METAGENOMICS
Cikakken, rufaffiyar kwayoyin halittar kwayan cuta daga microbiomes ta amfani da jerin nanopore
Tsarin Nanopore |Metagenomics |MAGANAR |Kwayoyin kwayoyin halitta circularization |Gut microbiota
Karin bayanai
1.An gabatar da wata sabuwar hanya don cire dogon gutsuttsura na DNA a cikin wannan binciken, wanda ya haifar da hakar microgram na tsarki, HMW DNA wanda ya dace da jerin dogon karantawa daga 300 MG na stool.
2.An gabatar da tsarin aikin taro, Lathe, a cikin wannan binciken, inda aka tattara MAGs ta hanyar dogon karantawa kuma an gyara ta ta gajerun karantawa.
3. An kimanta Lathe ta hanyar haɗin izgili.An samu nasarar hada 7 cikin 12 na kwayoyin cuta zuwa dunkule guda kuma 3 an hada su zuwa guda hudu ko kasa da haka.
4.Lathe aka kara amfani da stool samfurori, wanda ya haifar da 20 circularized genomes, ciki har da Prevotella copri da dan takarar Cibiobacter sp., waɗanda aka sani da kasancewa masu wadata a cikin abubuwan halitta ta hannu.
Babban Nasara
Ka'idar cirewa don HWM DNA
An daɗe ana sha wahala daga taurin ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta (HMW) DNA daga stool.A cikin wannan binciken, an gabatar da ka'idar cirewar enzyme mai tushe don guje wa yanke mai yawa ta hanyar bugun dutse a cikin hanyoyin gargajiya.Kamar yadda aka nuna a cikin adadi mai zuwa, an fara bi da samfurori tare da hadaddiyar giyar enzymes, ciki har da enzyme lytic, MetaPolyzyme, da dai sauransu don lalata ganuwar tantanin halitta.An fitar da DNA da aka saki ta tsarin Phenol-chloroform, sannan Proteinase K da RNase A narkewa, tsarkakewar tushen shafi da zaɓin girman SPRI.Wannan hanyar ta sami nasarar samar da micrograms na HMW DNA daga stool na mita 300, wanda ke cika buƙatun jeri na dogon lokaci dangane da inganci da yawa.
Hoto 1. HWM Tsarin hakar DNA
Tsarin tsarin Lathe
Kamar yadda aka bayyana a cikin adadi mai zuwa, Lathe yana ƙunshe da tsarin da ake amfani da shi na tsarin kiran tushe ta amfani da Guppy.Majalisu guda biyu da aka dade ana karantawa sannan Flye da Canu suka samar da su daban tare da gano kuskure da cirewa.An haɗa ƙananan majalisun biyu tare da haɗakar gaggawa.Bayan haɗuwa, manyan majalisu a matakin megabase ana duba su don kewayawa.Daga baya, ana aiwatar da gyare-gyaren yarjejeniya akan waɗannan majalisu tare da gajerun karatu.Ana sarrafa ƙwayoyin ƙwayoyin ƙwayoyin cuta na ƙarshe don ganowa da cirewa.
Hoto 2. Tsarin tsarin taron Lathe
Ƙimar Lathe tare da cakuda kwayoyin cuta
An yi amfani da daidaitaccen cakuda nau'ikan nau'ikan ATCC 12 wanda ya ƙunshi duka Gram-positive da Gram-negative kwayoyin don kimanta aikin dandamali na jerin nanopore da Lathe a cikin taro na MAG.Jimlar bayanan 30.3 Gb an samar da su ta hanyar dandalin nanopore tare da N50 na 5.9 kb.Lathe ya fi inganta taro N50 zuwa 1.6 zuwa 4 idan aka kwatanta da sauran kayan aikin taro da aka daɗe ana karantawa da ninki 2 zuwa 9 idan aka kwatanta da kayan aikin haɗakarwa.Daga cikin kwayoyin halittar kwayan cuta guda 12, bakwai sun taru zuwa guda guda (Hoto na 3. Circos tare da dige baki).An tara ƙarin guda uku zuwa guda huɗu ko ƙasa da haka, wanda mafi ƙarancin taro ya ƙunshi kashi 83% na kwayoyin halitta a cikin mazugi ɗaya.
Hoto 3. Genome taro a cikin ƙayyadaddun nau'ikan ƙwayoyin cuta iri-iri 12
Aikace-aikacen Lathe a cikin samfuran stool
An ƙara amfani da wannan hanyar akan samfuran stool na ɗan adam don kwatanta ganewar kwayoyin halitta da haɗin kai zuwa hanyoyin da ake da su, gajimaren karantawa da nazarin tushen karantawa.Daga samfurori guda uku da aka haɗa, sabon cirewar tushen enzyme ya haifar da aƙalla 1 μg a kowace 300 MG na yawan shigarwa.Jerin Nanopore na waɗannan HMW DNA ya haifar da dogon karantawa tare da N50 na 4.7 kb, 3.0kb da 3.0kb bi da bi.Musamman, hanyar da ake yanzu tana nuna babban yuwuwar gano ƙananan ƙwayoyin cuta idan aka kwatanta da hanyoyin da suka wanzu.An nuna bambance-bambancen nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in bambancin alpha anan idan aka kwatanta da gajeriyar karantawa da gajimare na karantawa.Haka kuma, duk wani nau'i na bincike na ɗan gajeren karantawa, har ma da yawancin kwayoyin halitta na Gram-positive lysis, an dawo dasu ta wannan hanyar.
Hoto 4. Bambance-bambancen Alpha da abubuwan haraji da aka ƙaddara ta Nanopore, gajerun karantawa da hanyoyin karanta-girgije.
Lathe ya samar da dogon lokaci-N50 gabaɗaya gabaɗaya fiye da taron gajeriyar karatu da karantawa, duk da ƙarancin shigar da ɗanyen bayanai mai ninki uku zuwa shida.An samar da daftarin kwayoyin halitta ta hanyar contig binning, wanda a cikinsa aka rarraba zane-zane zuwa "masu inganci" ko "bangare" bisa ga cikakke, gurɓatawa, kwafi guda ɗaya, da sauransu. zuwa ga gajeren karatu da karanta-girgije.
Hoto 5. Ƙaƙƙarwar haɗuwa ta kowace ƙungiya ta kowace hanya
Bugu da ƙari, tsarin haɗin kai na yanzu yana iya haifar da rufaffiyar, kwayoyin halitta.A cikin samfurorin stool, an haɗa nau'ikan kwayoyin halitta guda takwas masu inganci guda takwas kuma biyar daga cikin waɗannan sun sami daidaitaccen kewayawa.Hanyar da aka dade ana karantawa kuma ta nuna iyawa mai ban sha'awa wajen warware abubuwa masu maimaitawa a cikin kwayoyin halitta.Da'iraP. koprigenome ya samo asali ne ta wannan hanya, wanda aka sani yana dauke da babban mataki na maimaitawa.Mafi kyawun taro na wannan genome ta gajeriyar karantawa da karanta-girgije bai wuce N50 na 130 kb ba, har ma da zurfin ɗaukar hoto na 4800X.Wadannan manyan adadin abubuwan da aka kwafi an warware su ta hanyar dogon karantawa, waɗanda galibi ana samun su a wuraren ɓangarorin guntun karatu ko manyan taron girgije.An ba da rahoton wani rufaffiyar kwayar halitta a cikin wannan binciken, wanda aka yi imanin cewa memba ne na kwanan nan da aka kwatantaCibiobacterclade.An gano phage guda biyar a cikin wannan rufaffiyar taro, daga 8.5 zuwa 65.9 kb.
Hoto 6. Tsarin Circos na rufaffiyar genomes na P.copri da Cibiobacter sp.
Magana
Moss, EL, Maghini, DG, & Bhatt, AS (2020).Cikakken, rufaffiyar kwayoyin halittar kwayan cuta daga microbiomes ta amfani da jerin nanopore.Halitta fasahar halittu,38(6), 701-707.
Fasaha da Manyan Labarai yana da nufin raba mafi kyawun aikace-aikacen nasara na baya-bayan nan na fasahohi daban-daban na babban abin da ake aiwatarwa a fagen bincike daban-daban da kuma ƙwararrun dabaru a ƙirar gwaji da hakar bayanai.
Lokacin aikawa: Janairu-07-2022