Imikhiqizo

Ukulandelana kwe-Metagenomic-Nanopore

I-Metagenomic Sequencing-Nanopore Isithombe Esifakiwe

I-Metagenomics iyithuluzi lamangqamuzana elisetshenziselwa ukuhlaziya izinto ezixubile ze-genomic ezikhishwe kumasampula emvelo, ezihlinzeka ngolwazi oluningiliziwe ngokuhlukahluka kwezinhlobo kanye nobuningi, ukwakheka kwabantu, ubudlelwano be-phylogenetic, izakhi zofuzo ezisebenzayo kanye nenethiwekhi yokuxhumana enezici zemvelo, njll. Amapulatifomu okulandelana kwe-Nanopore asanda kwethulwa kwizifundo ze-metagenomic.Ukusebenza kwayo okuhle kakhulu kubude bokufunda kuthuthukise kakhulu ukuhlaziywa kwe-metagenomic yokusakaza, ikakhulukazi ukuhlanganiswa kwe-metagenome.Ngokuthatha izinzuzo zobude bokufunda, ucwaningo lwe-metagenomic olusekelwe ku-Nanopore luyakwazi ukuzuza umhlangano oqhubekayo uma uqhathaniswa ne-shot-gun metagenomics.Kushicilelwe ukuthi i-metagenomics esekwe e-Nanopore ikhiqize ngempumelelo ama-genomes ebhaktheriya aphelele futhi avaliwe avela kuma-microbiomes (Moss, EL, et. al,I-Nature Biotech, 2020)

Inkundla:I-Nanopore PromethION P48

Xhumana nathi

Imininingwane Yesevisi

Imiphumela yedemo

Icala le-BMK

Izinzuzo Zesevisi

● Ukuhlanganiswa kwekhwalithi ephezulu-Ukuthuthukisa ukunemba kokuhlonza izinhlobo zezilwane kanye nokubikezela kofuzo lwe-funcational

● Ukuhlukaniswa kwe-genome yebhaktheriya okuvaliwe

● Ukusetshenziswa okunamandla kakhulu futhi okunokwethenjelwa endaweni ehlukahlukene, isb ukutholwa kwamagciwane amancane noma izakhi zofuzo ezihlobene nokumelana nama-antibiotic.

● Ukuhlaziya i-metagenome yokuqhathanisa

Imininingwane Yesevisi

Inkundla	Ukulandelanisa	Idatha enconyiwe	Isikhathi sokushintsha
I-Nanopore	ONT	6 G/10 G	65 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

Izidingo zesampula nokulethwa

Izimfuneko zesampula:

NgobaIzingcaphuno ze-DNA:

Uhlobo lwesampula	Inani	Ukugxila	Ubumsulwa
Izingcaphuno ze-DNA	1-1.5 μg	＞ 20 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

Okwedlule: Ukulandelana okuphelele kwe-genome bisulfite

Olandelayo: Ukulandelana kwe-Metagenomic -NGS

1.I-Heatmap: Ukuhlanganisa ukunotha kwezinhlobo zezilwane2.Izakhi zofuzo ezisebenzayo ezichazelwe ezindleleni ze-KEGG ze-metabolicInethiwekhi ye-3.Species correlation4.Ama-Circos ezakhi zofuzo ze-CARD antibiotic resistance

Icala le-BMK

I-Nanopore metagenomics inika amandla ukuxilongwa okusheshayo komtholampilo kokutheleleka kwebhaktheriya okuphansi kokuphefumula

Kushicilelwe:I-Nature Biotechnology, 2019

Amaphuzu Avelele Ezobuchwepheshe
Ukulandelanisa: I-Nanopore MinION
I-Clinical metagenomics bioinformatics: Ukuncipha kwe-DNA ye-Host, i-WIMP kanye nokuhlaziywa kwe-ARMA
Ukutholwa ngokushesha: amahora angu-6
Ukuzwela okuphezulu: 96.6%

Imiphumela esemqoka

Ngo-2006, ukutheleleka okuphansi kokuphefumula (LR) kwabangela ukufa kwabantu abayizigidi ezi-3 emhlabeni jikelele.Indlela ejwayelekile yokutholwa kwe-pathogen ye-LR1 ukutshalwa, okungezwani kahle, isikhathi eside futhi entula isiqondiso ekwelashweni kwangaphambi kwesikhathi kwama-antibiotic.Ukuxilongwa okusheshayo nokunembile kwamagciwane sekuyisikhathi eside kuyisidingo esiphuthumayo.UDkt. Justin waseNyuvesi yase-East Anglia kanye nabalingani bakhe basungula ngempumelelo indlela ye-metagenomic esekelwe e-Nanopore ukuze kutholwe igciwane lesandulela ngculaza.Ngokuhamba komsebenzi wabo, i-99.99% ye-DNA yokusingatha ingaqedwa.Ukutholwa kumagciwane kanye nezakhi zofuzo ezimelana nama-antibiotic kungaqedwa emahoreni ayi-6.

Ireferensi
Charalampous, T. , Kay, GL , Richardson, H. , Aydin, A. , & O'Grady, J. .(2019).I-Nanopore metagenomics inika amandla ukuxilongwa okusheshayo komtholampilo kokutheleleka kwebhaktheriya okuphansi kokuphefumula.I-Nature Biotechnology, 37(7), 1.