Iimveliso

Ulandelelwano lweMetagenomic-Nanopore

IMetagenomic Sequencing-Nanopore Featured Image

I-Metagenomics sisixhobo se-molecular esetyenziselwa ukuhlalutya izinto ezixubileyo ze-genomic ezikhutshwe kwiisampuli zendalo, ezinikezela ngolwazi olucacileyo kwiintlobo ezahlukeneyo zeentlobo kunye nobuninzi, isakhiwo sabantu, ubudlelwane be-phylogenetic, izakhi zofuzo ezisebenzayo kunye nenethiwekhi yokulungelelanisa kunye nezinto ezisingqongileyo, njl njl. kwizifundo zemetagenomic.Ukusebenza kwayo okugqwesileyo kubude bokufunda kuphuculwe kakhulu uhlalutyo lwemetagenomic esezantsi, ngakumbi indibano yemetagenome.Ukuthatha izibonelelo zobude bokufunda, i-Nanopore-based based metagenomic study iyakwazi ukuphumeza indibano eqhubekayo ngokuthelekisa ne-shot-gun metagenomics.Kuye kwapapashwa ukuba i-metagenomics esekwe kwi-Nanopore yenze ngempumelelo i-genomes yebhakteria epheleleyo kunye nevaliweyo ukusuka kwi-microbiomes (Moss, EL, et. al.I-Biotech yendalo, 2020)

Iqonga:Nanopore PromethION P48

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Iziphumo zeDemo

Ityala le-BMK

Iinkonzo eziluncedo

● Idibano ekumgangatho ophezulu-Ukuphucula ukuchaneka kokuchongwa kweentlobo kunye noqikelelo lwemfuza esebenzayo

● Ukwahlukaniswa kwegenome yebhaktiriya evaliweyo

● Usetyenziso olunamandla ngakumbi noluthembekileyo kwiindawo ezahlukeneyo, umz. ukufunyanwa kweentsholongwane ze-pathogenic okanye iigenes ezinxulumene nokumelana namayeza okubulala iintsholongwane.

● Uhlalutyo lothelekiso lwemetagenome

IiNgcaciso zeNkonzo

Iqonga	Ukulandelelanisa	Idatha ecetyiswayo	Ixesha lokuguqula
Nanopore	ONT	6 G/10 G	65 Iintsuku zokusebenza

Uhlalutyo lwe-Bioinformatics

● Ulawulo lomgangatho wedatha ekrwada

● Indibano yeMetagenome

● Iseti yemfuza engafunekiyo kunye nenkcazo

● Uhlalutyo lweentlobo ngeentlobo zezilwanyana

● Uhlalutyo lweyantlukwano yemfuza

● Uhlalutyo lwamaqela ngamaqela

● Uhlalutyo lombutho ngokuchasene nemiba yovavanyo

IiMfuno zeSampulu kunye nokuhanjiswa

Iimfuno zesampulu kunye nokuhanjiswa

IiMfuno zeSampulu:

KubaIzicatshulwa zeDNA:

Uhlobo lweSampuli	Isixa	Ukugxila	Ubunyulu
Izicatshulwa zeDNA	1-1.5 μg	＞ 20 ng/μl	OD260/280= 1.6-2.5

Ngeesampuli zokusingqongileyo:

Uhlobo lwesampuli	Inkqubo yesampulu ecetyiswayo
Umhlaba	Isixa sesampulu: malunga.5 g;Into eseleyo ebunayo kufuneka isuswe kumphezulu;Gaya iinqununu ezinkulu kwaye udlule kwi-2 mm filter;Iisampulu ze-Aliquot kwi-EP-tube engcolileyo okanye i-cyrotube yogcino.
Ilindle	Isixa sesampulu: malunga.5 g;Qokelela kwaye iisampulu ze-aliquot kwi-EP-tube engcolileyo okanye i-cryotube yokubhukisha.
Iziqulatho zamathumbu	Iisampuli kufuneka ziqwalaselwe phantsi kwemeko ye-aseptic.Hlamba izicubu eziqokelelweyo nge-PBS;I-Centrifuge i-PBS kwaye uqokelele i-precipitant kwi-EP-tubes.
Udaka	Isixa sesampulu: malunga.5 g;Qokelela kwaye isampuli ye-aliquot ye-sludge kwi-EP-tube engcolileyo okanye i-cryotube yokugcinelwa
Amanzi	Kwisampulu enesixa esilinganiselweyo se-microbial, njengamanzi epompo, amanzi omthombo, njl., Qokelela ubuncinci be-1 L yamanzi kwaye udlule kwi-0.22 μm yokucoca ukutyebisa i-microbial kwi-membrane.Gcina inwebu kwityhubhu enyumba.
Ulusu	Khulula ngononophelo umphezulu wolusu ngeswab yomqhaphu engcolileyo okanye iblade yotyando kwaye uyibeke kwityhubhu engenanto.

Unikezelo lweSampuli eCetyisiweyo

Umkhenkce iisampulu kulwelo nitrogen iiyure 3-4 kwaye ugcine kulwelo nitrogen okanye -80 degree ukuya ugcino ixesha elide.Isampulu yokuthumela ngenqanawe eyomileyo iyafuneka.

Ukuhamba komsebenzi weNkonzo

Ukuhanjiswa kwesampuli

Ukwakhiwa kwethala leencwadi

Ukulandelelanisa

Uhlalutyo lwedatha

Iinkonzo emva kokuthengisa

Ngaphambili: Ulandelelwano lwegenome bisulfite epheleleyo

Okulandelayo: Ulandelelwano lweMetagenomic -NGS

1.Heatmap: Ubutyebi beentlobo zeentloboI-2.Iijene ezisebenzayo ezichazwe kwiindlela ze-metabolic ze-KEGG3.Inethiwekhi yonxibelelaniso lweentloboI-4.I-Circos ye-CARD i-antibiotic resistance genenes

Ityala le-BMK

I-Nanopore metagenomics yenza ukuxilongwa ngokukhawuleza kweklinikhi yosulelo olusezantsi lwebhaktiriya yokuphefumla

Ipapashiwe:I-Nature Biotechnology, ngo-2019

Iimbalasane zobuGcisa
Ukulandelelana: I-Nanopore MinION
I-bioinformatics yeklinikhi ye-metagenomics: Ukuchithwa kwe-DNA ye-Host, i-WIMP kunye nohlalutyo lwe-ARMA
Ukubonwa ngokukhawuleza: iiyure ze-6
Uvakalelo oluphezulu: 96.6%

Iziphumo eziphambili

Ngo-2006, usulelo olusezantsi lokuphefumla (LR) lwabangela ukufa kwabantu abazizigidi ezi-3 kwihlabathi jikelele.Indlela eqhelekileyo yokufunyaniswa kwepathogen ye-LR1 kukuhlakula, enovakalelo olulambathayo, ixesha elide lokujika-jika kunye nokunqongophala kwesikhokelo kunyango lwakwangoko lonyango lokubulala iintsholongwane.Uxilongo olukhawulezileyo noluchanekileyo lwe-microbial kudala iyimfuneko engxamisekileyo.UDkt Justin waseYunivesithi yase-East Anglia kunye nabalingane bakhe baphuhlise ngempumelelo indlela ye-metagenomic esekelwe kwi-Nanopore yokufumanisa i-pathogen.Ngokutsho kokuhamba komsebenzi wabo, i-99.99% ye-DNA yokusingatha inokupheliswa.Ukufunyanwa kwi-pathogens kunye ne-antibiotic resistant genes kunokugqitywa kwiiyure ze-6.

Isalathiso
Charalampous, T. , Kay, GL , Richardson, H. , Aydin, A. , & O'Grady, J.(2019).I-Nanopore metagenomics yenza ukuxilongwa ngokukhawuleza kweklinikhi yosulelo olusezantsi lwebhaktiriya yokuphefumla.I-Nature Biotechnology, 37(7), 1.