China Metagenomic Sequencing (NGS) fakitale ndi opanga |BMK

Metagenomic Sequencing (NGS)

Metagenome imatanthawuza kusonkhanitsa kwa chibadwa chamagulu osakanikirana a zamoyo monga chilengedwe, metagenome yaumunthu, ndi zina zotero. Lili ndi ma genome a tizilombo tomwe timalima komanso tosalimidwa.Metagenomic sequencing ndi chida cha mamolekyu chomwe chimagwiritsidwa ntchito posanthula zinthu zosakanikirana zamtundu wotengedwa kuchokera ku zitsanzo zachilengedwe, zomwe zimapereka chidziwitso chambiri pamitundu yosiyanasiyana ndi kuchuluka kwake, kuchuluka kwa anthu, ubale wa phylogenetic, majini ogwira ntchito komanso maukonde olumikizana ndi chilengedwe.

nsanja:Illumina NovaSeq6000

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Nkhani Yophunzira

Ubwino wa Utumiki

ØKudzipatula komanso kulima kopanda mbiri yamagulu ang'onoang'ono

ØKusamvana kwakukulu pakuzindikira mitundu yocheperako mu zitsanzo zachilengedwe

ØLingaliro la "meta-" limagwirizanitsa zinthu zonse zamoyo pamlingo wogwirira ntchito, mlingo wa mitundu ndi jini, zomwe zimasonyeza malingaliro amphamvu omwe ali pafupi ndi zenizeni.

ØBMK imadziunjikira zambiri mumitundu yosiyanasiyana yokhala ndi zitsanzo zopitilira 10,000 zokonzedwa.

Mafotokozedwe a Utumiki

Kutsatansanja	Library	Zokolola zovomerezeka	Nthawi yoyerekeza yozungulira
Illumina NovaSeq 6000	Chithunzi cha PE250	50K/100K/300K Tags	Masiku 30

Bioinformatics kusanthula

üKuwongolera khalidwe la data yaiwisi

üMsonkhano wa Metagenome

üMa jini osagwirizana ndi ma jini ndi ndemanga

üKusanthula kwamitundu yosiyanasiyana

üKusanthula kwamitundu yosiyanasiyana ya ma genetic

üKusanthula kwamagulu

üKusanthula kwamagulu motsutsana ndi zinthu zoyeserera

Zitsanzo Zofunika ndi Kutumiza

Zitsanzo Zofunika:

ZaZithunzi za DNA:

Mtundu Wachitsanzo	Ndalama	Kukhazikika	Chiyero
Zithunzi za DNA	＞ 30 ng	＞ 1 ng/μl	OD260/280= 1.6-2.5

Za zitsanzo zachilengedwe:

Mtundu wachitsanzo	Njira yoyeserera yolangizidwa
Nthaka	Kuchuluka kwa zitsanzo: pafupifupi.5 g pa;Chotsalira chofota chiyenera kuchotsedwa pamwamba;Pewani zidutswa zazikulu ndikudutsa fyuluta ya 2 mm;Zitsanzo za Aliquot mu chubu chosabala cha EP kapena cyrotube kuti zisungidwe.
Ndowe	Kuchuluka kwa zitsanzo: pafupifupi.5 g pa;Sonkhanitsani ndi aliquot zitsanzo mu EP-chubu wosabala kapena cryotube kuti musungidwe.
Zomwe zili m'matumbo	Zitsanzo ziyenera kukonzedwa pansi pa chikhalidwe cha aseptic.Sambani minofu yosonkhanitsidwa ndi PBS;Centrifuge the PBS ndi kusonkhanitsa precipitant mu EP-machubu.
Sludge	Kuchuluka kwa zitsanzo: pafupifupi.5 g pa;Sungani ndi aliquot sludge zitsanzo mu wosabala EP-chubu kapena cryotube kusungitsa
Madzi	Kwa zitsanzo zokhala ndi tizilombo tating'onoting'ono, monga madzi apampopi, madzi apachitsime, ndi zina zotero, Sonkhanitsani madzi osachepera 1 L ndikudutsa 0.22 μm fyuluta kuti mulemeretse tizilombo toyambitsa matenda pa nembanemba.Sungani nembanemba mu chubu chosabala.
Khungu	Pala pamwamba pa khungu ndi thonje wosabala kapena tsamba la opaleshoni ndikuyika mu chubu wosabala.

Kutumiza Kwachitsanzo Kovomerezeka

Ikani zitsanzo mu nayitrogeni wamadzimadzi kwa maola 3-4 ndikusunga mu nayitrogeni wamadzimadzi kapena -80 digiri kuti musungidwe nthawi yayitali.Kutumiza kwachitsanzo kokhala ndi ayezi wowuma ndikofunikira.

Kuyenda kwa Ntchito Yantchito

Kutumiza kwachitsanzo

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Zam'mbuyo: Metagenomic Sequencing-Nanopore

Ena: Chromatin Immunoprecipitation Sequencing (ChIP-seq)

1.Histogram: Kugawa kwamitundu

2.Majini ogwira ntchito ofotokozera njira za metabolic za KEGG

Mapu a 3.Kutentha: Ntchito zosiyana zotengera kuchuluka kwa majini4.Circos of CARD antibiotic resistance genenes

BMK Case

Kuchuluka kwa majini olimbana ndi maantibayotiki ndi tizilombo toyambitsa matenda m'mphepete mwa mizu ya mangrove

Lofalitsidwa:Journal of Hazardous Equipment, 2021

Njira yotsatirira:

Zipangizo: Zotulutsa za DNA za zidutswa zinayi za mizu ya mangrove yokhudzana ndi zitsanzo: nthaka yosabzalidwa, rhizosphere, episphere ndi endosphere compartments
Pulatifomu: Illumina HiSeq 2500
Zolinga: Metagenome
16S rRNA jini V3-V4 dera

Zotsatira zazikulu

Tsatanetsatane wa metagenomic ndi mbiri ya metabarcoding pamizu yopitilira muzu wa mitengo ya mangrove adakonzedwa kuti aphunzire kufalitsa ma antibiotic resistance genes (ARGs) kuchokera kunthaka kupita ku zomera.Deta ya Metagenomic inavumbulutsa kuti 91.4% ya majini olimbana ndi maantibayotiki amadziwika bwino m'zigawo zonse zinayi za dothi zomwe zatchulidwa pamwambapa, zomwe zimasonyeza kachitidwe kosalekeza.16S rRNA amplicon sequencing adapanga 29,285 motsatira, kuyimira mitundu 346.Kuphatikizana ndi mbiri ya zamoyo pogwiritsa ntchito ma amplicon sequencing, kufalitsa kumeneku kunapezeka kuti sikunachokere ku microbiota yokhudzana ndi mizu, komabe, ikhoza kuthandizidwa ndi mafoni amtundu.Kafukufukuyu adazindikira kutuluka kwa ma ARGs ndi tizilombo toyambitsa matenda kuchokera m'nthaka kulowa muzomera kudzera munjira yolumikizana ndi mizu ya nthaka.

Buku

Wang, C. , Hu, R. , Strong, PJ , Zhuang, W. , & Shu, L. .(2020).Kuchuluka kwa majini olimbana ndi maantibayotiki ndi tizilombo toyambitsa matenda m'nthaka-mizu ya mangrove mosalekeza.Journal of Hazardous Materials, 408, 124985.