I-METAGENOMICS
Ama-genome ebhaktheriya aphelele, avaliwe avela kuma-microbiomes esebenzisa ukulandelana kwe-nanopore
I-Nanopore Sequencing |I-Metagenomics |IZITHOMBE |I-bacterium genome circularization |Amathumbu microbiota
Amaphuzu avelele
1. Indlela yenoveli yokukhipha izingcezu ezinde ze-DNA yethulwe kulolu cwaningo, eyazuza ukukhishwa kwe-microgram ye-pure, i-HMW DNA elungele ukulandelana okufunde isikhathi eside kusuka ku-300 mg wesihlalo.
I-2.Ukuhamba komsebenzi womhlangano, i-Lathe, yethulwa kulolu cwaningo, lapho ama-MAG ahlanganiswa ngokufundwa okude futhi alungiswa ngokufundwa okufushane.
3.I-Lathe yahlolwa ngengxube mbumbulu.Amagciwane ayi-7 kwayi-12 ahlanganiswe ngempumelelo aba yi-contigs eyodwa futhi ama-3 ahlanganiswa aba ama-contigs amane noma ngaphansi.
4.I-Lathe yaphinde yasetshenziswa kumasampula endle, akhiqiza ama-genome ayindilinga angama-20, okuhlanganisa i-Prevotella copri kanye nekhandidethi elithi Cibiobacter sp., ezazaziwa ngokucebile kuzakhi zofuzo ezihambayo.
Impumelelo Eyinhloko
Iphrothokholi yokukhipha ye-HWM DNA
Izifundo ze-metagenomic ezisekelwe ku-gut ezifundwe isikhathi eside sekuyisikhathi eside zihlushwa ubulukhuni ekukhipheni isisindo samangqamuzana aphezulu (HMW) DNA esitokisini.Kulolu cwaningo, i-enzyme-based extraction protocol yethulwa ukuze kugwenywe ukugunda okubanzi ngokushaya ubuhlalu ngezindlela zendabuko.Njengoba kuboniswe esithombeni esilandelayo, amasampula aqale aphathwa nge-cocktail yama-enzyme, okuhlanganisa i-lytic enzyme, i-MetaPolyzyme, njll.I-DNA ekhishiwe yakhishwa ngohlelo lwe-Phenol-chloroform, lulandelwa yi-Proteinase K ne-RNase A digestion, ukuhlanzwa okusekelwe kukholomu kanye nokukhethwa kosayizi we-SPRI.Le ndlela ikwazile ukukhiqiza ama-micrograms e-HMW DNA ukusuka ku-300 m wesihlalo, esifeza izimfuneko zokulandelana ezifundwe isikhathi eside ngokokubili ikhwalithi kanye nenani.
Umfanekiso 1. HWM DNA extraction scheme
Ukugeleza kweSkimu seLathe
Njengoba kuchazwe esithombeni esilandelayo, i-Lathe iqukethe inqubo ekhona yenqubo ye-basecalling eluhlaza kusetshenziswa i-Guppy.Ama-assemblies amabili afunde isikhathi eside abe esekhiqizwa i-Flye ne-Canu ngokuhlukana okulandelwa ukutholwa nokususwa kwe-mis-assembly.Ama-sub-assemblies amabili ahlanganiswe nokuhlanganisa okusheshayo.Lapho ehlanganiswa, ama-assemblies amakhulu ezingeni le-megabase abe esehlolelwa ukuzungeza.Kamuva, ukulungiswa kokuvumelana kule mihlangano kucubungula ngokufundwa okufushane.Ama-genome ebhaktheriya aqoqiwe ekugcineni acutshungulwa ukuze kutholwe futhi kususwe i-mis-assembly yokugcina.
Umfanekiso 2. Ukugeleza kwesu lomhlangano weLathe
Ukuhlolwa kweLathe ngengxube yebhaktheriya mbumbulu
Ingxube evamile ye-ATCC 12-species ehlanganisa kokubili amagciwane e-Gram-positive kanye ne-Gram-negative yasetshenziswa ukuze kuhlolwe ukusebenza kweplathifomu yokulandelana kwe-nanopore kanye ne-Lathe ekuhlanganiseni kwe-MAG.Ingqikithi yedatha engu-30.3 Gb yenziwe inkundla ye-nanopore ene-N50 ye-5.9 kb.I-Lathe ithuthukise kakhulu ukuhlanganiswa kwe-N50 kuya ku-1.6 kuya kwezingu-4 uma kuqhathaniswa namanye amathuluzi omhlangano afundeke isikhathi eside kanye nokuphindwe kabili kuya kweziyi-9 uma kuqhathaniswa namathuluzi okuhlanganisa ayingxube.Kuma-genome ebhaktheriya ayi-12, ayisikhombisa ahlanganiswe abe yi-contigs eyodwa(Umfanekiso 3. Ama-Circo anechashazi elimnyama).Ezinye ezintathu zahlanganiswa zaba ama-contig amane noma ambalwa, lapho umhlangano ongaphelele wawuqukethe u-83% we-genome ku-contig eyodwa.
Umfanekiso 3. Ama-genome assemblies engxubeni yebhaktheriya yezinhlobo eziyi-12 echaziwe
Ukusetshenziswa kwe-Lathe kumasampuli esitulo
Le ndlela yaphinde yasetshenziswa kumasampula endle yomuntu ukuze kuqhathaniswe ukuhlonzwa kwento ephilayo kanye nokuhlangana komhlangano nezindlela ezikhona, ifu lokufunda nokuhlaziya okusekelwe ekufundeni okufushane.Kusukela kumasampuli amathathu ahilelekile, isizinda esisha esisekelwe ku-enzyme sikhiqize okungenani i-1 μg ngo-300 mg wesisindo sokufaka.Ukulandelana kwe-Nanopore kwale HMW DNA kukhiqize ukufunda okude nge-N50 ye-4.7 kb, 3.0kb kanye no-3.0kb ngokulandelanayo.Ngokuphawulekayo, indlela yamanje ibonise amandla amakhulu ekutholweni kwe-microbial uma kuqhathaniswa nezindlela ezikhona.Ukuhlukahluka kweleveli yezinhlobo zezinhlobo okuphezulu ngokuqhathaniswa kuboniswe lapha uma kuqhathaniswa nefu elifushane elifundwayo nelifundwayo.Ngaphezu kwalokho, zonke izinhlobo ezivela ekuhlaziyweni okufushane, ngisho nezinto eziphilayo ezikwazi ukumelana ne-lysis ze-Gram-positive, zatholwa ngale ndlela.
Umfanekiso 4. Ukuhlukahluka kwe-alpha kanye nezingxenye ze-taxonomic ezinqunywa i-Nanopore, izindlela zokufunda okufushane nokufunda-amafu
I-Lathe ikhiqize i-N50 ye-all-assembly ende kakhulu kunomhlangano wamafu ofundeka kancane futhi ofundwe, naphezu kokufaka okuphansi okuphindwe kathathu kuya kasithupha kwedatha eluhlaza.Ama-genomes asalungiswa akhiqizwa i-contig binning, lapho okusalungiswa kwahlukaniswa kwaba “ikhwalithi ephezulu” noma “ingxenye” ngokusekelwe ekupheleleni, ekungcolisweni, kuzakhi zofuzo zekhophi eyodwa, njll. Ukuhlanganiswa okufundwe isikhathi eside kuboniswe ukuhambisana okuphezulu kakhulu ngezindleko eziphansi uma kuqhathaniswa. ukufunda okufushane nokufunda-ifu.
Umfanekiso 5. Ukuhlangana kokuhlangana kwento ngayinye yendlela ngayinye
Ngaphezu kwalokho, indlela yokuhlanganisa yamanje iyakwazi ukukhiqiza ama-genome avaliwe, ayindilinga.Kumasampula ezihlalo, ama-genomes ayisishiyagalombili ekhwalithi ephezulu, e-single-contig aqoqwa futhi amahlanu alawa azuza ukujikeleza kwe-percise.Indlela yokufunda isikhathi eside iphinde yabonisa umthamo omangalisayo ekuxazululeni izici eziphindaphindayo kuma-genome.IseyingiP. coprii-genome yenziwa yile ndlela, eyaziwa ukuthi iqukethe izinga eliphezulu lokuphindaphinda ukulandelana.Ukuhlanganiswa okuhamba phambili kwalolu genome ngokufunda-ifu okufushane futhi okufundwe akuzange kudlule i-N50 ye-130 kb, ngisho nokujula kokufakwa okungu-4800X.Lezi zakhi zenombolo yekhophi ephezulu zaxazululwa ngokugcwele ngendlela yokufunda isikhathi eside, evame ukutholakala ezindaweni ezinqamulayo zemihlangano yokufunda okufushane noma efundwayo.Olunye uhlobo lofuzo oluvaliwe lubikwe kulolu cwaningo, okukholakala ukuthi luyilungu lokuchazwa muva njeI-Cibiobacterclade.I-putive phage emihlanu ikhonjwe kulo mhlangano ovaliwe, osukela ku-8.5 kuya ku-65.9 kb.
Umfanekiso 6. Umdwebo we-Circos wama-genomes avaliwe we-P.copri ne-Cibiobacter sp.
Ireferensi
U-Moss, EL, Maghini, DG, & Bhatt, AS (2020).Ama-genome ebhaktheriya aphelele, avaliwe avela kuma-microbiomes esebenzisa ukulandelana kwe-nanopore.I-biotechnology yemvelo,38(6), 701-707.
Ubuchwepheshe Nokugqama ihlose ukwabelana ngohlelo lokusebenza oluyimpumelelo lwakamuva kakhulu lobuchwepheshe bokulandelana komphumela ophezulu ezinkundleni ezihlukahlukene zokucwaninga kanye nemibono ekhaliphile ekwakhiweni kokuhlola kanye nokumbiwa kwedatha.
Isikhathi sokuthumela: Jan-07-2022