METAGENOMICS
Indwara ya bagiteri yuzuye, ifunze mikorobe ikoresheje nanopore ikurikirana
Urukurikirane rwa Nanopore |Metagenomics |MAGs |Indwara ya bagiteri ikwirakwizwa |Gutera microbiota
Ingingo z'ingenzi
1.Uburyo bushya bwo gukuramo ibice birebire bya ADN byatanzwe muri ubu bushakashatsi, bwatanze gukuramo microgramu ya ADN yera, HMW ikwiranye nigihe kirekire cyo gusoma kuva mg 300 zintebe.
2.Mu gikorwa cyo guterana, Lathe, cyatangijwe muri ubu bushakashatsi, aho MAGs zateranijwe no gusoma birebire kandi bigakosorwa nigihe gito.
3.Ibihe byasuzumwe no kuvanga urwenya.7 kuri 12 ya bagiteri yakusanyirijwe hamwe muburyo bumwe naho 3 ikusanyirizwa hamwe muri bane cyangwa nkeya.
4.Lathe yongeye gukoreshwa mubitereko byintebe, byabyaye genome 20 zizunguruka, harimo na prevotella copri hamwe numukandida Cibiobacter sp., byari bizwiho kuba bikize mubintu bigendanwa.
Ibyagezweho
Gukuramo protocole ya ADN ya HWM
Ubushakashatsi bumaze igihe kirekire bushingiye ku nda ya metagenomic ubushakashatsi bumaze igihe kinini bubabazwa no gukuramo ADN ifite uburemere buke (HMW) ADN.Muri ubu bushakashatsi, hashyizweho protocole ishingiye kuri enzyme kugirango yirinde gukata cyane no gukubita amasaro muburyo gakondo.Nkuko bigaragara ku gishushanyo gikurikira, ibyitegererezo byabanje kuvurwa hakoreshejwe cocktail ya enzymes, harimo enzyme ya lytic, MetaPolyzyme, nibindi kugirango bitesha agaciro inkuta za selile.ADN yasohotse yakuwe muri sisitemu ya Phenol-chloroform, ikurikirwa na Proteinase K na RNase A igogora, gushingira ku nkingi no guhitamo ingano ya SPRI.Ubu buryo bwashoboye gutanga microgrammes za ADN ya HMW kuva m 300 zintebe, zuzuza ibisabwa bisomwa igihe kirekire ukurikije ubwiza nubwinshi.
Igicapo 1. Gahunda yo gukuramo ADN ya HWM
Gahunda ya Lathe
Nkuko byasobanuwe mubishushanyo bikurikira, Lathe ikubiyemo inzira ihari ya basecalling progaramu ikoresheje Guppy.Inteko ebyiri zimaze igihe kinini zisomwa noneho zikorwa na Flye na Canu ukundi gukurikirwa no gutahura nabi no gukuraho.Inteko ebyiri-zahujwe hamwe na yihuta.Iyo uhujwe, inteko nini kurwego rwa megabase noneho zigenzurwa kugirango zizenguruke.Ibikurikiraho, kunonosora ibyumvikanyweho kuri izi nteko bitunganywa no gusoma bigufi.Indwara ya bagiteri ya nyuma ikusanyirijwe hamwe kugirango ikorwe nabi kandi ikurweho.
Igishushanyo 2. Gahunda yimigambi yinteko
Isuzuma rya Lathe hamwe na bagiteri ivanze
Ikoreshwa rya ATCC risanzwe ryubwoko 12 rigizwe na Gram-positif na Gram-mbi ya bagiteri yakoreshejwe kugirango isuzume imikorere ya nanopore ikurikirana hamwe na Lathe mu nteko ya MAG.Igiteranyo cya 30.3 Gb cyatanzwe na nanopore platform hamwe na N50 ya 5.9 kb.Ubwiherero bwateje imbere inteko N50 kugeza kuri 1,6 kugeza kuri 4 ugereranije nibindi bikoresho byo gusoma bimaze igihe kirekire hamwe ninshuro 2 kugeza kuri 9 ugereranije nibikoresho byo guteranya hybridd.Muri genoside 12 za bagiteri, zirindwi zateranijwe hamwe (Ishusho 3. Circos ifite akadomo kirabura).Ibindi bitatu byakusanyirijwe mu matsinda ane cyangwa make, aho inteko ituzuye yarimo 83% ya genome mugice kimwe.
Igicapo 3. Inteko za genome zisobanuwe zubwoko 12 bwavanze na bagiteri
Gukoresha Lathe mucyitegererezo
Ubu buryo bwakoreshejwe no mubitereko byabantu kugirango bagereranye ibinyabuzima hamwe no guterana hamwe nuburyo buriho, gusoma-ibicu hamwe nisesengura rito-rishingiye.Uhereye kuri ubwo buryo butatu burimo, gushya gushya gushingiye kuri enzyme byatanze byibuze 1 μg kuri 300 mg yinjiza.Nanopore ikurikirana ya ADN ya HMW yabyaye gusoma-hamwe na N50 ya 4.7 kb, 3.0kb na 3.0kb.Ikigaragara ni uko ubu buryo bwerekanye imbaraga zikomeye mugutahura mikorobe ugereranije nuburyo buriho.Ugereranije amoko yo murwego rwohejuru ya alpha yerekanwe hano ugereranije no gusoma-gusoma no gusoma-igicu.Byongeye kandi, genera zose zivuye mubisesengura-bigufi, ndetse mubisanzwe ibinyabuzima birwanya Gram-positif, byagarutsweho nubu buryo.
Igicapo 4. Ibinyuranyo bya Alpha nibice bya tagisi byagenwe na Nanopore, gusoma-bigufi no gusoma-bicu
Umusarani watanze igihe kirekire-inteko N50 kuruta gusoma-gusoma no gusoma-igicu, nubwo inshuro eshatu kugeza kuri esheshatu zinjiza amakuru yibanze.Draft genomes yakozwe na contig binning, aho ibishushanyo byashyizwe mubice "byiza-byiza" cyangwa "igice" bishingiye kubwuzuye, kwanduza, ingirabuzimafatizo imwe, nibindi. Kuri Bigufi-Gusoma no Gusoma-Igicu.
Igicapo 5. Iteraniro ryibinyabuzima kuri buri buryo
Byongeye kandi, uburyo bwo guterana ubu bushobora gutanga genome zifunze, zizunguruka.Mu byitegererezo by'intebe, umunani zujuje ubuziranenge, genoside imwe ihuriweho hamwe na bitanu muri byo byagezweho.Uburyo bwasomwe burigihe kandi bwerekanye ubushobozi butangaje mugukemura ibintu bisubiramo muri genome.KuzengurukaP. coprigenome yakozwe nubu buryo, buzwiho kuba burimo urwego rwo hejuru rusubiramo.Iteraniro ryiza ryiyi genome ukoresheje gusoma-gusoma no gusoma-igicu nticyigeze kirenga N50 ya 130 kb, nubwo gifite uburebure bwa 4800X.Izi kopi ndende yibintu byakemuwe byuzuye nuburyo bwo gusoma-burigihe, wasangaga akenshi bumena ingingo zo gusoma-gusoma cyangwa gusoma-ibicu.Indi genome ifunze yavuzwe muri ubu bushakashatsi, ikekwa ko ari umunyamuryango uherutse gusobanurwaCibiobacterclade.Ibyiciro bitanu byashyizwe ahagaragara muriyi nteko ifunze, kuva kuri 8.5 kugeza kuri 65.9 kb.
Igicapo 6. Igishushanyo mbonera cya genoside zifunze za P.copri na Cibiobacter sp.
Reba
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Indwara ya bagiteri yuzuye, ifunze mikorobe ikoresheje nanopore ikurikirana.Kamere y'ibinyabuzima,38(6), 701-707.
Ikoranabuhanga hamwe nibyingenzi igamije gusangira ibyagezweho vuba muburyo butandukanye bwo gukoresha tekinoroji ikurikirana muburyo butandukanye hamwe nibitekerezo byiza mugushushanya no gucukura amakuru.
Igihe cyo kohereza: Mutarama-07-2022