Zogulitsa

Metagenomic Sequencing-Nanopore

Metagenomics ndi chida cha molekyulu chomwe chimagwiritsidwa ntchito pofufuza zinthu zosakanikirana zamtundu wamtundu zomwe zimatengedwa kuchokera ku zitsanzo zachilengedwe, zomwe zimapereka chidziwitso chambiri pamitundu yosiyanasiyana ya zamoyo ndi kuchuluka kwake, kuchuluka kwa anthu, ubale wa phylogenetic, majini ogwirira ntchito ndi maukonde ogwirizana ndi zinthu zachilengedwe, etc. Mapulatifomu a Nanopore adayambitsa posachedwa ku maphunziro a metagenomic.Kuchita kwake kwabwino pakuwerenga kwanthawi yayitali kunapititsa patsogolo kusanthula kwa metagenomic, makamaka msonkhano wa metagenome.Kutengera ubwino wowerengera kutalika, kafukufuku wa Nanopore-based metagenomic amatha kukwaniritsa msonkhano wopitilirabe poyerekeza ndi kuwombera mfuti.Zasindikizidwa kuti metagenomics yochokera ku Nanopore idapanga bwino ma genome athunthu komanso otsekedwa a bakiteriya kuchokera ku ma microbiomes (Moss, EL, et. al,Nature Biotech, 2020)

nsanja:Nanopore PromethION P48

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

BMK Case

Ubwino wa Utumiki

● Kusonkhanitsa kwapamwamba-Kupititsa patsogolo kulondola kwa kuzindikiritsa mitundu ya zamoyo ndi kuneneratu za majini

● Kutsekeka kwa mabakiteriya a genome

● Kugwiritsa ntchito mwamphamvu kwambiri komanso kodalirika m'madera osiyanasiyana, mwachitsanzo, kuzindikira tizilombo toyambitsa matenda kapena tizilombo toyambitsa matenda.

● Kuyerekeza kwa metagenome

Mafotokozedwe a Utumiki

nsanja	Kutsata	Deta yovomerezeka	Nthawi yosinthira
Nanopore	ONT	6G/10G	65 Masiku ogwira ntchito

Bioinformatics kusanthula

● Kuwongolera khalidwe la data yaiwisi

● Msonkhano wa Metagenome

● Ma jini osagwiritsidwa ntchito mowonjezera ndi mawu ake

● Kusanthula zamitundu yosiyanasiyana

● Kusanthula kwa mitundu yosiyanasiyana ya ma genetic

● Kusanthula magulu

● Kusanthula kwamagulu motsutsana ndi zinthu zoyesera

Zitsanzo Zofunika ndi Kutumiza

Zitsanzo zofunika ndi kupereka

Zitsanzo Zofunika:

ZaZithunzi za DNA:

Mtundu Wachitsanzo	Ndalama	Kukhazikika	Chiyero
Zithunzi za DNA	1-1.5 μg	＞ 20 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 a m'chitsime, 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

Kupereka zitsanzo

Kumanga laibulale

Kutsata

Kusanthula deta

Pambuyo pogulitsa ntchito

Zam'mbuyo: Kutsatizana konse kwa genome bisulfite

Ena: Metagenomic Sequencing -NGS

1.Heatmap: Mitundu yolemera yamitundu2.Majini ogwira ntchito ofotokozera njira za metabolic za KEGG3.Species correlation network4.Circos ya CARD antibiotic resistance genes

BMK Case

Nanopore metagenomics imathandizira kuzindikira mwachangu matenda a bakiteriya otsika kupuma

Lofalitsidwa:Nature Biotechnology, 2019

Mfundo Zaumisiri
Kutsata: Nanopore MinION
Clinical metagenomics bioinformatics: Host DNA depletion, WIMP ndi ARMA kusanthula
Kuzindikira mwachangu: maola 6
Kuzindikira kwakukulu: 96.6%

Zotsatira zazikulu

Mu 2006, matenda otsika a kupuma (LR) adapha anthu 3 miliyoni padziko lonse lapansi.Njira yodziwika bwino yodziwira tizilombo toyambitsa matenda a LR1 ndi kulima, komwe sikumva bwino, kutembenukira nthawi yayitali komanso kusowa chitsogozo pamankhwala oyambilira a maantibayotiki.Kuzindikira msanga ndi kolondola kwa tizilombo toyambitsa matenda kwakhala kofunikira kwambiri.Dr. Justin wochokera ku yunivesite ya East Anglia ndi anzake adapanga bwino njira ya Nanopore-based metagenomic kuti azindikire tizilombo toyambitsa matenda.Malinga ndi mayendedwe awo, 99.99% ya DNA yolandila imatha kuthetsedwa.Kuzindikira tizilombo toyambitsa matenda ndi majini osamva maantibayotiki kumatha kutha pakatha maola 6.

Buku
Charalampous, T. , Kay, GL , Richardson, H. , Aydin, A. , & O'Grady, J. .(2019).Nanopore metagenomics imathandizira kuzindikira mwachangu matenda a bakiteriya otsika kupuma.Nature Biotechnology, 37(7), 1.