esemqoka
Ukulandelana kwe-Metagenomic kanye ne-metabarcoding profiling ekuqhubekeni kwezimpande zenhlabathi yezithombo zomhlume kwacutshungulwa ukuze kucwaningwe ukusabalalisa izakhi zofuzo zokulwa namagciwane (ama-ARG) kusuka emhlabathini kuya ezitshalweni.Idatha ye-Metagenomic yembula ukuthi u-91.4% wezakhi zofuzo zokumelana nama-antibiotic zazivame ukuhlonzwa kuzo zonke izingxenye ezine zenhlabathi ezishiwo ngenhla, ezibonisa imfashini eqhubekayo.Ukulandelana kweamplicon ye-16S rRNA kukhiqize ukulandelana okungu-29,285, okumelela izinhlobo ezingama-346.Kuhlanganiswe nephrofayili yezinhlobo ngokulandelana kwe-amplicon, lokhu kusatshalaliswa kwatholakala kuzimele ku-microbiota ehlotshaniswa nezimpande, nokho, kungenziwa lula ukuhamba kwezakhi zofuzo.Lolu cwaningo luhlonze ukugeleza kwama-ARG namagciwane asuka enhlabathini aye ezitshalweni ngokuqhubeka okuxhumene kwezimpande zenhlabathi.
Ireferensi
Wang, C. , Hu, R. , Strong, PJ , Zhuang, W. , & Shu, L. .(2020).Ukuvama kwezakhi zofuzo zokumelana nama-antibiotic kanye namagciwane amagciwane eduze kwezimpande zenhlabathi-umhlume.Ijenali Yezinto Eziyingozi, 408, 124985.
