China Metagenomic Sequencing-Nanopore fakitale ndi opanga |BMK

Metagenomic Sequencing-Nanopore

Metagenomics ndi chida cha mamolekyu chomwe chimagwiritsidwa ntchito pofufuza zinthu zosakanikirana zamtundu wamtundu zomwe zimatengedwa kuchokera ku zitsanzo zachilengedwe, zomwe zimapereka chidziwitso chatsatanetsatane cha mitundu yosiyanasiyana ya zamoyo ndi kuchuluka kwake, chiwerengero cha anthu, ubale wa phylogenetic, majini ogwira ntchito ndi maukonde ogwirizana ndi chilengedwe, ndi zina zotero. ku maphunziro a metagenomic.Kuchita kwake kwapadera 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

ØKusonkhana kwapamwamba-Kupititsa patsogolo kulondola kwa chidziwitso cha mitundu ndi kuneneratu kwa jini funcational

ØKupatula kwa bakiteriya matupi athu

ØKugwiritsa ntchito mwamphamvu komanso kodalirika m'malo osiyanasiyana, mwachitsanzo, kuzindikira tizilombo toyambitsa matenda kapena majini okhudzana ndi maantibayotiki.

ØKuyerekeza kwa metagenome

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 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: 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 of CARD antibiotic resistance genenes

BMK Case

Nanopore metagenomics imathandizira kuzindikira matenda a bakiteriya m'munsi mwa kupuma

Lofalitsidwa:Nature Biotechnology, 2019

Mfundo Zaumisiri
Kutsata: Nanopore MinION
Clinical metagenomics bioinformatics: Host DNA depletion, WIMP ndi kusanthula kwa ARMA
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, kutembenuka kwanthawi 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 yochokera ku metagenomic kuti azindikire tizilombo toyambitsa matenda.Malinga ndi kayendedwe kawo, 99.99% ya DNA yolandila imatha kuthetsedwa.Kuzindikira tizilombo toyambitsa matenda ndi majini osamva maantibayotiki kumatha kutha pakadutsa maola 6.

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