METAGENOMICS
Mkpụrụ ndụ nje bacteria mechiri emechi site na microbiomes na-eji usoro nanopore
Usoro Nanopore |Metagenomics |MAG |Mgbasa genome nke nje bacteria |Ọrịa microbiota
Isi ihe
1. E gosipụtara usoro ọhụụ iji wepụta iberibe DNA ogologo n'ọmụmụ ihe a, bụ nke rụpụtara mmịpụta microgram nke dị ọcha, DNA HMW dabara adaba maka usoro ọgụgụ ogologo site na 300 mg nke stool.
2.E webatara usoro ọrụ mgbakọ, Lathe, n'ọmụmụ ihe a, ebe a na-achịkọta MAGs site na ogologo oge na-agụ ma mezie site na mkpirisi ọgụgụ.
3. A na-enyocha Lathe site na ngwakọta mock.Achịkọtara nje bacteria 7 n'ime 12 nke ọma n'ime otu contigs na 3 gbakọtara n'ime akụkụ anọ ma ọ bụ obere.
4.Lathe e tinyekwuru na stool samples, nke mere 20 circularized genomes, gụnyere Prevotella copri na nwa akwukwo Cibiobacter sp., nke a maara maka ịba ụba nke mkpụrụ ndụ ihe nketa mkpanaka.
Isi Mmeri
Usoro mmịpụta maka HWM DNA
Ọmụmụ ihe ọmụmụ metagenomic nke gbadoro ụkwụ na-agụ ogologo oge ka a na-ata ahụhụ site na isi ike n'iwepụta DNA dị arọ nke dị elu (HMW) na stool.N'ime ọmụmụ ihe a, ewebata usoro mmịpụta nke sitere na enzyme iji zere ịkpacha ajị dị ukwuu site na iti bead n'ụzọ ọdịnala.Dị ka egosiri na ọnụ ọgụgụ na-esonụ, a na-ebu ụzọ emeso ihe atụ ndị ahụ na mmanya mmanya nke enzymes, gụnyere lytic enzyme, MetaPolyzyme, wdg iji weda mgbidi cell.E weputara DNA ewepụtara site na sistemu Phenol-chloroform, sochiri Proteinase K na RNase A mgbaze, ịdị ọcha dabere na kọlụm na nhọrọ nha SPRI.Usoro a jisiri ike wepụta microgram nke DNA HMW site na stool dị mita 300, nke na-emezu ihe achọrọ usoro nhazi ogologo oge n'ihe gbasara ma ogo na ọnụọgụ.
Ọgụgụ 1. Atụmatụ mmịpụta DNA HWM
Usoro nhazi nke Lathe
Dịka akọwara na ọnụ ọgụgụ na-esonụ, Lathe nwere usoro dị adị nke usoro ịkpọ oku site na iji Guppy.Flye na Canu ga-emepụta ọgbakọ abụọ a gụchara ogologo oge wee chọpụta na mwepụ na-ezighi ezi.A na-ejikọta ọgbakọ abụọ ahụ na ngwa ngwa.Mgbe ha jikọtara ọnụ, a na-enyocha nnukwu mgbakọ dị na ọkwa megabase maka ịgbasa.N'ikpeazụ, a na-eji mkpiri agụ hazi mmezi nkwenye na mgbakọ ndị a.A na-ahazi mkpụrụ ndụ nje bacteria gbakọtara ikpeazụ maka nchọpụta na mwepụ ikpeazụ.
Ọgụgụ 2. Usoro nhazi nke mgbakọ Lathe
Nlele nke Lathe na mock bacteria ngwakọta
Ngwakọta agwakọta ụdịdị ATCC 12 ọkọlọtọ nke gụnyere ma nje bacteria Gram-positive na gram-negative ejiri rụọ ọrụ iji nyochaa arụmọrụ nke ikpo okwu nanopore sequencing na Lathe in MAG.Ngụkọta nke 30.3 Gb data sitere na ikpo okwu nanopore nwere N50 nke 5.9 kb.Mgbakọ N50 ka mma nke ukwuu ruo 1.6 ruo okpukpu anọ ma e jiri ya tụnyere ngwaọrụ mgbakọ ndị ọzọ a na-agụ ogologo oge yana 2 ruo 9 okpukpu ma e jiri ya tụnyere ngwaọrụ mgbakọ ngwakọ.N'ime mkpụrụ ndụ nje bacteria iri na abụọ, asaa ka achịkọtara n'ime otu contigs (Njirimara 3. Circos nwere ntụpọ ojii).Akpọkọtara atọ ọzọ n'ime anọ ma ọ bụ obere contigs, nke mgbakọ na-ezughị ezu nwere 83% nke genome n'ime otu contig.
Ọgụgụ 3. Genome na-ezukọta na ngwakọta nje nje 12 akọwapụtara
Ngwa nke Lathe na stool samples
A na-etinyekwa usoro a n'ihe atụ nke stool nke mmadụ iji tụnyere njirimara organism na mgbakọ contiguity na usoro ndị dị ugbu a, igwe ojii na-agụ na nyocha nke dị mkpirikpi.Site n'omume atọ a metụtara, mmịpụta nke sitere na enzyme ọhụrụ wepụtara opekata mpe 1 μg kwa 300 mg nke ntinye ntinye.Usoro Nanopore nke DNA HMW ndị a mepụtara ogologo-agụ na N50 nke 4.7 kb, 3.0kb na 3.0kb n'otu n'otu.N'ụzọ doro anya, usoro dị ugbu a gosipụtara ike dị ukwuu na nchọpụta microbial ma e jiri ya tụnyere ụzọ ndị dị adị.E gosipụtara ụdịdị dị iche iche nke ụdị dị elu dị elu ma e jiri ya tụnyere obere ọgụgụ na igwe ojii.Ọzọkwa, usoro a enwetara mkpụrụ ndụ niile sitere na nyocha agụghị nkenke, ọbụnadị ihe dị ndụ na-eguzogide ọgwụ gram-positive.
Ọnụọgụ 4. Ụdị Alfa dị iche iche na ihe taxanomic kpebisiri ike site na Nanopore, ụzọ mkpirisi na-agụ na ịgụ-igwe ojii.
Lathe wepụtara ogologo oge nzuko zuru ezu N50 karịa mgbakọ igwe mkpirisi ọgụgụ na ịgụ, n'agbanyeghị ntinye data dị ala nwere okpukpu atọ ruo isii.A na-emepụta genomes nke Draft site na contig binning, nke a na-ekewapụta ihe ndị ahụ n'ime "dị elu" ma ọ bụ "ele mmadụ anya n'ihu" dabere na izu oke, mmetọ, mkpụrụ ndụ ihe nketa nke otu mkpụrụ ndụ ihe nketa, wdg. Mgbakọ ogologo oge na-agụ gosipụtara nnukwu contiguity dị elu na ọnụ ala dị ala ma e jiri ya tụnyere ya. ka mkpiri-agụ na ịgụ-igwe ojii.
Ọgụgụ 5. Per-organism mgbakọ contiguity nke usoro ọ bụla
Ọzọkwa, usoro mgbakọ dị ugbu a nwere ike iwepụta genome mechiri emechi, okirikiri.N'ihe atụ stool, a chịkọtara mkpụrụ ndụ ihe nketa asatọ dị elu, nke nwere otu ihe na ise n'ime ndị a mezuru oke okirikiri.Usoro ọgụgụ nke ogologo oge gosikwara ike dị egwu n'ịdozi ihe ndị na-emegharị ugboro ugboro na genome.Kpụrụ okirikiriP. akwụkwọgenome sitere na usoro a, nke amarala na ọ nwere ogo dị elu nke usoro nkwughachi.Mgbakọ kacha mma nke genome a site na mkpirisi ọgụgụ na ịgụ igwe anaghị agafe N50 nke 130 kb, ọbụlagodi na mkpuchi mkpuchi nke 4800X.A na-edozi ihe ndị a dị elu nke ọnụ ọgụgụ dị elu site na usoro ọgụgụ dị ogologo, bụ nke a na-ahụkarị n'ebe ndị na-emebi emebi nke mgbakọ dị mkpirikpi ma ọ bụ ịgụ akwụkwọ.A kọrọ genome ọzọ mechiri emechi na ọmụmụ ihe a, nke a kwenyere na ọ bụ onye otu nke akọwapụtara n'oge na-adịbeghị anyaCibiobacterkpuchiri ekpuchi.Achọpụtara phage putative ise na ọgbakọ mechiri emechi, sitere na 8.5 ruo 65.9 kb.
Ọgụgụ 6. Circos eserese nke genomes mechiri emechi nke P.copri na Cibiobacter sp.
Ntụaka
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Mkpụrụ ndụ nje bacteria mechiri emechi site na microbiomes na-eji usoro nanopore.Biotechnology okike,38(6), 701-707.
Teknụzụ na isi ihe bu n'obi kesaa ngwa kacha aga nke ọma na nso nso a nke teknụzụ usoro usoro dị elu dị iche iche na mpaghara nyocha dị iche iche yana echiche mara mma na nhazi nnwale na ngwuputa data.
Oge nzipu: Jan-07-2022