METAGENOMICS
Hoʻopiha piha, pani ʻia nā genomes bacteria mai nā microbiome me ka hoʻohana ʻana i ke ʻano nanopore
Nanopore Sequencing |Metagenomics |MAG |ʻO ka pōʻaiapili genome bacteria |ʻAno microbiota
Nā mea nui
1. Ua hōʻike ʻia kahi ala hou e unuhi i nā ʻāpana lōʻihi o ka DNA i kēia haʻawina, kahi i loaʻa ai ka unuhi ʻana o ka microgram o ka maʻemaʻe, HMW DNA kūpono no ka heluhelu lōʻihi ʻana mai ka 300 mg o ka waihona.
2. Ua hoʻokomo ʻia kahi kaʻina hana hui, Lathe, i loko o kēia haʻawina, kahi i ʻākoakoa ai nā MAG e nā heluhelu lōʻihi a hoʻoponopono ʻia e nā heluhelu pōkole.
3. Ua loiloi ʻia ka Lathe e ka hui hoʻohenehene.7 mai loko o 12 mau huakini i hoʻohui maikaʻi ʻia i hoʻokahi contigs a 3 i hōʻuluʻulu ʻia i ʻehā a liʻiliʻi paha contigs.
4. Ua hoʻohana hou ʻia ka Lathe i nā laʻana stool, kahi i hana ai i 20 circularized genomes, me Prevotella copri a me ka moho Cibiobacter sp., i ʻike ʻia no ka waiwai ʻana i nā mea genetic mobile.
Hana Nui
ʻO ka protocol unuhi no ka HWM DNA
Ua lōʻihi ka loaʻa ʻana o nā haʻawina metagenomic gut metagenomic ma muli o ka paʻakikī i ka unuhi ʻana i ke kaumaha molekala kiʻekiʻe (HMW) mai ka waihona.I loko o kēia haʻawina, ua hoʻokomo ʻia kahi kaʻina hoʻoheheʻe e pili ana i ka enzyme no ka pale ʻana i ka ʻako nui ʻana me ka hahau ʻana i ka bead ma nā ʻano kuʻuna.E like me ka mea i hōʻike ʻia ma ke kiʻi aʻe, ua mālama mua ʻia nā laʻana me ka cocktail o nā enzymes, me ka lytic enzyme, MetaPolyzyme, etc. no ka hoʻohaʻahaʻa ʻana i nā paia cell.Ua unuhi ʻia ka DNA i hoʻokuʻu ʻia e ka ʻōnaehana Phenol-chloroform, a ukali ʻia e Proteinase K a me RNase A digestion, ka hoʻomaʻemaʻe ʻana i ke kolamu a me ke koho ʻana i ka nui SPRI.Ua hoʻokō kēia ʻano hana i nā micrograms o HMW DNA mai 300 m o ka stool, kahi e hoʻokō ai i nā koi sequencing lōʻihi e pili ana i ka maikaʻi a me ka nui.
Kiʻi 1. HWM DNA unuhi
Ke kahe o ka papahana o ka Lathe
E like me ka mea i wehewehe ʻia ma ke kiʻi aʻe, loaʻa i ka Lathe ke kaʻina hana o ke kaʻina hana kumu kumu me Guppy.Hoʻopuka ʻia ʻelua mau hui heluhelu lōʻihi e Flye a me Canu ma hope o ka ʻike ʻana a me ka wehe ʻana.Hoʻohui ʻia nā ʻāpana ʻelua me ka quickmerge.Ma ka hui ʻana, nānā ʻia nā hui nui ma ka pae megabase no ka circularization.A laila, hoʻoponopono ʻia ka ʻae ʻana i kēia mau hui me ka heluhelu pōkole.Hoʻoponopono ʻia nā genome bacteria i hui hope ʻia no ka ʻike ʻana a me ka wehe ʻana i ka hui hewa ʻole.
Kiʻi 2. ʻO ke kahe o ka papahana o ka hui Lathe
ʻO ka loiloi o ka Lathe me ka hui ʻana o nā hua bacteria hoʻohenehene
Ua hoʻohana ʻia kahi huila ATCC 12-species maʻamau i loaʻa i nā bacteria Gram-positive a me Gram-negative e loiloi i ka hana o ka papa kuhikuhi nanopore sequencing a me Lathe i ka hui MAG.ʻO ka huina o 30.3 Gb ʻikepili i hana ʻia e ka nanopore platform me N50 o 5.9 kb.Ua hoʻomaikaʻi nui ka Lathe i ka hui N50 i ka 1.6 a i ka 4-fold i hoʻohālikelike ʻia me nā mea hana hui lōʻihi heluhelu ʻia a me 2 a 9-fold i hoʻohālikelike ʻia me nā mea hana hui hybridd.ʻO 12 mau huakino bacteria, ʻehiku i hōʻuluʻulu ʻia i hoʻokahi contigs (Figure 3. Circos me kahi kiko ʻeleʻele).ʻEkolu hou aʻe i hōʻuluʻulu ʻia i ʻehā a i ʻole ka liʻiliʻi o nā contigs, kahi o ka hui piha ʻole i loaʻa ka 83% o ka genome i hoʻokahi contig.
Kiʻi 3. Nā hui genome i loko o kahi hui bacteria 12-species i wehewehe ʻia
Ka hoʻohana ʻana i ka Lathe i loko o nā kumu hoʻohālike
Ua hoʻohana hou ʻia kēia ʻano hana i nā laʻana o ka pahu kanaka i mea e hoʻohālikelike ai i ka ʻike ʻana o ke kino a me ka pili ʻana o ka hui ʻana i nā ʻano i loaʻa, heluhelu-kapu a me ka heluhelu pōkole.Mai nā hōʻailona ʻekolu i hoʻopili ʻia, ua loaʻa ka 1 μg ma kahi o 300 mg o ka nui hoʻokomo.Hoʻokumu ʻo Nanopore sequencing o kēia HMW DNA i nā heluhelu lōʻihi me N50 o 4.7 kb, 3.0kb a me 3.0kb.ʻO ka mea nui, ua hōʻike ʻia ke ʻano o kēia manawa i ka mana nui i ka ʻike microbial i hoʻohālikelike ʻia me nā ʻano hana.Ua hōʻike ʻia ka ʻokoʻa alpha kiʻekiʻe o nā ʻano ʻano-level i hoʻohālikelike ʻia me ka heluhelu pōkole a me ka ao heluhelu.Eia kekahi, ua hoʻihoʻi ʻia nā genera a pau mai ka nānā ʻana i ka heluhelu pōkole, ʻo ia hoʻi nā mea ola kino Gram-positive lysis-resistant, ma kēia ʻano hana.
Kiʻi 4. ʻO ka ʻokoʻa Alpha a me nā ʻāpana ʻauhau i hoʻoholo ʻia e Nanopore, nā ʻano heluhelu pōkole a me ka heluhelu ʻana i ke ao.
ʻOi aku ka lōʻihi o ka hāʻawi ʻana o ka Lathe i ka hui holoʻokoʻa N50 ma mua o ka heluhelu pōkole a me ka heluhelu ʻana i ke ao, ʻoiai he ʻekolu a ʻeono mau manawa haʻahaʻa o ka ʻikepili maka.Hoʻokumu ʻia nā genomes kikoʻī e contig binning, kahi i hoʻokaʻawale ʻia ai nā kikoʻī i ka "kiʻekiʻe" a i ʻole "ʻāpana" e pili ana i ka hoʻopiha piha ʻana, ka hoʻohaumia ʻana, nā genes kumu hoʻokahi kope, a me nā mea ʻē aʻe. i ka heluhelu pōkole a me ke ao heluhelu.
Kiʻi 5. ʻO ka hui pū ʻana o kēlā me kēia ʻano hana
Eia kekahi, hiki i ke ala hui o kēia manawa ke hāʻawi i nā genomes i pani ʻia.I loko o nā hōʻailona stool, ʻewalu kiʻekiʻe kiʻekiʻe, hoʻokahi-contig genomes i hōʻuluʻulu ʻia a ʻelima o kēia i loaʻa i ka percise circularization.Ua hōʻike pū ʻia ke ala heluhelu lōʻihi i ka hiki ke hoʻoholo i nā mea hou i loko o nā genome.Hoʻopuni ʻiaP. copriUa hoʻokumu ʻia ka genome e kēia ala, kahi i ʻike ʻia he kiʻekiʻe kiʻekiʻe o ka hana hou ʻana.ʻO ka hui maikaʻi loa o kēia genome ma ka heluhelu pōkole a me ke ao heluhelu ʻaʻole i ʻoi aku ma mua o N50 o 130 kb, ʻoiai me ka hohonu o ka uhi o 4800X.Ua hoʻoholo piha ʻia kēia mau mea helu kope kiʻekiʻe e ke ala heluhelu lōʻihi, i ʻike pinepine ʻia ma nā wahi haʻihaʻi o nā hui heluhelu pōkole a heluhelu-kapua paha.Ua hōʻike ʻia kekahi genome paʻa i loko o kēia haʻawina, i manaʻo ʻia he lālā ia o ka mea i wehewehe ʻiaCibiobacterclade.ʻElima putative phage i ʻike ʻia i kēia hui paʻa, mai 8.5 a 65.9 kb.
Kiʻi 6. Kiʻi Circos o nā genome paʻa o P.copri a me Cibiobacter sp.
Kuhikuhi
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Hoʻopiha piha, pani ʻia nā genomes bacteria mai nā microbiome me ka hoʻohana ʻana i ke kaʻina nanopore.Nature biotechnology,38(6), 701-707.
ʻenehana a me nā mea koʻikoʻi manaʻo e kaʻana like i nā noi holomua hou loa o nā ʻenehana hoʻokaʻina kiʻekiʻe-throughput like ʻole ma nā kahua ʻimi noiʻi like ʻole a me nā manaʻo maikaʻi loa i ka hoʻolālā hoʻokolohua a me ka ʻimi ʻikepili.
Ka manawa hoʻouna: Jan-07-2022