METAGENOMIKA
Gqibezela, i-bacterial genomes evaliweyo evela kwi-microbiomes usebenzisa i-nanopore sequencing
Ulandelelwano lweNanopore |Metagenomics |Iimaski |Ibhaktheriya genome circularization |Amathumbu microbiota
Iimbalasane
I-1.Indlela yenoveli yokukhupha amaqhekeza amade e-DNA yaboniswa kolu phononongo, oluphumelele ukutsalwa kwe-microgram ecocekileyo, i-HMW DNA ilungele ukulandelelana kwexesha elide ukusuka kwi-300 mg yesitulo.
I-2.I-workflow workflow, i-Lathe, yaziswa kolu cwaningo, apho ii-MAGs zahlanganiswa ngokufundwa okude kwaye zilungiswe ngokufundwa okufutshane.
3.I-Lathe yavavanywa ngumxube ohlekisayo.Iibhaktheriya ezisi-7 kwezili-12 ziye zadityaniswa ngempumelelo zaba yi-contigs enye kwaye ezi-3 zadityaniswa zaba zii-contigs ezine okanye ezimbalwa.
4. ILathe yaphinda yasetyenziswa kwiisampuli zestool, ezavelisa i-20 circularized genomes, kubandakanywa ne-Prevotella copri kunye nomviwa uCibiobacter sp., ezaziwa ngokuba zizityebi kwizinto zofuzo ezihambayo.
Impumelelo engundoqo
Iprotocol yokutsalwa kwe-HWM DNA
Izifundo ze-metagenomic esele zifundwe ixesha elide kudala zibandezeleka ngenxa yobunzima ekukhupheni ubunzima obuphezulu bemolekyuli (HMW) DNA kwisitulo.Kolu phononongo, iprotocol ye-enzyme-based extraction protocol yaziswa ukuphepha ukucheba okubanzi ngokubetha amaso kwiindlela zemveli.Njengoko kuboniswe kulo mfanekiso ulandelayo, iisampulu zaphathwa okokuqala nge-cocktail ye-enzymes, kubandakanywa i-enzyme ye-lytic, i-MetaPolyzyme, njl njl ukuthobisa iindonga zeseli.I-DNA ekhutshweyo ikhutshwe yinkqubo ye-Phenol-chloroform, ilandelwa yiProteinase K kunye ne-RNase A digestion, ukuhlanjululwa kwekholomu kunye nokukhethwa kobukhulu be-SPRI.Le ndlela ikwazile ukuvelisa ii-micrograms ze-HMW DNA ukusuka kwi-300 m yesitulo, ezalisekisa iimfuno zokulandelelana kwexesha elide ngokomgangatho kunye nobuninzi.
Umzobo 1. HWM DNA extraction scheme
Ukuhamba kweSkimu seLathe
Njengoko kuchaziwe kulo mzobo ulandelayo, iLathe iqulethe inkqubo ekhoyo yenkqubo ekrwada ye-basecalling usebenzisa i-Guppy.Iindibano ezimbini ezifunda ixesha elide emva koko ziveliswa nguFlye kunye neCanu ngokwahlukeneyo zilandelwa kukubhaqwa kwe-mis-assembly kunye nokususwa.Ii-sub-assemblies ezimbini zidibaniswe kunye nokudibanisa ngokukhawuleza.Emva kokudibanisa, iindibano ezinkulu kwinqanaba le-megabase zijongwa ukuba zisetyhula.Emva koko, ukulungiswa kwemvumelwano kwezi ndibano kwenziwa ngokufundwa okufutshane.Iigenome zebhaktiriya ezidityanisiweyo zokugqibela zicutshungulwa ukuze kufunyaniswe i-mis-assembly yokugqibela kunye nokususwa.
Umzobo 2. Ukuhamba kweskimu sendibano yeLathe
Uvavanyo lweLathe kunye nomxube weebhaktheriya ezihlekisayo
Umxube oqhelekileyo we-ATCC we-12-species umxube oquka zombini iibhaktheriya ze-Gram-positive kunye ne-Gram-negative zaqeshwa ukuvavanya ukusebenza kwe-nanopore sequencing platform kunye neLathe kwindibano ye-MAG.Ingqikithi yedatha ye-30.3 Gb yenziwe yi-platform ye-nanopore kunye ne-N50 ye-5.9 kb.I-Lathe iphuculwe kakhulu indibano ye-N50 ukuya kwi-1.6 ukuya kwi-4-fold xa kuthelekiswa nezinye izixhobo zokuhlanganisa ezifundwe ixesha elide kunye ne-2 ukuya kwi-9-fold xa kuthelekiswa nezixhobo zokuhlanganisa ezixutywe.Kwiigenomes zebhaktiriya ezili-12, ezisixhenxe zadityaniswa zaba yi-contigs enye (Umfanekiso 3. IiSekethe ezinechaphaza elimnyama).Ezinye ezithathu zadityaniswa zaba zii-contigs ezine okanye ezimbalwa, apho eyona ndibano ingaphelelanga iqulethe i-83% ye-genome kwi-contig enye.
Umzobo 3. Iindibano zeGenome kumxube webhaktiriya ochaziweyo weentlobo ezili-12
Ukusetyenziswa kweLathe kwiisampuli zesitulo
Le ndlela iphinde isetyenziswe kwiisampuli zesitulo somntu ukwenzela ukuthelekisa ukuchongwa kwezinto eziphilayo kunye nokudibana kwendibano kwiindlela ezikhoyo, ukufunda-ilifu kunye nohlalutyo olufutshane olusekelwe.Ukususela kwiisampuli ezintathu ezibandakanyekayo, i-enzyme entsha-based extraction ivelise ubuncinci i-1 μg nge-300 mg ye-input mass.Ukulandelelana kwe-Nanopore kwezi HMW DNA zenze ukufundwa kwexesha elide kunye ne-N50 ye-4.7 kb, 3.0kb kunye ne-3.0kb ngokulandelanayo.Ngokucacileyo, indlela yangoku ibonise amandla amakhulu ekubhaqweni kwe-microbial xa kuthelekiswa neendlela ezikhoyo.Ngokuthelelelekayo iintlobo eziphezulu zenqanaba leyantlukwano yealpha ibonisiwe apha xa kuthelekiswa nokufundwa okufutshane kunye nelifu elifundwayo.Ngaphezu koko, yonke igenera evela kuhlalutyo lofundo olufutshane, nkqu ne-lysis-resistant organisms Gram-positive, yafunyanwa ngale ndlela.
Umzobo 4. Ukwahluka kwe-alpha kunye namacandelo e-taxanomic agqitywe nguNanopore, iindlela zokufunda ezimfutshane kunye nokufunda-ilifu
I-Lathe ivelise i-N50 ende yendibano epheleleyo kune-short-read and read-cloud assembly, ngaphandle kwegalelo eliphantsi eliphindwe kathathu ukuya kwisithandathu ledatha ekrwada.Idrafti ye-genomes yaveliswa yi-contig binning, apho iidrafti zahlelwa "kwizinga eliphezulu" okanye "inxenye" ngokusekwe ekugqibeleleni, ekungcolisekeni, kwikopi enye yemfuza engundoqo, njl. njl. ukufunda-mfutshane kunye nokufunda-ilifu.
Umzobo 5. Ukudibanisa kwe-per-organism yendlela nganye
Ngaphezu koko, indlela yokuhlanganisa yangoku iyakwazi ukuvelisa i-genomes evaliweyo, ejikelezayo.Kwiisampulu zesitulo, ii-genomes ezisibhozo ezikumgangatho ophezulu, i-single-contig zahlanganiswa kwaye ezintlanu kwezi ziphumelele i-percise circularization.Indlela yokufunda ixesha elide ikwabonise amandla achukumisayo ekusombululeni izinto eziphindaphindwayo kwiigenomes.IsetyhulaP. coprii-genome yenziwe yile ndlela, eyaziwayo ukuba iqulethe i-high-degree yokuphindaphinda ulandelelwano.Eyona ndibano ingcono yale genome ngokufunda-mfutshane kunye nelifu elifundwayo ayizange igqithe kwi-N50 ye-130 kb, nokuba kubunzulu obuyi-4800X.Ezi zinto ziphezulu zeekopi zamanani zasonjululwa ngokupheleleyo yindlela yokufunda ixesha elide, ehlala ifumaneka kwiindawo zokuqhawula iindibano ezimfutshane zokufunda okanye ezifundwayo.Enye i-genome evaliweyo yaxelwa kolu phando, ekukholelwa ukuba lilungu elichazwe kutshanjeI-Cibiobacterclade.Intlanu yokubeka phage ichongiwe kule ndibano ivaliweyo, ukusuka kwi-8.5 ukuya kwi-65.9 kb.
Umzobo 6. Umzobo we-Circos we-genomes evaliweyo ye-P.copri kunye ne-Cibiobacter sp.
Isalathiso
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Gqibezela, i-bacterial genomes evaliweyo evela kwi-microbiomes usebenzisa i-nanopore sequencing.I-biotechnology yendalo,38(6), 701-707.
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Ixesha lokuposa: Jan-07-2022