Metagenomic Sequencing -NGS

Metagenome e bolela pokello ea kakaretso ea liphatsa tsa lefutso tsa sehlopha se tsoakaneng sa lintho tse phelang, tse kang metagenome ea tikoloho, metagenome ea batho, joalo-joalo. E na le liphatsa tsa lefutso tsa likokoana-hloko tse lengoang le tse ke keng tsa lengoa.Metagenomic sequencing ke sesebelisoa sa limolek'hule se sebelisetsoang ho hlahloba lisebelisoa tse tsoakiloeng tsa genomic tse ntšitsoeng lisampoleng tsa tikoloho, tse fanang ka boitsebiso bo qaqileng ka mefuta-futa ea mefuta le bongata, sebopeho sa baahi, kamano ea phylogenetic, liphatsa tsa lefutso tse sebetsang le marang-rang a amanang le maemo a tikoloho.

Sethala:Illumina NovaSeq6000

Iteanye le rona

Lintlha tsa Tšebeletso

Demo Results

Thuto-pale

Melemo ea Tšebeletso

● Ho itšehla thajana le ho lema ntle ho tefo bakeng sa profiling ea sechaba sa likokoana-hloko

● Qeto e phahameng ea ho fumana mefuta e mengata e fokolang lisampoleng tsa tikoloho

● Khopolo ea "meta-" e kopanya likarolo tsohle tsa likokoana-hloko boemong ba ts'ebetso, boemo ba mefuta le liphatsa tsa lefutso, tse bontšang pono e matla e haufi le 'nete.

● BMK e bokella boiphihlelo bo bongata mefuteng e fapaneng ea lisampole ka lisampole tse fetang 10,000 tse sebetsitsoeng.

Litlhaloso tsa Tšebeletso

Sethala	Tatelano	Lintlha tse khothalelitsoeng	Nako ea ho fetoha
Illumina NovaSeq 6000	PE150	6 G/10 G/20 G	45 Matsatsi a ho sebetsa

Litlhahlobo tsa Bioinformatics

● Taolo ea boleng ba data e tala

● Kopano ea Metagenome

● Liphatsa tsa lefutso tse sa hlokahaleng le tlhaloso

● Tlhahlobo ea mefuta-futa ea mefuta

● Tlhahlobo ea ho se tšoane ha liphatsa tsa lefutso

● Tlhahlobo ea lihlopha

● Tlhahlobo ea mokhatlo khahlanong le lintlha tsa liteko

Litlhoko tsa Mohlala le Phano

Litlhoko tsa Mohlala:

Bakeng saLintlha tsa DNA:

Mofuta oa Mohlala	Chelete	Ho tsepamisa maikutlo	Bohloeki
Lintlha tsa DNA	＞ 100 ng	＞ 1 ng/μl	OD260/280= 1.6-2.5

Bakeng sa mehlala ea tikoloho:

Mofuta oa mohlala	Mokhoa o khothaletsoang oa sampole
Mobu	Chelete ea mohlala: hoo e ka bang.5 g;Lintho tse setseng tse omeletseng li hloka ho tlosoa holimo;Sila likotoana tse kholo 'me u fete ka har'a filthara ea 2 mm;Lisampole tsa Aliquot ka har'a EP-tube kapa cyrotube bakeng sa ho behelloa.
Mantle	Chelete ea mohlala: hoo e ka bang.5 g;Bokella le lisampole tsa aliquot ho EP-tube kapa cryotube e sa sebetseng bakeng sa ho behelloa.
Likahare tsa mala	Mehlala e hloka ho sebetsoa tlas'a boemo ba aseptic.Hlatsoa lithane tse bokeletsoeng ka PBS;Hlakola PBS 'me u bokelle precipitant ka EP-tubes.
Sludge	Chelete ea mohlala: hoo e ka bang.5 g;Bokella le aliquot sludge sampole ho EP-tube e hloekisitsoeng kapa cryotube bakeng sa ho behelloa
Metsi	Bakeng sa sampole e nang le palo e lekanyelitsoeng ea likokoana-hloko, joalo ka metsi a pompo, metsi a seliba, joalo-joalo, Bokella bonyane metsi a 1 L 'me u fete ka 0.22 μm filthara ho ntlafatsa likokoana-hloko tse holim'a lera.Boloka lera ka har'a tube e hloekisitsoeng.
Letlalo	Hlakola bokaholimo ba letlalo ka hloko ka swab e hloekisitsoeng ea k'hothone kapa lehare la ho buoa 'me u le kenye ka har'a tube e sa hloekang.

Tlhahiso ea Mehlala e khothalelitsoeng

Emisa lisampole ka har'a naetrojene e metsi bakeng sa lihora tse 3-4 'me u li boloke ka har'a metsi a naetrojene kapa -80 degree ho boloka nako e telele.Ho hlokahala ho romelloa ka sekepe se nang le leqhoa le omileng.

Phallo ea Mosebetsi oa Ts'ebeletso

Mohlala oa thomello

Kaho ea laebrari

Tatelano

Tlhahlobo ea lintlha

Litšebeletso tsa ka mor'a thekiso

E fetileng: Metagenomic Sequencing-Nanopore

E 'ngoe: Sequencing ea Chromatin Immunoprecipitation (ChIP-seq)

1.Histogram: Kabo ea mefuta

2.Liphatsa tsa lefutso tse sebetsang tse hlalositsoeng ho KEGG metabolic pathways

'Mapa oa 3.Heat: Mesebetsi e fapaneng e itšetlehileng ka bongata ba liphatsa tsa lefutso4.Li-Circos tsa liphatsa tsa lefutso tsa CARD tsa lithibela-mafu

Taba ea BMK

Ho ata ha liphatsa tsa lefutso tsa lithibela-mafu le likokoana-hloko tsa baktheria hammoho le motso oa mobu oa mangrove

E hatisitsoe:Journal of Hazardous Materials, 2021

Leano la tatellano:

lintho tse bonahalang: Likaroloana tsa DNA tsa likaroloana tse 'nè tsa lisampole tse amanang le motso oa mangrove: mobu o sa lengoang, rhizosphere, episphere le endosphere compartments.
Sethala: Illumina HiSeq 2500
Sepheo: Metagenome
16S rRNA mofuta V3-V4 sebakeng

Liphetho tsa bohlokoa

Metagenomic sequencing le metabarcoding profiling mabapi le ho tsoela pele ka metso ea mobu oa lifate tsa mangrove li ile tsa sebetsoa molemong oa ho ithuta ka phatlalatso ea liphatsa tsa lefutso tsa lithibela-mafu (ARGs) ho tloha mobung ho ea ho limela.Lintlha tsa Metagenomic li senotse hore 91.4% ea liphatsa tsa lefutso tsa lithibela-mafu li ne li atisa ho tsejoa likarolong tsohle tse 'nè tsa mobu tse boletsoeng ka holimo, tse bontšang mokhoa o tsoelang pele.16S rRNA amplicon sequencing e hlahisitse tatelano ea 29,285, e emelang mefuta e 346.Ho kopanngoa le profiling ea mefuta ka tatellano ea li-amplicon, phatlalatso ena e fumanoe e ikemetse ho microbiota e amanang le metso, leha ho le joalo, e ka nolofalloa ke mobile ea likarolo tsa lefutso.Boithuto bona bo hloaile phallo ea li-ARG le likokoana-hloko tse tsoang mobung ho ea limeleng ka ho tsoela pele ho hokahantsoeng ha metso ea mobu.

Referense

Wang, C. , Hu, R. , Strong, PJ , Zhuang, W. , & Shu, L. .(2020).Ho ata ha liphatsa tsa lefutso tsa lithibela-mafu le likokoana-hloko tsa baktheria ho latela metso ea mobu oa mangrove.Journal of Hazardous Materials, 408, 124985.