Zogulitsa

16S/18S/ITS Amplicon Sequencing-PacBio

Subunit pa 16S ndi 18S rRNA yomwe ili ndi zigawo zonse zotetezedwa kwambiri komanso zosinthika kwambiri ndi chala chabwino kwambiri cha molekyulu yozindikiritsa zamoyo za prokaryotic ndi eukaryotic.Kutengera mwayi wotsatizana, ma amplicon awa amatha kuyang'ana pazigawo zosungidwa ndipo madera osinthika amatha kudziwika bwino kuti tizindikire tizilombo toyambitsa matenda zomwe zimathandizira pamaphunziro okhudza kusanthula kwamitundu yosiyanasiyana, taxonomy, phylogeny, ndi zina. ) kutsatizana kwa nsanja ya PacBio kumathandizira kupeza zowerengera zolondola kwambiri, zomwe zimatha kuphimba ma amplicons aatali (pafupifupi 1.5 KB).Kuwona kokulirapo kwa ma genetic kumathandizira kwambiri kusintha kwa mitundu ya mabakiteriya kapena bowa.

nsanja:PacBio Sequel II

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Nkhani Yophunzira

Ubwino wa Utumiki

● Kuwerenga kwautali kumawonetsa kutsatizana konse kwa 16S/18S/ITS

● Kuyimba kolondola kwambiri ndi kutsatizana kwa PacBio CCS mode

● Kusasinthika kwamtundu wamtundu mu ndemanga za OTU/ASV

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

● 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	Deta yovomerezeka	Nthawi Yosinthira
PacBio Sequel II	SMRT-belu	5K/10K/20K Tags	44 Masiku ogwira ntchito

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	＞ 1 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: 16S/18S/ITS Amplicon Sequencing-NGS

Ena: Kuyesa kwa Chromatin Yopezeka ndi Transposase yokhala ndi Sequencing High Throughput (ATAC-seq)

1.Annotation rate of V3+V4(Illumina)-based microbial community profiling vs. Full-length (PacBio)-based profiling.
(Zambiri zama projekiti 30 osankhidwa mwachisawawa zidagwiritsidwa ntchito pazowerengera)

2. Mlingo wofotokozera wa kutsatizana kwa matalikidwe aatali-kutalika pamitundu yamitundu yosiyanasiyana

3.Kugawa kwamitundu

4.Mtengo wa Phylogenetic

BMK Case

Kuwonekera kwa arsenic kumapangitsa kuwonongeka kwa matumbo otchinga m'matumbo ndikuyambitsa kuyambika kwa matumbo-chiwindi chomwe chimatsogolera ku kutupa ndi pyroptosis ya chiwindi mu abakha.

Lofalitsidwa:Sayansi ya chilengedwe chonse,2021

Njira yotsatirira:

Zitsanzo: Control vs 8 mg/kg ATO poyera gulu
Kutsata zokolola za data: 102,583 ma CCS aiwisi amatsatizana onse
Kuwongolera: 54,518 ± 747 CCS yogwira mtima
Kuwonetsedwa kwa ATO: 45,050 ± 1675 CCS yogwira mtima

Zotsatira zazikulu

Kusiyanasiyana kwa Alpha:Kuwonekera kwa ATO kunasintha kwambiri kuchuluka kwa tizilombo ta m'mimba komanso kusiyanasiyana kwa abakha.

Kusanthula kwa Metastats:
Mu phylum level: 2 bacterial phyla amapezeka m'magulu olamulira
Pagulu lamagulu: 6 mibadwo idapezeka yosiyana kwambiri ndi kuchuluka kwake
M'magulu amitundu: Mitundu 36 idadziwika yonse, ndipo 6 mwa iyo yosiyana kwambiri ndi kuchuluka kwa relavive

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).