TS'ELISO
Li-genome tsa baktheria tse felletseng, tse koetsoeng tse tsoang ho li-microbiomes li sebelisa tatellano ea nanopore
Nanopore Sequencing |Metagenomics |MAKA |Bakteria genome circularization |Gut microbiota
Lintlha-khōlō
1. Mokhoa o mocha oa ho ntša likhechana tse telele tsa DNA o hlahisitsoe thutong ena, o fihletseng ho ntšoa ha microgram ea pure, HMW DNA e loketseng tatellano ea nako e telele ho tloha ho 300 mg ea setulo.
2.Tlhahiso ea mosebetsi oa kopano, Lathe, e ile ea hlahisoa thutong ena, moo MAGs e neng e bokelloa ka ho bala nako e telele le ho lokisoa ka ho bala nako e khutšoanyane.
3.Lathe e ile ea hlahlojoa ka motsoako oa mock.Libaktheria tse 7 ho tse 12 li ile tsa kopanngoa ka katleho hore e be li-contig tse le 'ngoe 'me tse 3 li ile tsa kopanngoa ho li-contig tse 'nè kapa tse seng kae.
4.Lathe e ile ea sebelisoa hape ho lisampole tsa mantle, tse ileng tsa hlahisa liphatsa tsa lefutso tse 20 tse pota-potileng, ho kenyelletsa Prevotella copri le mokhethoa Cibiobacter sp., tse neng li tsebahala ka ho rua liphatsa tsa lefutso tsa mobile.
Katleho e ka Sehloohong
Protocol ea ho hula bakeng sa HWM DNA
Lithuto tsa nako e telele tsa tatellano ea metagenomic ea mala e 'nile ea hlokofatsoa ke bothata ba ho ntša boima ba limolek'hule tse phahameng (HMW) DNA ho tloha setulong.Thutong ena, ho ile ha hlahisoa mokhoa oa ho ntša li-enzyme ho qoba ho kuta haholo ka ho otla ka lifaha ka mekhoa ea setso.Joalo ka ha ho bonts'itsoe setšoantšong se latelang, lisampole li ile tsa qala ho phekoloa ka "cocktail" ea li-enzyme, ho kenyeletsoa enzyme ea lytic, MetaPolyzyme, joalo-joalo ho senya marako a lisele.DNA e lokollotsoeng e ile ea ntšoa ke Phenol-chloroform system, e lateloa ke Proteinase K le RNase A tshilo ea lijo, tlhoekiso e thehiloeng ho mela le khetho ea boholo ba SPRI.Mokhoa ona o atlehile ho hlahisa li-micrograms tsa HMW DNA ho tloha ho 300 m ea setuloana, se phethahatsang litlhoko tsa tatellano ea nako e telele ho latela boleng le bongata.
Setšoantšo sa 1. Leano la ho ntša DNA ea HWM
Ho phalla ha morero oa Lathe
Joalo ka ha ho hlalositsoe setšoantšong se latelang, Lathe e na le ts'ebetso e teng ea ts'ebetso e tala ea basecalling e sebelisang Guppy.Likopano tse peli tse baloang ka nako e telele li hlahisoa ke Flye le Canu ka ho fapana tse lateloang ke ho lemoha le ho tlosoa ha li-mis-assembly.Likopano tse peli tse nyane li kopantsoe le ho kopanya kapele.Ha li kopantsoe, likopano tse kholo tse boemong ba megabase li hlahlojoa hore na li potoloha.Ka mor'a moo, ntlafatso ea tumellano ea likopano tsena e sebetsoa ka ho bala ha nako e khutšoanyane.Liphatsa tsa lefutso tsa baktheria tsa ho qetela li sebetsoa bakeng sa ho fumanoa le ho tlosoa ha phoso ea ho qetela.
Setšoantšo sa 2. Phallo ea moralo oa kopano ea Lathe
Tlhahlobo ea Lathe e nang le motsoako oa libaktheria tsa mock
Ho ile ha sebelisoa motsoako o tloaelehileng oa mefuta ea ATCC 12 o nang le libaktheria tsa Gram-positive le Gram-negative ho lekola tšebetso ea sethala sa tatellano ea nanopore le Lathe kopanong ea MAG.Kakaretso ea data ea 30.3 Gb e hlahisitsoe ke sethala sa nanopore se nang le N50 ea 5.9 kb.Lathe e ntlafalitse haholo kopano ea N50 ho isa ho 1.6 ho makhetlo a 4 ha e bapisoa le lisebelisoa tse ling tsa kopano tse baloang nako e telele le makhetlo a 2 ho isa ho a 9 ha a bapisoa le lisebelisoa tsa kopano tse nyalisitsoeng.Ho liphatsa tsa lefutso tse 12 tsa baktheria, tse supileng li ile tsa bokelloa ho ba li-contigs tse le 'ngoe (Setšoantšo sa 3. Li-Circos tse nang le letheba le letšo).Tse ling tse tharo li ile tsa bokelloa ho li-contig tse 'nè kapa tse fokolang, moo kopano e sa fellang ka ho fetisisa e neng e na le 83% ea genome ho contig e le' ngoe.
Setšoantšo sa 3. Likopano tsa Genome ka motsoako oa baktheria oa mefuta e 12.
Tšebeliso ea Lathe ka lisampole tsa mantle
Mokhoa ona o ile oa boela oa sebelisoa ho lisampole tsa setulo sa motho e le ho bapisa ho tsebahatsa lintho tse phelang le ho kopana ha kopano le mekhoa e teng, ho bala-leru le tlhahlobo e thehiloeng ho nako e khutšoanyane.Ho tsoa ho disampole tse tharo tse amehang, tlhahiso e ncha e thehiloeng ho enzyme e hlahisitse bonyane 1 μg ka 300 mg ea boima ba ho kenya.Tatelano ea Nanopore ea HMW DNA ena e hlahisitse lipalo tse telele ka N50 ea 4.7 kb, 3.0kb le 3.0kb ka ho latellana.Haholo-holo, mokhoa oa hona joale o bonts'a monyetla o moholo oa ho lemoha likokoana-hloko ha o bapisoa le mekhoa e teng.Ho ipapisitsoe le mefuta e fapaneng ea alpha ea boemo bo holimo e bontšitsoe mona ha e bapisoa le ea ho bala leru le lekhutšoane.Ho feta moo, mefuta eohle e tsoang ho tlhahlobo e baloang ka nako e khuts'oane, esita le likokoana-hloko tse hanyetsanang le Gram-positive, li ile tsa fumanoa ka mokhoa ona.
Setšoantšo sa 4. Phapang ea alpha le likarolo tsa taxanomic tse khethiloeng ke Nanopore, mekhoa e khutšoanyane ea ho bala le ho bala-leru
Lathe e hlahisitse N50 e telele haholo ho feta kopano e baloang le ho baloa leru, leha ho bile le tlhahiso e tlase ka makhetlo a mararo ho isa ho a tšeletseng ea data e tala.Liphatsa tsa lefutso tse hlophisitsoeng li ne li hlahisoa ke contig binning, moo lingoloa li neng li arotsoe ka "boleng bo phahameng" kapa "karolo" ho latela botlalo, tšilafalo, liphatsa tsa lefutso tsa kopi e le 'ngoe, joalo-joalo. Kopano e baloang nako e telele e bonts'itsoe khokahano e phahameng haholo ka litšenyehelo tse tlase ho bala nako e kgutshwane le leru la ho bala.
Setšoantšo sa 5. Khokahano ea kopano ea motho ka mong ea mokhoa o mong le o mong
Ho feta moo, mokhoa oa hona joale oa kopano o khona ho hlahisa liphatsa tsa lefutso tse koetsoeng, tse chitja.Ka lisampole tsa setulo, liphatsa tsa lefutso tse robeli tsa boleng bo holimo, tsa single-contig li ile tsa bokelloa 'me tse hlano tsa tsona li ile tsa finyella percise circularization.Mokhoa oa ho bala nako e telele o boetse o bontšitse bokhoni bo tsotehang ba ho rarolla likarolo tse pheta-phetoang liphatseng tsa lefutso.SedikadikweP. coprigenome e ile ea hlahisoa ke mokhoa ona, oo ho tsejoang hore o na le ho pheta-pheta ha tatellano e phahameng.Kopano e ntle ka ho fetisisa ea genome ena ka ho bala hakhutšoanyane le leru ha ho mohla e kileng ea feta N50 ea 130 kb, leha e le ka botebo ba 4800X.Lintlha tsena tsa linomoro tse phahameng tsa kopi li ile tsa rarolloa ka botlalo ka mokhoa o baloang ka nako e telele, oo hangata o neng o fumanoa libakeng tse robehileng tsa likopano tse baloang ka nako e khuts'oane kapa tsa maru.E 'ngoe ea genome e koetsoeng e tlalehiloe thutong ena, eo ho lumeloang hore ke setho sa se sa tsoa hlalosoaCibiobacterclade.Phage e mehlano ea putative e ile ea khethoa kopanong ena e koetsoeng, ho tloha ho 8.5 ho isa ho 65.9 kb.
Setšoantšo sa 6. Setšoantšo sa Circos sa liphatsa tsa lefutso tse koetsoeng tsa P.copri le Cibiobacter sp.
Referense
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Li-genome tsa baktheria tse felletseng, tse koetsoeng tse tsoang ho li-microbiomes li sebelisa tatellano ea nanopore.Theknoloji ea tlhaho ea tlhaho,38(6), 701-707.
Theknoloji le Lintlha-khōlō e ikemiselitse ho arolelana ts'ebeliso ea morao-rao e atlehileng ea mekhoa e fapaneng ea tatellano ea maemo a holimo mabaleng a fapaneng a liphuputso hammoho le mehopolo e bohlale moralong oa liteko le ho rafa data.
Nako ea poso: Jan-07-2022