Iimveliso

Ulandelelwano lweMetagenomic -NGS

I-Metagenome ibhekiselele kwingqokelela yemathiriyeli yemfuzo iyonke yoluntu oluxubeneyo lwezinto eziphilayo, ezifana ne-metagenome yokusingqongileyo, i-metagenome yabantu, njl njl.Ukulandelelana kwe-Metagenomic sisixhobo se-molecular esetyenziselwa ukuhlalutya izinto ezixubeneyo ze-genomic ezikhutshwe kwiisampuli zendalo, ezinikezela ngolwazi olucacileyo kwiintlobo zeentlobo zeentlobo kunye nobuninzi, ubume benani labantu, ubudlelwane be-phylogenetic, izakhi zofuzo ezisebenzayo kunye nenethiwekhi yokulungelelanisa kunye nezinto ezingqongileyo.

Iqonga:Illumina NovaSeq6000

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Iziphumo zeDemo

Into yokuphonononga

Iinkonzo eziluncedo

● Ukwahlukaniswa kunye nokulinywa simahla ukulungiselela iprofayili yentsholongwane yoluntu

● Isisombululo esiphezulu ekufumaneni iintlobo eziphantsi kobuninzi kwiisampuli zokusingqongileyo

● Ingcamango ye "meta-" idibanisa zonke iimpawu zebhayoloji kwinqanaba lokusebenza, inqanaba leentlobo kunye nenqanaba lemfuza, ebonisa imbono eguquguqukayo esondele kwinyani.

● I-BMK iqokelela amava amakhulu kwiintlobo ezahlukeneyo zeesampulu ezineisampulu ezingaphezulu kwe-10,000 ezicutshungulweyo.

IiNgcaciso zeNkonzo

Iqonga	Ukulandelelanisa	Idatha ecetyiswayo	Ixesha lokutshintsha
Illumina NovaSeq 6000	PE150	6 G/10 G/20 G	45 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:

KubaIzicatshulwa zeDNA:

Uhlobo lweSampuli	Isixa	Ukugxininisa	Ubunyulu
Izicatshulwa zeDNA	＞ 30 ng	＞ 1 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 lweMetagenomic-Nanopore

Okulandelayo: I-Chromatin Immunoprecipitation Sequencing (ChIP-seq)

1.I-Histogram: Ukuhanjiswa kweentlobo

I-2.Iijene ezisebenzayo ezichazwe kwiindlela ze-metabolic ze-KEGG

3.Imephu yobushushu: Imisebenzi eyohlukeneyo esekelwe kubuninzi bemfuzaI-4.I-Circos ye-CARD i-antibiotic resistance genenes

Ityala le-BMK

Ukuxhaphaka kofuzo lokuxhathisa amayeza okubulala iintsholongwane kunye neentsholongwane zebhaktiriya ecaleni kwengcambu yommango

Ipapashiwe:Ijenali Yezinto Eziyingozi, 2021

Iqhinga lokulandelelanisa:

Izinto: Izicatshulwa ze-DNA zamaqhekeza amane eengcambu zemangrove ezinxulumene neisampulu: umhlaba ongatyalwanga, irhizosphere, i-episphere kunye ne-endosphere compartments
Iqonga: Illumina HiSeq 2500
Ujoliso: Metagenome
16S rRNA gene V3-V4 ummandla

Iziphumo eziphambili

Ulandelelwano lwe-Metagenomic kunye ne-metabarcoding profiling kwi-root-root continuum ye-mangrove saplings yalungiswa ukuze kufundwe ukusasazwa kofuzo lwe-antibiotic resistance (ARGs) ukusuka emhlabeni ukuya kwizityalo.Idatha ye-Metagenomic ibonise ukuba i-91.4% yeejeneli zokuxhathisa izibulala-ntsholongwane ziye zachongwa ngokuqhelekileyo kuwo onke amacandelo omhlaba akhankanywe ngasentla, abonisa ifashoni eqhubekayo.Ulandelelwano lweamplicon ye-16S rRNA yenze i-29,285 ulandelelwano, emele iintlobo ze-346.Ukudibanisa neentlobo zeprofayili ngokulandelelana kwe-amplicon, oku kusasazwa kwafunyaniswa ukuba kuzimeleyo kwi-microbiota ehambelana neengcambu, nangona kunjalo, inokuququzelelwa yi-mobile ye-genetic elements.Olu phononongo luchonge ukuhamba kwee-ARGs kunye neentsholongwane ezibangela izifo ezisuka emhlabeni zisiya kwizityalo ngokudityaniswa kweengcambu zomhlaba.

Isalathiso

Wang, C. , Hu, R. , Strong, PJ , Zhuang, W. , & Shu, L. .(2020).Ukuxhaphaka kwejini yokuxhathisa amayeza okubulala iintsholongwane kunye neentsholongwane zebhaktiriya ecaleni kwengcambu yomhlaba-ummango.Ijenali yeZixhobo ezinobungozi, 408, 124985.