16S/18S/ITS Amplicon Sequencing-NGS
Uru ọrụ
● Nchọpụta enweghị ikewapụ na ngwa ngwa nke ihe mejupụtara microbial na nlele gburugburu
● Mkpebi dị elu na ihe ndị dị ala na-aba ụba na ihe atụ gburugburu ebe obibi
● Ntụle QIIME2 kacha ọhụrụ nwere nyocha dị iche iche n'ihe gbasara nchekwa data, nkọwa, OTU/ASV.
● Ntinye dị elu, izi ezi dị elu
● 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 | Mmụba data akwadoro | Atụmatụ ngbanwe oge |
| Illumina NovaSeq Platform | PE250 | 50K/100K/300K Tags | Ụbọchị 30 |
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 | 30 nng | 1 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
1.Species nkesa
2.Okpomọkụ map: Ụdị akụ na ụba nke ụyọkọ
3.Rare akụkụ okirikiri
4.NMDS nyocha
5.Lefse nyocha
Okwu BMK
Ndị nwere oke ibu nwere yana enweghị ụdị ọrịa shuga 2 na-egosi ikike ịrụ ọrụ microbial dị iche iche na nhazi.
Ebipụtara:Onye ọbịa Cell & Microbe, 2019
Atụmatụ usoro:
Ọrịa shuga na-adịghị ala ala (n=633);Oke ibu na-abụghị ọrịa shuga (n=494);Obese-Ụdị ọrịa shuga 2 (n=153);
Mpaghara ebumnuche: 16S rDNA V1-V2
Platform: Illumina Miseq (usoro amplicon dabere na NGS)
A na-edobere akụkụ nke mmịpụta DNA na usoro metagenomic na Illumina Hiseq
Nsonaazụ igodo
Achọpụtara profaịlụ microbial nke ọrịa metabolic ndị a nke ọma.
Site n'iji njirimara microbial emepụtara site na usoro 16S, a hụrụ oke ibu na-ejikọta ya na mgbanwe nke microbial mejupụtara, njirimara onye ọ bụla, karịsịa mbelata dị ukwuu na Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, wdg. Na mgbakwunye, a hụrụ T2D jikọtara ya na mmụba nke Escherichia / shigella. .
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).
