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16S/18S/ITS Amplicon Sequencing-PacBio

Mkpụrụ ndụ ihe nketa 16S na 18S rRNA, yana mpaghara Internal Transcribed Spacer (ITS), na-eje ozi dị ka akara akara mkpịsị aka mkpụrụ ndụ ihe dị mkpa n'ihi nchikota ha nke mpaghara echekwara nke ukwuu yana hyper-agbanwe agbanwe, na-eme ka ha bụrụ ngwa bara uru maka ịkọwapụta ihe dị ndụ prokaryotic na eukaryotic. Mmụba na usoro nke mpaghara ndị a na-enye ụzọ na-enweghị ikewapụ maka nyocha ihe mejupụtara microbial na ụdị dị iche iche n'ofe gburugburu ebe obibi dị iche iche. Ọ bụ ezie na usoro Illumina na-elekwasịkarị anya mpaghara hypervariable dị mkpụmkpụ dị ka V3-V4 nke 16S na ITS1, egosipụtara na nkọwa taxonomic dị elu bụ nke a ga-enweta site n'ịhazi ogologo 16S, 18S na ITS. Ụzọ a zuru oke na-ebute pasentị dị elu nke usoro nkewa nke ọma, na-enweta ọkwa nke mkpebi na-agbatị ruo n'ichepụta ụdị. PacBio's Single-Molecule Real-Time (SMRT) usoro usoro ihe omume pụtara ìhè site n'inye ogologo ọgụgụ dị ogologo (HiFi) nke na-ekpuchi amplicon ogologo zuru oke, na-emegide izi ezi nke usoro Illumina. Ikike a na-enye ndị na-eme nchọpụta ohere inweta uru na-enweghị atụ - echiche panoramic nke ọdịdị mkpụrụ ndụ ihe nketa. Mkpuchi agbatịkwuru na-ebuli mkpebi a dị na nkọwapụta ụdịrị, ọkachasị n'ime obodo nje ma ọ bụ fungal, na-eme ka nghọta miri emi banyere mgbagwoju anya nke ụmụ nje.

Kpọtụrụ anyị

Nkọwa ọrụ

Bioinformatics

Nsonaazụ ngosi

Akwụkwọ ndị egosipụtara

Atụmatụ ọrụ

● Usoro nhazi usoro: PacBio Revio

● Usoro usoro: CCS (HiFi na-agụ)

● Mmụba nke mpaghara ebumnuche sochiri njikọ tandem nke amplicon tupu nkwadebe ọba akwụkwọ mgbịrịgba HiFi SMRT.

Uru ọrụ

●Mkpebi taxonomic dị elu: Than short-amplicon usoro,na-eme ka ọnụego nhazi OTU dị elu na ọkwa ụdị.

●Oku ntọala ziri ezi nke ukwuuUsoro usoro PacBio CCS (HiFi na-agụ).

●Enweghị ikewapụ onwe ya: Nchọpụta ngwa ngwa nke ihe mejupụtara microbial na ihe atụ gburugburu ebe obibi.

●Ọdabara nke ukwuu: Ọmụmụ obodo dị iche iche microbial.

●Nyocha bioinformatic zuru oke: Ngwungwu QIIME2 kachasị ọhụrụ (nghọta ọnụ ọgụgụ nke microbial ecology) nwere nyocha dị iche iche n'ihe gbasara nchekwa data, nkọwa, OTU/ASV.

●Ọkachamara sara mbara: Site na puku kwuru puku amplicon sequencing oru ngo a na-eme kwa afọ, BMKGENE na-eweta ahụmahụ afọ iri, otu nyocha nke nwere nkà dị ukwuu, ọdịnaya zuru oke, na nkwado ahịa ahịa dị mma.

Nkọwapụta ọrụ

Ụlọ akwụkwọ	Usoro usoro	akwadoro data
Amplicon	PacBio Revio	10/30/50 K mkpado (CCS)

Ihe atụ chọrọ

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

Nnyefe nlele akwadoro

Kpochapụ ihe nlele ahụ na nitrogen mmiri mmiri maka awa 3-4 ma chekwaa na nitrogen mmiri mmiri ma ọ bụ -80 ogo na ndoputa ogologo oge. Achọrọ mbupu ihe atụ na ice-akọrọ.

Usoro ọrụ ọrụ

Nbufe ihe atụ

Ịrụ ụlọ akwụkwọ

Usoro

Nyocha data

Mgbe-ire ọrụ

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

Osote: Nyocha maka ntụgharị-Chromatin nwere ike ịnweta ya na usoro ntinye dị elu (ATAC-seq)

Ọ gụnyere nyocha ndị a:

● Njikwa ogo data raw

●OTU ụyọkọ/De-noise(ASV)

●OTU nkọwa

●Ntụle dị iche iche nke Alpha: ọtụtụ indexes, gụnyere Shannon, Simpson na ACE.

●Beta diversity analysis

●Nyocha n'etiti otu

●Ntụle njikọ: n'etiti ihe gbasara gburugburu ebe obibi na OUT mejupụtara na iche iche

●16S amụma mkpụrụ ndụ ihe nketa

Histogram nke nkesa taxonomic

Osisi phylogenetic nkesa obodo

Nyocha Alfa dị iche iche: ACE

index

Nyocha ụdị dị iche iche beta: PCoA

Nyocha otu: ANOVA

Nyochaa ọganihu nke BMKGene's amplicon sequencing ọrụ na PacBio kwadoro site na nchịkọta mbipụta.

Gao, X. na Wang, H. (2023) 'Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to different Phenology (Regreen vs. Grassy) na Alpine Merino Sheep with Two Growing Stages on Alpine Meadow', Fermentation, 9 1), p. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. et al. (2023) 'iji were metagenomics dabere na culturomics na nyocha dị elu', npj Biofilms na Microbiomes 2023 9:1, 9(1), p. 1–14. doi: 10.1038/s41522-023-00439-8.

M, L. et al. (2022) 'Mmetụta nke fatty acid salts on gbaa ụka e ji mara, nje di iche iche na aerobic kwụsie ike nke gwakọtara silage kwadebere na alfalfa, osikapa ahịhịa na ọka wit bran', Journal of the Science of Food and Agriculture, 102(4), p. 1475- 1487. doi: 10.1002/JSFA.11482.

Yang, J. et al. (2023) 'Mmekọrịta dị n'etiti Oxidative Stress Biomarkers na Gut Microbiota na Mmetụta Antioxidant nke Mwepụta sitere na Sonchus brachyotus DC. na Nchegbu Oxidative Intestinal Oxazolone kpalitere na Zebrafish okenye', Antioxidants 2023, Vol. 12, Peeji nke 192, 12 (1), p. 192. doi: 10.3390/ANTIOX12010192.