16S/18S/ITS Amplicon Sequencing-PacBio
Melemo ea Tšebeletso
● Ho bala nako e telele ho senola tatellano ea bolelele bo felletseng ea 16S/18S/ITS
● Mehala e nepahetseng haholo e nang le tatellano ea mokhoa oa PacBio CCS
● Qeto ea boemo ba mefuta ho tlhaloso ea OTU/ASV
● QIIME2 ea morao-rao e hlahlobang phallo e nang le litlhahlobo tse fapaneng ho latela bobolokelo, litlhaloso, OTU/ASV.
● E sebetsa lithutong tse fapaneng tsa sechaba ka likokoana-hloko
● BMK e na le boiphihlelo bo batsi ka disampole tse fetang 100,000/selemo, e kwahelang mobu, metsi, kgase, seretse, mantle, mala, letlalo, moro o bedisang, dikokonyana, dimela, jj.
● BMKCloud e nolofalitse tlhaloso ea data e nang le lisebelisoa tse 45 tsa ho hlahloba motho ka mong
Litlhaloso tsa Tšebeletso
| TatelanoSethala | Laebrari | Lintlha tse khothalelitsoeng | Nako ea ho fetola |
| PacBio Sequel II | SMRT-tloloko | 5K/10K/20K Ditheke | 44 Matsatsi a ho sebetsa |
Litlhahlobo tsa Bioinformatics
● Taolo ea boleng ba data e tala
● OTU clustering/De-noise(ASV)
● Tlhaloso ea OTU
● Mefuta e sa tšoaneng ea alpha
● Ho fapana ha Beta
● Tlhahlobo ea lihlopha
● Tlhahlobo ea mokhatlo khahlanong le lintlha tsa liteko
● Ho bolela esale pele ka liphatsa tsa lefutso
Litlhoko tsa Mohlala le Phano
Litlhoko tsa Mohlala:
Bakeng saLintlha tsa DNA:
| Mofuta oa Mohlala | Chelete | Ho tsepamisa maikutlo | Bohloeki |
| Lintlha tsa DNA | ＞ 1 μg | ＞ 20 ng/μl | OD260/280= 1.6-2.5 |
Bakeng sa mehlala ea tikoloho:
| Mofuta oa mohlala | Mokhoa o khothaletsoang oa sampole |
| Mobu | Chelete ea mohlala: hoo e ka bang.5 g;Lintho tse setseng tse omeletseng li hloka ho tlosoa holimo;Sila likotoana tse kholo 'me u fete ka har'a filthara ea 2 mm;Lisampole tsa Aliquot ka har'a EP-tube kapa cyrotube bakeng sa ho behelloa. |
| Mantle | Chelete ea mohlala: hoo e ka bang.5 g;Bokella le lisampole tsa aliquot ho EP-tube kapa cryotube e sa sebetseng bakeng sa ho behelloa. |
| Likahare tsa mala | Mehlala e hloka ho sebetsoa tlas'a boemo ba aseptic.Hlatsoa lithane tse bokeletsoeng ka PBS;Hlakola PBS 'me u bokelle precipitant ka EP-tubes. |
| Sludge | Chelete ea mohlala: hoo e ka bang.5 g;Bokella le aliquot sludge sampole ho EP-tube e hloekisitsoeng kapa cryotube bakeng sa ho behelloa |
| Metsi | Bakeng sa sampole e nang le palo e lekanyelitsoeng ea likokoana-hloko, joalo ka metsi a pompo, metsi a seliba, joalo-joalo, Bokella bonyane metsi a 1 L 'me u fete ka 0.22 μm filthara ho ntlafatsa likokoana-hloko tse holim'a lera.Boloka lera ka har'a tube e hloekisitsoeng. |
| Letlalo | Hlakola bokaholimo ba letlalo ka hloko ka swab e hloekisitsoeng ea k'hothone kapa lehare la ho buoa 'me u le kenye ka har'a tube e sa hloekang. |
Tlhahiso ea Mehlala e khothalelitsoeng
Emisa lisampole ka har'a naetrojene e metsi bakeng sa lihora tse 3-4 'me u li boloke ka har'a metsi a naetrojene kapa -80 degree ho boloka nako e telele.Ho hlokahala ho romelloa ka sekepe se nang le leqhoa le omileng.
Phallo ea Mosebetsi oa Ts'ebeletso
Mohlala oa thomello
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
1.Sekhahla sa tlhaloso ea V3 + V4(Illumina)-based microbial community profiling vs. Full-length (PacBio)-based profiling.
(Lintlha tsa merero e 30 e khethiloeng ka mokhoa o sa reroang li sebelisitsoe bakeng sa lipalo-palo)
2. Sekhahla sa tatellano ea amplicon ea bolelele bo felletseng maemong a mefuta ka mefuta e fapaneng ea lisampole.
3.Kabo ea mefuta
4.Sefate sa Phylogenetic
Taba ea BMK
Ho pepeseha ha arsenic ho baka tšenyo e thibelang mala le ho kenya ts'ebetsong ha sebete sa sebete se lebisang ho ruruha le pyroptosis ea sebete ka matata.
E hatisitsoe:Saense ea Kakaretso ea Tikoloho,2021
Leano la tatellano:
Mehlala: Taolo vs 8 mg / kg sehlopha se pepesitsoeng sa ATO
Ho lekanya tlhahiso ea data: 102,583 tatelano e tala ea CCS ka kakaretso
Taolo: 54,518 ± 747 CCS e sebetsang
ATO-e pepesitsoe : 45,050 ± 1675 CCS e sebetsang
Liphetho tsa bohlokoa
Mefuta e fapaneng ea alpha:Ho pepeseha ha ATO ho fetotse haholo bongata ba likokoana-hloko tsa mala le mefuta-futa ea matata.
Tlhahlobo ea Metastats:
Boemong ba phylum: 2 bacterial phyla e fumanoa feela ka lihlopha tsa taolo
Boemong ba leloko: Meloko e 6 e fumanoe e fapane haholo ka bongata
Boemong ba mefuta: mefuta e 36 e ile ea khetholloa ka kakaretso, 'me e 6 ea eona e fapane haholo ka bongata ba tlhaho.
Referense
Thingholm, LB , et al."Batho ba Batenya ba nang le Lefu la tsoekere la Mofuta oa 2 le ba se nang bona ba Bontša Matla le Sebopeho se Fapaneng sa Maleng a Microbial."Cell Host & Microbe26.2(2019).
