16S/18S/ITS Amplicon Sequencing-PacBio
Izinzuzo Zesevisi
● Ukufunda isikhathi eside kuveza ukulandelana kobude obugcwele be-16S/18S/ITS
● Ukushaya kwesisekelo okunembe kakhulu okulandelanayo kwemodi ye-PacBio CCS
● Ukulungiswa kwezinga lezinhlobo kusichasiselo se-OTU/ASV
● Ukugeleza kokuhlaziya kwe-QIIME2 kwakamuva nokuhlaziya okuhlukahlukene ngokuya kusizindalwazi, isichasiselo, i-OTU/ASV.
● 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 | Idatha enconyiwe | Isikhathi sokushintsha |
| I-PacBio Sequel II | Insimbi ye-SMRT | 5K/10K/20K Omaka | 44 Izinsuku zokusebenza |
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 | ＞ 1 μg | ＞ 20 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.Isilinganiso sesichasiselo se-V3+V4(Illumina)-based microbial community profileing vs. Full-length (PacBio)-based profileing.
(Idatha yamaphrojekthi angu-30 akhethwe ngokungahleliwe afakwe kwizibalo)
2.Isilinganiso sesichasiselo sokulandelana kwe-amplicon yobude obugcwele ezingeni lezinhlobo zezinhlobo zesampuli ezihlukene
3.Ukusatshalaliswa kwezinhlobo zezilwane
4.Isihlahla se-Phylogenetic
Icala le-BMK
Ukuchayeka kwe-arsenic kubangela ukulimala kwesithiyo samathumbu kanye nokwenza kusebenze i-axis yesibindi se-gut okuholela ekuvuvukeni kanye ne-pyroptosis yesibindi kumadada.
Kushicilelwe:Isayensi Yendawo Yonke,2021
Isu lokulandelanisa:
Amasampuli: Ukulawula vs 8 mg/kg iqembu eliveziwe le-ATO
Ilandelanisa isivuno sedatha: 102,583 ukulandelana kwe-CCS eluhlaza sekukonke
Ukulawula: 54,518 ± 747 CCS esebenzayo
I-ATO-eveziwe : 45,050 ± 1675 i-CCS esebenzayo
Imiphumela esemqoka
Ukuhlukahluka kwe-Alpha:Ukuchayeka kwe-ATO kushintshe kakhulu ukunotha kwamagciwane emathunjini kanye nokwehluka kwamadada.
Ukuhlaziywa kwe-Metastats:
Ezingeni le-phylum: I-bacterial phyla engu-2 itholakala kuphela emaqenjini okulawula
Ezingeni lohlobo: Izinhlobo eziyisi-6 zitholwe zihluke kakhulu ngobuningi obuhlobene
Ngokwezinga lezinhlobo zezilwane: izinhlobo ezingama-36 zikhonjwe sezizonke, eziyisi-6 zazo zihluke kakhulu ngobuningi bokuphila.
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).
