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16S/18S/ITS Amplicon Sequencing-PacBio

Mkpụrụ ndụ ihe nketa rRNA nke 16S na 18S, yana mpaghara Internal Transcribed Spacer (ITS), na-eje ozi dị ka ihe nrịbama mkpịsị aka molekul dị mkpa n'ihi njikọta ha nke mpaghara ndị a na-echekwa nke ọma na ndị nwere oke mgbanwe, na-eme ka ha bụrụ ngwaọrụ bara uru maka ịkọwapụta ihe ndị dị ndụ prokaryotic na eukaryotic. Ịgbasawanye na nhazi nke mpaghara ndị a na-enye ụzọ na-enweghị iche maka inyocha ihe mejupụtara nje na ụdị dị iche iche n'ofe gburugburu ebe obibi dị iche iche. Ọ bụ ezie na nhazi Illumina na-elekwasị anya na mpaghara ndị nwere obere mgbanwe dị ka V3-V4 nke 16S na ITS1, egosila na nkọwa taxonomic dị elu nwere ike iru site na ịhazi ogologo zuru oke nke 16S, 18S, na ITS. Usoro a zuru oke na-ebute pasentị dị elu nke usoro nhazi nke ọma, na-enweta ọkwa mkpebi nke gbasaa na njirimara ụdị. Ikpo okwu nhazi nke PacBio nke Single-Molecule Real-Time (SMRT) pụtara ìhè site n'inye ọgụgụ ogologo ziri ezi (HiFi) nke na-ekpuchi amplicons ogologo zuru oke, na-asọmpi nkenke nke nhazi Illumina. Ike a na-enye ndị nchọpụta ohere inweta uru na-enweghị atụ - echiche zuru oke nke ọdịdị mkpụrụ ndụ ihe nketa. Mkpuchi ahụ gbasaara na-eme ka mkpebi dị na nkọwa ụdị anụmanụ dịkwuo elu nke ukwuu, ọkachasị n'ime obodo nje bacteria ma ọ bụ fungal, na-eme ka a ghọtakwuo ihe mgbagwoju anya nke ọnụ ọgụgụ nje bacteria.

Kpọtụrụ anyị

Nkọwa Ọrụ

Bioinformatics

Nsonaazụ ngosi

Mbipụta ndị a ma ama

Atụmatụ Ọrụ

● Ikpo okwu nhazi: PacBio Revio

● Ụdị usoro nhazi: CCS (HiFi na-agụ)

● Mmụba nke mpaghara ebumnuche na-esote njikọ amplicons otu ugboro tupu nkwadebe ọbá akwụkwọ HiFi SMRT mgbịrịgba

Uru Ọrụ

●Mkpebi ọkwa dị elu: Tusoro iheomume mkpụmkpụ nke han,na-eme ka ọnụego nhazi OTU dị elu na ọkwa ụdị.

●Oku ntọala ziri ezi nke ukwuu: Usoro nhazi PacBio CCS (na-agụ HiFi).

●Enweghị ikewapụ onwe: Nchọpụta ngwa ngwa nke ihe mejupụtara nje bacteria na ihe nlele gburugburu ebe obibi.

●Ọ bara uru nke ukwuu: Ọmụmụ ihe dị iche iche gbasara nje bacteria.

●Nyocha bioinformatik zuru oke: Ngwugwu QIIME2 kachasị ọhụrụ (nghọta ọnụọgụgụ n'ihe gbasara gburugburu ebe obibi nje) nwere nyocha dị iche iche n'ihe gbasara nchekwa data, nkọwa, OTU/ASV.

●Ọkachamara sara mbara: Site na ọtụtụ puku ọrụ nhazi usoro amplicon kwa afọ, BMKGENE na-eweta ahụmịhe karịrị afọ iri, otu nyocha nwere nkà dị elu, ọdịnaya zuru oke, na nkwado dị mma mgbe eresịrị ahịa.

Nkọwapụta Ọrụ

Ọbá akwụkwọ	Atụmatụ Usoro Nhazi	Data akwadoro
Amplicon	PacBio Revio	Akara 10/30/50 K (CCS)

Ihe Nlereanya Dị Mkpa

Ntinye uche (ng/µL)	Mkpokọta ego (µg)	Olu (µL)
≥5	≥0.3	≥20

Nnyefe Ihe Nlereanya Akwadoro

Debe ihe nlele ndị ahụ n'ime mmiri nitrogen ruo awa 3-4 ma chekwaa ha n'ime mmiri nitrogen ma ọ bụ -80 degrees ruo mgbe ha ga-adịru ogologo oge. A chọrọ mbupu ihe nlele ahụ na ice kpọrọ nkụ.

Usoro Ọrụ Ọrụ

Nnyefe ihe nlele

Iwu ụlọ akwụkwọ

Usoro nhazi

Nyocha data

Ọrụ mgbe erechara

Nke gara aga: 16S/18S/ITS Usoro Amplicon-NGS

Osote: Nnwale maka Chromatin a na-enweta site na Transposase na usoro nhazi dị elu (ATAC-seq)

Gụnyere nyocha ndị a:

●Njikwa mma data dị mfe

●Ịhazi/Ịbelata mkpọtụ (ASV)

●Nkọwa OTU

●Nyocha nke ụdịdị Alfa: ọtụtụ ndeksi, gụnyere Shannon, Simpson na ACE.

●Nyocha nke ụdị Beta

●Nyocha otu dị iche iche

●Nyocha njikọ: n'etiti ihe ndị metụtara gburugburu ebe obibi na ihe mejupụtara OUT na ụdịdị dị iche iche

●Amụma mkpụrụ ndụ ihe nketa ọrụ nke 16S

Histogram nke nkesa taxonomic

Osisi phylogenetic nkesa obodo

Nyocha nke ụdịdị Alfa: ACE

ndeksi

Nyocha nke ụdị Beta: PCoA

Nyocha otu dị iche iche: ANOVA

Chọpụta ọganihu ndị ọrụ nhazi usoro amplicon nke BMKGene na PacBio mere site na nchịkọta mbipụta ahaziri ahazi.

Gao, X. na Wang, H. (2023) 'Nyocha ntụnyere nke profaịlụ nje na ọrụ Rumen n'oge mgbanwe na usoro dị iche iche (Regreen vs. Grassy) na Alpine Merino Atụrụ nwere usoro uto abụọ n'elu Alpine Meadow', Ịgbacha, 9(1), p. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. na ndị otu ya (2023) 'Ịjide ihe gbara ọchịchịrị nje bacteria n'ime ala ọzara site na iji nyocha nke ihe ndị sitere na omenala na mkpebi dị elu', npj Biofilms and Microbiomes 2023 9:1, 9(1), pp. 1–14. doi: 10.1038/s41522-023-00439-8.

Mu, L. et al. (2022) 'Mmetụta nke nnu abụba na-akpata na njirimara ịgbaze, ụdị nje bacteria na nkwụsi ike aerobic nke silage agwakọtara agwakọta nke e ji alfalfa, ahịhịa osikapa na ọka wit kwadebe', Journal of the Science of Food and Agriculture, 102(4), pp. 1475–1487. doi: 10.1002/JSFA.11482.

Yang, J. et al. (2023) 'Mmekọrịta dị n'etiti ihe nrịbama Oxidative Stress Biomarkers na Gut Microbiota na Mmetụta Antioxidant nke Extracts sitere na Sonchus brachyotus DC. na Oxazolone-Induced Intestinal Oxidative Stress in the Adult Zebrafish', Antioxidants 2023, Mpịakọta 12, Peeji nke 192, 12(1), p. 192. doi: 10.3390/ANTIOX12010192.