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Imikhiqizo

Ukulandelana kwe-Metagenomic -NGS

I-Metagenome ibhekisela eqoqweni lengqikithi yezinto zofuzo zomphakathi oxubile wezinto eziphilayo, njenge-metagenome yemvelo, i-metagenome yomuntu, njll. Iqukethe ama-genome akho kokubili amagciwane aphilayo nangatshalwa.Ukulandelana kwe-Metagenomic iyithuluzi lamangqamuzana elisetshenziselwa ukuhlaziya izinto ezixubile ze-genomic ezikhishwe kumasampula emvelo, okunikeza ulwazi oluningiliziwe ngokuhlukahluka kwezinhlobo kanye nobuningi, ukwakheka kwabantu, ubudlelwano be-phylogenetic, izakhi zofuzo ezisebenzayo kanye nenethiwekhi yokuxhumana nezici zemvelo.

Inkundla:Illumina NovaSeq6000


Imininingwane Yesevisi

Imiphumela yedemo

Ucwaningo lwesigameko

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

liuchengtu11

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

ilogo_02

Ukulethwa kwesampula

ilogo_04

Ukwakhiwa komtapo wolwazi

ilogo_05

Ukulandelanisa

ilogo_06

Ukuhlaziywa kwedatha

ilogo_07

Amasevisi angemuva kokuthengisa


  • Okwedlule:
  • Olandelayo:

  • 1.I-Histogram: Ukusatshalaliswa kwezinhlobo zezilwane

    3

    2.Izakhi zofuzo ezisebenzayo ezichazelwe ezindleleni ze-KEGG ze-metabolic

    4

    3.Imephu yokushisa: Imisebenzi ehlukile esekelwe kubuningi bofuzo oluhlobene54.Ama-Circos we-CARD antibiotic resistance genes

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    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.

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