Ngwaahịa

16S/18S/ITS Amplicon Sequencing-PacBio

Nke a dị na 16S na 18S rRNA nwere ma mpaghara echekwara nke ukwuu yana hyper-variable bụ akara mkpisiaka mkpụrụ ndụ zuru oke maka njirimara prokaryotic na eukaryotic.Na-ewere uru nke sequencing, ndị a amplicons nwere ike ezubere iche dabere na ndị echekwara akụkụ na hyper-variable mpaghara nwere ike n'ụzọ zuru ezu ji maka microbial njirimara na-enye aka ọmụmụ ekpuchi microbial diversity analysis, taxonomy, phylogeny, wdg Single-molecule ozugbo (SMRT) ) usoro nke PacBio ikpo okwu na-enyere aka inweta nke ukwuu ziri ezi ogologo agụ, nke nwere ike na-ekpuchi zuru-ogologo amplicon (ihe dị ka 1.5 Kb).Echiche sara mbara nke mkpụrụ ndụ ihe nketa mere ka mkpebi siri ike dị na nkọwa ụdịdị na nje bacteria ma ọ bụ fungi.

Platform:PacBio Sequel II

Kpọtụrụ anyị

Nkọwa ọrụ

Nsonaazụ ngosi

Ọmụmụ ihe

Uru ọrụ

● Ọgụgụ ogologo na-ekpughe usoro ogologo nke 16S/18S/ITS

● Oku ntọala zuru oke na usoro PacBio CCS

● Mkpebi ọkwa ọkwa dị iche iche na nkọwa OTU/ASV

● Ntụle QIIME2 kacha ọhụrụ nwere nyocha dị iche iche n'ihe gbasara nchekwa data, nkọwa, OTU/ASV.

● Ọdabara maka ọmụmụ ihe ọmụmụ obodo dị iche iche

● BMK nwere ọtụtụ ahụmahụ na n'elu 100,000 samples / afọ, ekpuchi ala, mmiri, gas, sludge, feces, eriri afọ, akpụkpọ, gbaa ụka efere, ụmụ ahụhụ, osisi, wdg.

● BMKCloud kwadoro nkọwa data nwere ngwaọrụ nyocha 45 ahaziri iche

Nkọwapụta ọrụ

UsoroPlatform	Ụlọ akwụkwọ	Data akwadoro	Oge ntụgharị
PacBio Sequel II	SMRT-mgbịrịgba	5K/10K/20K mkpado	44 ụbọchị ọrụ

Nyocha bioinformatics

● Njikwa ogo data raw

● Ụyọkọ OTU/De-noise(ASV)

● Nkọwa OTU

● Alfa dị iche iche

● Ụdị beta dị iche iche

● Nyocha n'etiti otu

● Nyocha mkpakọrịta megide ihe nnwale

● Amụma mkpụrụ ndụ ihe nketa ọrụ

Ihe nlele chọrọ na nnyefe

Ihe atụ chọrọ:

MakaDNA wepụ:

Ụdị Nlereanya	Ọnụ ego	Ntinye uche	Ịdị ọcha
DNA wepụ	1 μg	20 ng/μl	OD260/280 = 1.6-2.5

Maka ihe atụ gburugburu ebe obibi:

Ụdị ihe atụ	Usoro nlele akwadoro
Ala	Ọnụ ego nlele: ihe ruru.5 g;Ọ dị mkpa ka ewepụ ihe fọdụrụ akpọnwụ n'elu;Ghichaa nnukwu iberibe ma gafee nzacha 2 mm;Ihe nlele Aliquot na EP-tube ma ọ bụ cyrotube na-adịghị mma maka ndoputa.
Mpekere	Ọnụ ego nlele: ihe ruru.5 g;Chịkọta ma were ihe atụ na EP-tube ma ọ bụ cryotube na-adịghị mma maka ndoputa.
Ihe dị n'ime eriri afọ	Ekwesịrị ịhazi ihe nlele n'okpuru ọnọdụ aseptic.Jiri PBS saa anụ ahụ anakọtara;Centrifuge PBS ma kpokọta ihe na-asọpụta na EP-tube.
Sludge	Ọnụ ego nlele: ihe ruru.5 g;Chịkọta ma were ihe nlele sludge dị na EP-tube ma ọ bụ cryotube na-adịghị mma maka ndoputa
Mmiri mmiri	Maka ihe nlele nwere obere obere microbial, dị ka mmiri mgbata, mmiri olulu mmiri, wdg, Na-anakọta ma ọ dịkarịa ala mmiri 1 L wee gafee nzacha 0.22 μm iji mee ka microbial dị na akpụkpọ ahụ.Chekwaa akpụkpọ ahụ na tube na-adịghị mma.
Akpụkpọ anụ	Jiri nlezianya hichaa elu akpụkpọ ahụ site na iji swab na-adịghị mma ma ọ bụ agụba ịwa ahụ ma tinye ya na tube na-adịghị mma.

Nnyefe nlele akwadoro

Debe 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)

1.Nkọwa ọnụ ọgụgụ nke V3 + V4 (Illumina) dabeere microbial obodo profiling vs. Full-ogologo (PacBio) dabeere profaịlụ.
(Edebere data nke ọrụ 30 na-akpaghị aka maka ọnụ ọgụgụ)

2.Annotation ọnụego nke zuru-ogologo amplicon sequencing na ụdị-ọkwa dị iche iche sample ụdị.

3.Species nkesa

4.Phylogenetic osisi

Okwu BMK

Mkpughe nke arsenic na-ebute mmebi ihe mgbochi eriri afọ na n'ihi ya ịgbalite axis gut-imeju na-eduga na mbufụt na pyroptosis nke imeju na ọbọgwụ.

Ebipụtara:Sayensị nke mkpokọta gburugburu ebe obibi,2021

Atụmatụ usoro:

Ihe atụ: Control vs 8 mg/kg ATO kpughere otu
Usoro ntinye data: 102,583 raw CCS usoro na mkpokọta
Njikwa: 54,518 ± 747 CCS dị irè
ATO-ekpughere: 45,050 ± 1675 CCS dị irè

Nsonaazụ igodo

Ọdịiche Alfa:Mkpughe ATO gbanwere nke ukwuu ụbara microbial nke eriri afọ na ụdị dị iche iche dị na ọbọgwụ.

Nyocha metastats:
N'ọkwa phylum: phyla nje bacteria 2 ka ahụrụ naanị n'ime otu njikwa
Na genus larịị: 6 genera ka a hụrụ dị nnọọ iche na ikwu n'ụba
N'ọkwa ụdị: Achọpụtara ụdị 36 n'ozuzu ya, ebe 6 n'ime ha dị iche na nke ukwuu

Ntụaka

Thingholm, LB, et al."Ndị buru oke ibu nwere na ndị na-enweghị ụdị ọrịa shuga 2 na-egosi ike na ihe mejupụtara gut microbial dị iche iche."Cell Host & Microbe26.2 (2019).