16S/18S/ITS Amplicon Sequencing-NGS
Izinzuzo Zesevisi
● Ukuhlonza okungahlukanisiwe nokusheshayo kokwakheka kwamagciwane kumasampuli emvelo
● Ukulungiswa okuphezulu ezingxenyeni ezinenala ephansi kumasampuli emvelo
● Ukugeleza kokuhlaziya kwe-QIIME2 kwakamuva nokuhlaziya okuhlukahlukene ngokuya kusizindalwazi, isichasiselo, i-OTU/ASV.
● Ukusebenza okuphezulu, ukunemba okuphezulu
● Kusebenza ezifundweni ezihlukahlukene zomphakathi ezincane
● I-BMK inokuhlangenwe nakho okubanzi okunamasampula angaphezu kuka-100,000/ngonyaka, emboza inhlabathi, amanzi, igesi, udaka, indle, amathumbu, isikhumba, umhluzi obilayo, izinambuzane, izitshalo, njll.
● I-BMKCloud iqondise ukuhunyushwa kwedatha equkethe amathuluzi okuhlaziya omuntu angama-45
Imininingwane Yesevisi
| UkulandelanisaInkundla | Umtapowolwazi | Inzuzo yedatha enconyiwe | Isikhathi sokujika esilinganiselwe |
| I-Illumina NovaSeq Platform | I-PE250 | 50K/100K/300K Omaka | Izinsuku ezingama-30 |
I-Bioinformatics ihlaziya
● Ukulawulwa kwekhwalithi yedatha eluhlaza
● I-OTU clustering/De-noise(ASV)
● Isichasiselo se-OTU
● Ukuhlukahluka kwe-alpha
● Ukuhlukahluka kwe-Beta
● Ukuhlaziya kwamaqembu
● Ukuhlaziywa kwenhlangano ngokumelene nezici zokuhlola
● Isibikezelo sofuzo lomsebenzi
Izidingo Zesampula Nokulethwa
Izimfuneko zesampula:
NgobaIzingcaphuno ze-DNA:
| Uhlobo lwesampula | Inani | Ukugxila | Ubumsulwa |
| Izingcaphuno ze-DNA | ＞ 30 ng | ＞ 1 ng/μl | OD260/280= 1.6-2.5 |
Ngamasampuli emvelo:
| Uhlobo lwesampula | Inqubo yokusampula enconyiwe |
| Umhlabathi | Inani lesampula: cishe.5 g;Izinto ezisele ezibunile zidinga ukususwa phezulu;Gaya izingcezu ezinkulu futhi udlule ngesihlungi esingu-2 mm;Amasampuli e-Aliquot ku-EP-tube eyinyumba noma i-cyrotube ukuze abhukhe. |
| Indle | Inani lesampula: cishe.5 g;Qoqa futhi amasampula e-aliquot ku-EP-tube eyinyumba noma i-cryotube ukuze ubhukhe. |
| Okuqukethwe kwamathumbu | Amasampula adinga ukucutshungulwa ngaphansi kwesimo se-aseptic.Geza izicubu eziqoqiwe nge-PBS;I-Centrifuge i-PBS futhi uqoqe i-precipitant kumashubhu e-EP. |
| Udaka | Inani lesampula: cishe.5 g;Qoqa futhi isampula ye-aliquot sludge ku-EP-tube eyinyumba noma i-cryotube ukuze ubhukhe |
| Amanzi | Ukuze uthole isampula enenani elilinganiselwe le-microbial, njengamanzi kampompi, amanzi omthombo, njll., Qoqa okungenani amanzi angu-1 L futhi udlule kusihlungi esingu-0.22 μm ukuze unothise i-microbial kulwelwesi.Gcina ulwelwesi kushubhu oyinyumba. |
| Isikhumba | Hlanza ngokucophelela indawo yesikhumba ngeswabhu kakotini oyinyumba noma i-blade yokuhlinza bese uyibeka eshubhuni eliyinyumba. |
Ukulethwa Kwesampula Okunconyiwe
Friza amasampula ku-nitrogen ewuketshezi amahora angu-3-4 futhi uwagcine ku-nitrogen ewuketshezi noma -80 degree ekubhukeni isikhathi eside.Kudingeka isampula yokuthunyelwa ngeqhwa elomile.
Ukugeleza Komsebenzi Wesevisi
Ukulethwa kwesampula
Ukwakhiwa komtapo wolwazi
Ukulandelanisa
Ukuhlaziywa kwedatha
Amasevisi angemuva kokuthengisa
1.Ukusabalalisa kwezinhlobo zezilwane
2.Imephu yokushisa: Ukuhlanganisa ukunotha kwezinhlobo zezilwane
3.Ijika leqembu elingavamile
4.Ukuhlaziywa kwe-NMDS
5.I-Lefse analysis
Icala le-BMK
Abantu abakhuluphele abanesifo sikashukela sohlobo 2 nabangenalo babonisa amandla nokusebenza okuhlukile kwe-microbial yamathumbu
Kushicilelwe:I-Cell Host & Microbe, 2019
Isu lokulandelanisa:
I-Lean non-diabetes (n=633);Ukukhuluphala ngokweqile okungezona ushukela (n=494);Isifo sikashukela sohlobo 2 ezikhuluphele (n=153);
Isifunda okuhloswe ngaso: 16S rDNA V1-V2
I-Platform: I-Illumina Miseq (ukulandelana kwe-amplicon okusekelwe ku-NGS)
Isethi engaphansi yokukhishwa kwe-DNA ibe ngaphansi kokulandelana kwe-metagenomic ku-Illumina Hiseq
Imiphumela esemqoka
Iphrofayili ye-microbial yalezi zifo ze-metabolic zehlukaniswa ngempumelelo.
Ngokuqhathanisa izici ze-microbial ezikhiqizwa ukulandelana kwe-16S, ukukhuluphala kwatholakala ukuthi kuhlotshaniswa nezinguquko ekubunjweni kwamagciwane, izici zomuntu ngamunye, ukwehla okuphawulekayo ikakhulukazi ku-Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, njll. Ngaphezu kwalokho, i-T2D yatholakala ihlotshaniswa nokwanda kwe-Escherichia/shigella. .
Ireferensi
Thingholm, LB , et al."Abantu abakhuluphele abanesifo sikashukela sohlobo 2 nabangenaso babonisa amandla nokusebenza okuhlukile kwe-Gut Microbial Functional."I-Cell Host & Microbe26.2(2019).
