METAGENOMICS
Yakazara, yakavharwa mabhakitiriya genomes kubva ku microbiomes uchishandisa nanopore sequencing
Nanopore Sequencing |Metagenomics |MAGs |Bhakitiriya genome circularization |Gut microbiota
Pfungwa huru
1.Nzira itsva yekubvisa zvimedu zveDNA yakaratidzwa muchidzidzo ichi, iyo yakawana kutorwa kwemicrogram ye pure, HMW DNA yakakodzera kuverenga kwenguva refu kubva ku300 mg yechituru.
2.An assembly workflow, Lathe, yakatangwa muchidzidzo ichi, apo MAGS yakaunganidzwa nekuverenga kwenguva refu uye yakagadziriswa nepfupi-kuverenga.
3.Lathe yakaongororwa nemusanganiswa wekuseka.7 kubva mugumi nemaviri mabhakitiriya akaunganidzwa zvakabudirira kuita macontig mamwechete uye 3 akaunganidzwa kuita mana kana mashoma makonti.
4.Lathe yakashandiswa zvakare kune stool samples, iyo yakagadzira 20 circularized genomes, kusanganisira Prevotella copri uye mumiriri Cibiobacter sp., iyo yaizivikanwa nekupfuma mune mobile genetic elements.
Main Kubudirira
Kubvisa protocol yeHWM DNA
Kureba-kuverengera kutevedzana kwakavakirwa gut metagenomic zvidzidzo kwave kwenguva refu kutambura nekuomarara mukubvisa yakakwira molecular uremu (HMW) DNA kubva pachituru.Muchidzidzo ichi, enzyme-based extraction protocol yakaunzwa kudzivirira kuveura kwakakura nekurova bhiza nenzira dzechinyakare.Sezvinoratidzwa mumufananidzo unotevera, sampuli dzakatanga kurapwa necocktail yema enzymes, kusanganisira lytic enzyme, MetaPolyzyme, nezvimwewo kusvibisa madziro emasero.Yakabudiswa DNA yakatorwa nePhenol-chloroform system, ichiteverwa neProteinase K uye RNase A digestion, column-based purification uye SPRI saizi kusarudzwa.Iyi nzira yakakwanisa kuburitsa mamicrograms eHMW DNA kubva pa300 m yestool, iyo inozadzisa kwenguva yakareba kuverenga kutevedzana zvinodiwa maererano nemhando uye huwandu.
Mufananidzo 1. HWM DNA extraction scheme
Scheme kuyerera kweLathe
Sezvinotsanangurwa mumufananidzo unotevera, Lathe ine iripo maitiro eyakagadziriswa basecalling process uchishandisa Guppy.Magungano maviri akaverengeka kwenguva refu anozoburitswa naFlye neCanu zvakasiyana achiteverwa nekusaziva-assembly kuonekwa nekubviswa.Iwo maviri ma-sub-assemblies akabatanidzwa ne quickmerge.Pakubatanidzwa, magungano makuru pa megabase level anobva atariswa kuti atenderedzwa.Zvadaro, kugadzirisa kubvumirana pamagungano aya kunogadziriswa nekuverenga kupfupi.Yekupedzisira yakaunganidzwa mabhakitiriya genomes anogadziriswa kwekupedzisira mis-assembly kuonekwa uye kubviswa.
Mufananidzo 2. Scheme flow of Lathe assembly
Kuongororwa kweLathe ine mock bacteria musanganiswa
Yakajairwa ATCC 12-species musanganiswa inosanganisira ese Gram-positive uye Gram-negative mabhakitiriya akashandiswa kuongorora mashandiro e nanopore sequencing chikuva uye Lathe muMAG assembly.Huwandu hwe30.3 Gb data hwakagadzirwa nenanopore chikuva neN50 ye5.9 kb.Lathe yakanyanya kunatsiridza gungano N50 kusvika 1.6 kusvika 4-zvichienzaniswa nemamwe maturusi egungano akaverengwa kwenguva refu uye 2 kusvika 9-zvichienzaniswa nemidziyo yegungano yakasanganiswa.Pane gumi nemaviri genomes ebhakitiriya, manomwe akaunganidzwa kuita macontigs ega (Mufananidzo 3. Circos ine dot dema).Mamwe matatu akaunganidzwa kuita makontigi mana kana mashoma, umo musangano usina kukwana waive ne83% yegenome mune imwechete contig.
Mufananidzo 3. Magungano eGenome mune yakatsanangurwa 12-mhando yebhakitiriya musanganiswa
Kushandiswa kweLathe mumasampuli ezvigaro
Iyi nzira yakashandiswa zvakare kumasampuli ezvituro zvevanhu kuitira kuenzanisa kuzivikanwa kwechisikwa uye kusangana kwegungano kune nzira dziripo, kuverenga-gore uye pfupi-kuverenga-yakavakirwa ongororo.Kubva pamasampuri matatu anosanganisirwa, iyo enzyme-yakavakirwa dhizaini yakaburitsa ingangoita 1 μg pa300 mg yekupinza misa.Nanopore sequencing yeHMW DNA iyi yakagadzira kuverenga kwenguva refu neN50 ye4.7 kb, 3.0kb uye 3.0kb zvakateerana.Sezvineiwo, nzira iripo yakaratidza kugona kukuru mukuonekwa kwehutachiona kana ichienzaniswa nemaitiro aripo.Zvine mwero mhando dzemhando dzemhando dzealpha dzakaratidzwa pano zvichienzaniswa nepfupi-yekuverenga uye kuverenga-gore.Uyezve, genera rose kubva kupfupi-kuverenga ongororo, kunyangwe kazhinji lysis-resistant Gram-positive organisms, yakadzoserwa neiyi nzira.
Mufananidzo 4. Alpha siyana uye taxanomic zvikamu zvakatemwa naNanopore, pfupi-yekuverenga uye yekuverenga-gore nzira.
Lathe yakabereka yakareba-yakazara-gungano N50 pane pfupi-yekuverenga uye yekuverenga-gore musangano, zvisinei nekatatu- kusvika matanhatu-yakapetwa yakaderera yedata.Draft genomes dzakagadzirwa ne contig binning, umo zvinyorwa zvakaiswa mu "yemhando yepamusoro" kana "chikamu" zvichibva pakuzara, kusvibiswa, single-copy core genes, etc. Kuverengera kwenguva refu kuunganidzwa kwakaratidzwa zvakanyanya kukwirira contiguity pamutengo wakaderera zvichienzaniswa. kupfupi-kuverenga uye kuverenga-gore.
Mufananidzo 5. Per-organism assembly contiguity yeimwe neimwe nzira
Uyezve, nzira yazvino yegungano inokwanisa kuburitsa akavharika, akatenderera genome.Mune zvituru samples, masere emhando yepamusoro, single-contig genomes akaunganidzwa uye mashanu eaya akawana percise circularization.Nzira yekuverenga kwenguva refu yakaratidzawo kugona kunokatyamadza mukugadzirisa zvinhu zvinodzokororwa mumajenomes.CircularizedP. coprigenome yakagadzirwa nenzira iyi, iyo yave ichizivikanwa kuti ine yakakwirira-dhigirii yekudzokorora kutevedzana.Iko kuungana kwakanakisa kweiyi genome nekupfupi-kuverenga uye kuverenga-gore hakuna kumbopfuura N50 ye130 kb, kunyangwe nekudzika kwekuvhara kwe4800X.Aya makopi enhamba epamusoro zvinhu zvakagadziriswa zvizere nekuverenga-kurebesa nzira, iyo yaiwanzo wanikwa pakutyora-pfupi-yekuverenga kana kuverenga-gore magungano.Imwe genome yakavharwa yakashumwa muchidzidzo ichi, icho chaifungidzirwa kuti inhengo yechangobva kutsanangurwaCibiobacterclade.shanu putative phage yakaonekwa mugungano iri rakavharwa, kubva pa8.5 kusvika 65.9 kb.
Mufananidzo 6. Circos diagram yeakavharwa genomes yeP.copri uye Cibiobacter sp.
Reference
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Yakazara, yakavharwa mabhakitiriya genomes kubva ku microbiomes uchishandisa nanopore sequencing.Nature biotechnology,38(6), 701-707.
Tech uye Highlights inovavarira kugovera zvichangobva kuitika zvemashandisirwo akasiyana-siyana epamusoro-soro ekutevedzanisa matekinoroji munzvimbo dzakasiyana siyana dzekutsvagisa pamwe chete nemazano akajeka mukugadzira dhizaini nekuchera data.
Nguva yekutumira: Jan-07-2022