Zogulitsa

16S/18S/ITS Amplicon Sequencing-NGS

16S/18S/ITS amplicon sequencing ikufuna kuwulula kuchuluka kwa phylogeny, taxonomy, ndi mitundu yamitundu m'magulu ang'onoang'ono pofufuza zinthu za PCR za zolembera zanyumba zomwe zili ndi magawo omwe amakambirana kwambiri komanso osasinthika.Kuyambitsidwa kwa zala zabwino kwambiri zamamolekyulu olembedwa ndi Woeses et al, (1977) kumapereka mphamvu zodzipatula za microbiome.Kutsatizana kwa 16S (mabakiteriya), 18S (bowa) ndi Internal transcribed spacer(ITS, bowa) amalola kuzindikirika kwa mitundu yambirimbiri komanso mitundu yosowa komanso yosadziwika.Tekinoloje iyi yakhala chida chogwiritsidwa ntchito kwambiri komanso chachikulu pakuzindikiritsa mitundu yosiyanasiyana ya tizilombo m'malo osiyanasiyana, monga pakamwa pamunthu, matumbo, ndowe, ndi zina zambiri.

nsanja:Illumina NovaSeq Platform

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Nkhani Yophunzira

Ubwino wa Utumiki

● Kudzipatula kopanda kudzipatula komanso kuzindikira mwachangu za kapangidwe ka tizilombo tating'onoting'ono mu zitsanzo zachilengedwe

● Kusamvana kwakukulu m'zigawo zotsika kwambiri mu zitsanzo zachilengedwe

● Kusanthula kwaposachedwa kwa QIIME2 ndi kusanthula kosiyanasiyana malinga ndi nkhokwe, ndemanga, OTU/ASV.

● Zolondola kwambiri, zolondola kwambiri

● Imagwira ntchito pamaphunziro osiyanasiyana ammudzi

● BMK ali ndi zochitika zambiri ndi zitsanzo zoposa 100,000 / chaka, zophimba nthaka, madzi, mpweya, matope, ndowe, matumbo, khungu, fermentation msuzi, tizilombo, zomera, ndi zina zotero.

● BMKCloud inathandizira kutanthauzira kwa data komwe kumakhala ndi zida 45 zowunikira makonda

Mafotokozedwe a Utumiki

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

Bioinformatics kusanthula

● Kuwongolera khalidwe la data yaiwisi

● OTU clustering/De-noise(ASV)

● Mawu a OTU

● Kusiyanasiyana kwa alpha

● Mitundu yosiyanasiyana ya Beta

● Kusanthula magulu

● Kusanthula kwamagulu motsutsana ndi zinthu zoyesera

● Kuneneratu za majini

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 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: Mabakiteriya ndi fungal Yense ya Genome Yotsatizana

Ena: 16S/18S/ITS Amplicon Sequencing-PacBio

1.Kugawa kwamitundu

2.Kutentha mapu: Mitundu yolemera yamitundu

3.Kupindika kwamagulu osowa

4.NMDS kusanthula

5.Lefse kusanthula

BMK Case

Anthu onenepa kwambiri omwe ali ndi matenda amtundu wa 2 komanso omwe alibe matenda amtundu wa 2 amawonetsa magwiridwe antchito komanso kapangidwe kake

Lofalitsidwa:Cell Host & Microbe, 2019

Njira yotsatirira:

Woonda wopanda shuga (n=633);Wonenepa kwambiri wopanda shuga (n=494);Matenda a shuga a Type 2 onenepa kwambiri (n=153);
Chigawo chomwe mukufuna: 16S rDNA V1-V2
Platform: Illumina Miseq (NGS-based amplicon sequencing)
Magawo ang'onoang'ono a DNA adatsatiridwa ndi kutsatizana kwa metagenomic pa Illumina Hiseq

Zotsatira zazikulu

Mafotokozedwe a tizilombo ta matenda a kagayidwe kachakudyawa adasiyanitsidwa bwino.
Poyerekeza tizilombo tating'onoting'ono timene timapangidwa ndi kutsatizana kwa 16S, kunenepa kwambiri kunapezeka kuti kumagwirizana ndi kusintha kwa tizilombo toyambitsa matenda, maonekedwe a munthu aliyense, makamaka kuchepa kwakukulu kwa Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, ndi zina zotero. Kuwonjezera apo, T2D inapezeka kuti ikugwirizana ndi kuwonjezeka kwa Escherichia / shigella. .

Buku

Thingholm, LB, et al."Anthu onenepa kwambiri omwe ali ndi matenda amtundu wa 2 komanso omwe alibe matenda a shuga amawonetsa mphamvu komanso kapangidwe kake."Cell Host & Microbe26.2 (2019).