METAGENOMICS
Ma genome athunthu, otsekedwa a bakiteriya ochokera ku ma microbiomes pogwiritsa ntchito nanopore sequencing
Nanopore Sequencing |Metagenomics |MAWU |Bakiteriya genome circularization |Matenda a microbiota
Mfundo zazikuluzikulu
1. Njira yatsopano yochotsera zidutswa zazitali za DNA idawonetsedwa mu kafukufukuyu, yomwe idatulutsa microgram ya pure, HMW DNA yoyenera kutsatizana kwanthawi yayitali kuchokera ku 300 mg ya chopondapo.
2.Kuyenda kwa msonkhano, Lathe, kunayambika mu phunziro ili, kumene MAGs anasonkhanitsidwa ndi kuwerenga kwautali ndikuwongolera ndi kuwerenga kwachidule.
3.Lathe idawunikidwa ndi kusakaniza moseketsa.Mabakiteriya 7 mwa 12 adasonkhanitsidwa bwino kukhala ma contigs amodzi ndipo 3 adasonkhanitsidwa kukhala ma contigs anayi kapena kuchepera.
4.Lathe idagwiritsidwanso ntchito pazitsanzo za ndowe, zomwe zidapanga ma genome 20 ozungulira, kuphatikiza Prevotella copri ndi candidate Cibiobacter sp., omwe ankadziwika kuti anali olemera mu ma genetic elements.
Kupambana Kwakukulu
Protocol yotsitsa ya HWM DNA
Kafukufuku wowerengera kwanthawi yayitali wotsata m'matumbo a metagenomic akhala akuvutika ndi kuuma kwa mamolekyu apamwamba kwambiri (HMW) DNA kuchokera pachimbudzi.Mu kafukufukuyu, njira yochotsa ma enzyme idayambitsidwa kuti apewe kumeta ubweya wambiri pomenya mikanda m'njira zachikhalidwe.Monga momwe tawonetsera m'chithunzi chotsatirachi, zitsanzozo poyamba zinkagwiritsidwa ntchito ndi ma enzyme, kuphatikizapo lytic enzyme, MetaPolyzyme, ndi zina zotero kuti awononge makoma a maselo.DNA yotulutsidwa idatulutsidwa ndi Phenol-chloroform system, yotsatiridwa ndi Proteinase K ndi RNase A digestion, kuyeretsa kochokera pamizere ndi kusankha kwa SPRI.Njirayi idakwanitsa kutulutsa ma micrograms a HMW DNA kuchokera ku 300 m ya chopondapo, chomwe chimakwaniritsa zofunikira zowerengera nthawi yayitali malinga ndi mtundu ndi kuchuluka kwake.
Chithunzi 1. HWM DNA extraction scheme
Kutuluka kwa dongosolo la Lathe
Monga tafotokozera m'chithunzi chotsatirachi, Lathe ili ndi njira yomwe ilipo yogwiritsira ntchito Guppy.Misonkhano iwiri yowerengedwa kwanthawi yayitali imapangidwa ndi Flye ndi Canu mosiyana ndikutsatiridwa ndi kuzindikira ndi kuchotsa zolakwika.Mipingo iwiriyi ikuphatikizidwa ndi Quickmerge.Pakuphatikizana, misonkhano yayikulu pamlingo wa megabase imafufuzidwa kuti ikhale yozungulira.Pambuyo pake, kukongoletsedwa kwa mgwirizano pamisonkhanoyi kumakonzedwa ndi kuwerenga kwakufupi.Ma genome a bakiteriya omaliza omwe amasonkhanitsidwa amakonzedwa kuti azindikire ndikuchotsa zolakwika.
Chithunzi 2. Mayendedwe a dongosolo la msonkhano wa Lathe
Kuwunika kwa Lathe ndi kusakaniza kwa mabakiteriya onyoza
Zosakaniza zamtundu wa ATCC 12 zokhala ndi mabakiteriya a Gram-positive ndi Gram-negative adagwiritsidwa ntchito kuti awunikire momwe ma nanopore amatsatirira nsanja ndi Lathe mu MAG assembly.Deta yonse ya 30.3 Gb idapangidwa ndi nsanja ya nanopore yokhala ndi N50 ya 5.9 kb.Lathe idasintha kwambiri msonkhano wa N50 mpaka 1.6 mpaka 4 kuyerekeza ndi zida zina zowerengera nthawi yayitali komanso 2 mpaka 9 kuyerekeza ndi zida zophatikizira zosakanizidwa.Mwa ma genome a bakiteriya a 12, asanu ndi awiri adasonkhanitsidwa kukhala ma contigs amodzi (Chithunzi 3. Ma Circo okhala ndi dontho lakuda).Ena atatu adasonkhanitsidwa kukhala ma contigs anayi kapena ocheperapo, momwe msonkhano wosakwanira kwambiri unali ndi 83% ya genome mu contig imodzi.
Chithunzi 3. Magulu a genome mumitundu 12 yosakanikirana ndi mabakiteriya
Kugwiritsa ntchito Lathe mu zitsanzo za ndowe
Njirayi idagwiritsidwanso ntchito pazitsanzo za ndodo za anthu kuti zifananize chizindikiritso cha chamoyo ndi kulumikizana kwa msonkhano ndi njira zomwe zilipo, kuwerengera-mtambo ndi kusanthula kwakanthawi kochepa.Kuchokera pazitsanzo zitatu zomwe zikukhudzidwa, kutulutsa kwatsopano kwa enzyme kunatulutsa osachepera 1 μg pa 300 mg ya misa yolowera.Kutsata kwa Nanopore kwa HMW DNA iyi kunapanga zowerengera zazitali ndi N50 ya 4.7 kb, 3.0kb ndi 3.0kb motsatana.Zachidziwikire, njira yomwe ilipo tsopano ikuwonetsa kuthekera kwakukulu pakuzindikiritsa ma microbial poyerekeza ndi njira zomwe zidalipo kale.Kuchulukirachulukira kwa mitundu yamitundu yosiyanasiyana ya alpha yawonetsedwa pano poyerekeza ndi mtambo wowerengeka komanso wowerengera.Kuphatikiza apo, mitundu yonse yowerengera pang'ono, ngakhale zamoyo zolimbana ndi lysis ya Gram-positive, zidabwezedwa ndi njirayi.
Chithunzi 4. Kusiyanasiyana kwa alpha ndi zigawo za taxanomic zotsimikiziridwa ndi Nanopore, njira zowerengera mwachidule komanso zowerengera-mtambo.
Lathe idatulutsa gulu lonse la N50 lalitali kwambiri kuposa msonkhano wowerengeka pang'ono komanso wowerengeka wamtambo, mosasamala kanthu za kuyika katatu kapena kasanu ndi kutsika kwa data yaiwisi.Ma genomes okonzekera adapangidwa ndi ma contig binning, momwe zolembazo zidasankhidwa kukhala "zapamwamba" kapena "tsankho" kutengera kukwanira, kuipitsidwa, majini amtundu umodzi, ndi zina zambiri. kuti muwerenge mwachidule ndi kuwerenga-mtambo.
Chithunzi 5. Per-zamoyo msonkhano contiguity njira iliyonse
Kuphatikiza apo, njira yolumikiziranayi imatha kutulutsa ma genome otsekedwa, ozungulira.M'zitsanzo zazitsulo, ma genomes asanu ndi atatu apamwamba kwambiri, a single-contig anasonkhanitsidwa ndipo asanu mwa awa adapindula ndi percise circularization.Njira yowerengera nthawi yayitali idawonetsanso kuthekera kochititsa chidwi pakuthana ndi zinthu zobwerezabwereza mu ma genome.ZozunguliraP. coprigenome idapangidwa ndi njira iyi, yomwe imadziwika kuti ili ndi kubwerezabwereza kwapamwamba kwambiri.Kusonkhana kwabwino kwambiri kwa genome iyi powerenga pang'ono ndikuwerenga-mtambo sikunapitirire N50 ya 130 kb, ngakhale kuzama kwa 4800X.Nambala zapamwambazi zidathetsedwa kwathunthu ndi njira yowerengera nthawi yayitali, yomwe nthawi zambiri imapezeka pamisonkhano yowerengera mwachidule kapena yowerengera-mtambo.Gulu lina lotsekedwa linanenedwa mu kafukufukuyu, yemwe amakhulupirira kuti ndi membala wa posachedwapaCibiobacterclade.Phage zisanu zophatikizira zidadziwika pamsonkhano wotsekedwa, kuyambira 8.5 mpaka 65.9 kb.
Chithunzi 6. Circos chithunzi cha ma genome otsekedwa a P.copri ndi Cibiobacter sp.
Buku
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Ma genome athunthu, otsekedwa a bakiteriya ochokera ku ma microbiomes pogwiritsa ntchito nanopore sequencing.Nature biotechnology,38(6), 701-707.
Tech ndi Zowunikira Cholinga chake ndi kugawana njira zamakono zotsatirira bwino kwambiri m'mabwalo osiyanasiyana ofufuza komanso malingaliro anzeru pamapangidwe oyesera ndi migodi ya data.
Nthawi yotumiza: Jan-07-2022