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Taxanaha Metagenomic -NGS
Metagenome-ku waa ururinta wadarta walxaha hidde-sidaha ee bulshada isku dhafan ee noolaha, sida deegaanka iyo metagenom-yada aadanaha. Waxa uu ka kooban yahay genome-ka noolaha la beeran karo iyo kuwa aan la beeran karin labadaba. Isku-xigta metagenomic ee Shotgun ee NGS waxay awood u siinaysaa daraasadda muuqaalladan genomic-ga ah ee ku dhex-jira muunadaha deegaanka iyadoo la siinayo wax ka badan sifada taxonomic, iyadoo sidoo kale siinaya aragtiyo qaawan oo ku saabsan kala duwanaanshaha noocyada, dhaqdhaqaaqyada fara badan, iyo qaabdhismeedka dadweynaha ee kakan. Marka laga soo tago daraasaadka taxonomiga, metagenomics shotgun waxa kale oo ay bixisaa aragti genomics shaqaynaysa, taas oo awood u siinaysa sahaminta hiddo-sidaha la calaamadeeyay iyo doorkooda wax-ka-qabashada ee hababka deegaanka. Ugu dambayntii, samaynta isku xidhka isku xidhka walxaha hidde-sidaha iyo arrimaha deegaanka waxa ay gacan ka geysataa faham buuxa ee isdhexgalka adag ee ka dhexeeya bulshooyinka microbial iyo asalkooda deegaan. Gebagebadii, isku xigxiga metagenomic waxa uu u taagan yahay sidii qalab muhiim u ah daah-furka dhexgalka genomic ee bulshooyinka yaryar ee kala duwan, isaga oo iftiiminaya xidhiidhada dhinacyo badan leh ee ka dhexeeya hidde-sideyaasha iyo deegaanka ee ka jira hab-nololeedyadan kakan.
Platforms: Illumina NovaSeq iyo DNBSEQ-T7
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Taxanaha Metagenomic-TGS
Metagenome-ku waa ururinta walxaha hidde-sidaha ee bulshada isku dhafan ee noolaha, sida deegaanka iyo metagenom-yada aadanaha. Waxa uu ka kooban yahay genome-ka noolaha la beeran karo iyo kuwa aan la beeran karin labadaba. Isku xigxiga metagenomic ayaa awood u siinaya barashada muuqaalada genomic-ga adag ee ku dhex jira muunado deegaan iyadoo la siinayo wax ka badan astaanta taxonomic. Waxa kale oo ay soo bandhigaysaa aragtida genomics ee shaqaynaysa iyada oo sahamisa hidda-sidaha la calaamadeeyay iyo doorkooda wax-ka-qabashada ee hababka deegaanka. Iyadoo hababka qoryaha dhaqameed ee isku xigxiga Illumina si weyn loogu isticmaalay daraasadaha metagenomic, imaanshaha Nanopore iyo PacBio taxanaha akhriska dheer ayaa bedelay goobta. Nanopore iyo tignoolajiyada PacBio waxay kor u qaadaan falanqaynta bioinformatic-ka ee hoose, gaar ahaan isu-ururinta metagenome, iyagoo hubinaya shirar joogto ah. Warbixinadu waxay muujinayaan in Nanopore-ku-salaysan iyo metagenomics-ku-salaysan PacBio ay si guul leh u soo saareen genomy-bakteeriyo dhamaystiran oo xiran oo ka yimid microbiomes adag (Moss, EL, et al., Nature Biotech, 2020). Isku-dubbaridka Nanopore wax-akhrinta Illumina waxay siinaysaa hab istiraatijiyad ah oo lagu saxayo khaladaadka, yaraynta saxnaanta hoose ee Nanopore. Isku-dhafkan isku-dhafka ah wuxuu ka faa'iidaysanayaa awoodaha madal kasta oo isku xigxiga, isagoo bixinaya xal adag si looga gudbo xaddidaadaha suurtagalka ah iyo horumarinta saxnaanta iyo isku halaynta falanqaynta metagenomic.
Platform: Nanopore PromethION 48, Illumina iyo PacBio Revio
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Isku xigxiga genome bisulfite (WGBS)
Dhammaan Genome Bisulfite Sequencing (WGBS) waxay u taagan tahay habka heerka-dahabka ah ee sahaminta qoto dheer ee methyl DNA-da, gaar ahaan booska shanaad ee cytosine (5-mC), xakameynta muhiimka ah ee muujinta hiddaha iyo dhaqdhaqaaqa gacanta. Mabda'a ka hooseeya WGBS waxay ku lug leedahay daawaynta bisulfit, taasoo keenaysa beddelka cytosines-ka aan methylated ilaa uracil (C ilaa U), iyadoo ka tagaysa cytosines methylated oo aan isbeddelin. Farsamadani waxay bixisaa xallin hal-saldhig ah, taasoo u oggolaanaysa cilmi-baarayaasha inay si buuxda u baaraan methylome-ka oo ay ogaadaan qaababka methylation-ka ee aan caadiga ahayn ee la xiriira xaalado kala duwan, gaar ahaan kansarka. Shaqaalaysiinta WGBS, saynisyahannadu waxay heli karaan aragtiyo aan la qiyaasi karin oo ku saabsan muuqaallada methylation-ka ee genom-ballaaran, iyagoo siinaya faham qotodheer oo ku saabsan hababka epigenetic ee hoos yimaada hababka noole ee kala duwan iyo cudurrada.
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Qiimaynta chromatin-ka-wareejin-ka-hortagga leh ee la heli karo
ATAC-seq waa farsamo isku xigxig heersare ah oo loo isticmaalo falanqaynta gelitaanka chromatin-ballaaran ee genome-ga. Waxay adeegsataa waxay bixisaa faham qoto dheer oo ku saabsan hababka qalafsan ee xakamaynta epigenetic ee caalamiga ah ee muujinta hidda-wadaha. Habka wuxuu isticmaalaa transposase Tn5 firfircoon si uu isku mar u kala googooyo oo u sumadeeyo gobollada chromatin ee furan iyadoo la gelinayo iskuxirayaasha isku xigxiga. Kordhinta PCR ee soo socota waxay keenaysaa abuurista maktabad isku xigta, taas oo u oggolaanaysa aqoonsiga dhammaystiran ee gobollada chromatin-ka furan ee hoos yimaada xaalado waqti-meel gaar ah. ATAC-seq waxay bixisaa aragti guud ee muuqaalada chromatin ee la heli karo, si ka duwan hababka kaliya ee diirada saaraya goobaha xidhitaanka qodobka qoraalka ama gobolo gaar ah oo wax laga beddelay. Iyadoo la isku xigxigayo gobolladan chromatin-ka ah ee furan, ATAC-seq waxay muujinaysaa gobollada ay u badan tahay inay noqdaan taxane nidaamsan oo firfircoon iyo goobaha suurtagalka ah ee qoraalka qoraalka, iyagoo siinaya fikrado qiimo leh oo ku saabsan habaynta firfircoon ee muujinta hidda-socodka ee guud ahaan genome-ka.
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16S/18S/ITS Amplicon Sequencing-PacBio
Hidde-sidayaasha 16S iyo 18S rRNA, oo ay weheliyaan gudaha gudaha Transcribed Spacer (ITS), waxay u adeegaan calaamadaynta faraha molecular ee muhiimka ah sababtoo ah isku darka gobollada aadka loo ilaaliyo iyo kuwa kala duwan, iyaga oo ka dhigaya qalab qiimo leh oo lagu garto noolaha prokaryotic iyo eukaryotic. Kordhinta iyo isku xigxiga gobolladan waxay bixiyaan hab gooni-gooni ah oo ka xor ah baaritaanka ka kooban microbial iyo kala duwanaanshiyaha dhammaan nidaamyada deegaanka ee kala duwan. Iyadoo isku xigxiga Illumina ay caadi ahaan bartilmaameedsato gobollada gaagaaban ee kala duwanaansho leh sida V3-V4 ee 16S iyo ITS1, waxaa la muujiyay in taxonomic taxonomic sare ah lagu gaari karo iyadoo la isku xigxigayo dhererka buuxa ee 16S, 18S, iyo ITS. Habkan dhammaystiran waxa uu keenayaa boqolley sare oo taxane ah oo si sax ah loo kala saaray, iyada oo la gaadhayo heer xal ah oo ku fidsan aqoonsiga noocyada. PacBio's Single-Molecule Real-Time Real-Time (SMRT) isku xigxiga madal waxay u taagan tahay siinta akhrin aad u dheer oo sax ah (HiFi) kaas oo daboolaya amplicon-yada dhererka buuxa, iyadoo la tartameysa saxnaanta isku xigxiga Illumina. Awooddani waxay u oggolaanaysaa cilmi-baarayaasha inay helaan faa'iido aan la isku mid ahayn - aragtida muuqaalka muuqaalka hiddaha. Daboolista la dheereeyey ayaa si weyn kor ugu qaadeysa xallinta sharraxaadda noocyada, gaar ahaan gudaha bulshooyinka bakteeriyada ama fangaska, taas oo awood u siineysa faham qoto dheer oo ku saabsan qallafsanaanta dadka microbial-ka ah.
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16S/18S/ITS Amplicon Sequencing-NGS
Isku xigxiga Amplicon ee tignoolajiyada Illumina, si gaar ah loo beegsanayo 16S, 18S, iyo ITS calaamadaha hidde-sideyaasha, waa hab awood leh oo lagu daah-furo phylogeny, taxonomy, iyo noocyada tirada badan ee bulshooyinka microbial. Habkani wuxuu ku lug leeyahay isku xigxiga gobollada kala duwanaansho badan ee calaamadaha hidde-waynta. Asal ahaan waxa loo soo bandhigay sidii faraha molecular byWoeses iyo al1977dii, farsamadani waxa ay wax ka beddeshay sifada microbiome iyada oo awood u siisay baadhisyo gooni-gooni ah. Iyadoo la raacayo taxanaha 16S (bakteeriyada), 18S (fungi), iyo Internal Transcribed Spacer (ITS, fungi), cilmi-baarayaashu waxay aqoonsan karaan ma aha oo kaliya noocyada badan laakiin sidoo kale kuwa naadir ah iyo kuwa aan la aqoonsan. Si ballaaran loo qaatay sidii qalab muhiim ah, isku xigxiga amplicon wuxuu noqday qalab lagu garto ka kooban halabuurka microbial ee kala duwan, oo ay ku jiraan afka aadanaha, mindhicirrada, saxarada, iyo wixii ka dambeeya.
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Dib-u-habaynta Bakteeriyada iyo Fungal Genom Genome
Bakteeriyada iyo fangas-dhammaan mashaariicda dib-u-xidhiidhinta-genome-ga ayaa muhiim u ah horumarinta genomics-yada microbial iyadoo awood u siinaysa dhammaystirka iyo isbarbardhigga genome-yada microbial. Tani waxay sahlaysa injineernimada halsano, hagaajinta hababka warshadaha, iyo sahaminta dariiqyada dheef-shiid kiimikaadka sare. Intaa waxaa dheer, dib-u-ku-soo-celinta fungal iyo bakteeriyada ayaa muhiim u ah fahamka la-qabsiga deegaanka, hagaajinta noocyada, iyo muujinta dhaqdhaqaaqa kobcinta hidde-socodka, oo leh saameyn ballaaran oo ku saabsan daawada, beeraha, iyo sayniska deegaanka.
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PacBio-dhererka-buuxa 16S/18S/ITS Taxanaha Amplicon
Amplicon (16S / 18S / ITS) madal waxaa lagu horumariyaa iyadoo sano oo waayo-aragnimo ah ee falanqaynta mashruuca kala duwanaansho microbial, kaas oo ka kooban falanqaynta aasaasiga ah ee caadiga ah iyo falanqaynta shakhsiyeed: falanqaynta aasaasiga ah daboolayaa content falanqaynta caadiga ah ee cilmi microbial hadda, content falanqaynta waa qani iyo dhamaystiran. iyo natiijooyinka falanqaynta waxaa lagu soo bandhigay qaab warbixin mashruuc; Nuxurka falanqaynta shakhsi ahaaneed waa kala duwan. Tusaalooyinka waa la dooran karaa waxaana loo dejin karaa si dabacsan iyadoo loo eegayo warbixinta falanqaynta aasaasiga ah iyo ujeedada cilmi-baarista, si loo xaqiijiyo shuruudaha shakhsi ahaaneed. Nidaamka hawlgalka Windows, fudud oo degdeg ah.
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PacBio-Dhar-Full Transcriptome (Tixraac la'aan)
Qaadashada xogta isku xigxiga ee Baasifiga (PacBio) Isoform sida galinta, App-kan waxa uu awoodaa in uu aqoonsado taxanaha qoraalka qoraalka dhererka buuxa (la'aan la kulmin). Marka la sawiro taxanaha dhererka buuxa ee lidka ku ah genome tixraaca, qoraalada waxaa lagu hagaajin karaa hiddo-wadaha la yaqaan, qoraallada qoraalka, gobollada codeynta, iwm. Xaaladdan, aqoonsi sax ah oo ka badan qaab-dhismeedka mRNA, sida kala-bax kale, iwm, ayaa la gaari karaa. Falanqaynta wadajirka ah ee xogta isku xigxiga qoraalka ee NGS waxay awood u siinaysaa sharraxaad dhammaystiran iyo tiraab sax ah oo muujinta heerka qoraalka, taas oo inta badan faa'iido u leh muujinta kala duwanaanta hoose iyo falanqaynta shaqeynta.
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Taxanaha Bisulfite ee la dhimay (RRBS)
Matalaad la dhimay ee Bisulfite Sequencing (RRBS) ayaa u soo baxday sidii kharash-ool iyo wax-ku-ool ah beddelka Dhammaan Genome Bisulfite Sequencing (WGBS) ee cilmi-baarista methylation DNA. Iyadoo WGBS ay bixiso aragtiyo dhammaystiran iyadoo lagu eegayo genome-ga oo dhan hal xal oo sal ah, qiimaheeda sare wuxuu noqon karaa arrin xaddidaysa. RRBS waxay si xeeladaysan u yaraynaysaa caqabadan iyadoo si xushmad leh u falanqaynaysa qayb ka mid ah genome-ka. Habkani wuxuu ku tiirsan yahay kobcinta gobollada jasiiradaha hodanka ah ee CpG iyadoo la kala gooyo MspI oo ay ku xigto cabbirka xulashada 200-500/600 bps jajab. Sidaa darteed, gobollada u dhow jasiiradaha CpG oo keliya ayaa la isku xig-xigay, halka kuwa jasiiradaha CpG fog leh laga saaray falanqaynta. Nidaamkan, oo ay weheliso isku-dhafka bisulfit, wuxuu u oggolaanayaa in la ogaado xallinta sare ee methylation DNA, iyo habka isku xigxiga, PE150, ayaa si gaar ah diiradda u saaraya cidhifyada gelinta halkii dhexe, kordhinta waxtarka methylation profile. RRBS waa qalab aad u qiimo badan oo awood u siinaya cilmi-baarista methylation DNA ee qiimaha-wax-ku-ool ah iyo horumarinta aqoonta hababka epigenetic.
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Taxanaha RNA Prokaryotic
Taxanaha RNA waxa uu awood siinayaa sifaynta dhamaystiran ee dhammaan qoraalada RNA ee ku jira unugyada xaalado gaar ah. Tiknoolajiyadan casriga ah waxay u adeegtaa sidii qalab awood leh, oo daaha ka qaadaysa muuqaalada muujinta hidda-wadaha, qaab-dhismeedka hidda-wadaha, iyo hababka molecular ee la xidhiidha hababka noole ee kala duwan. Si ballaaran loo qaatay cilmi baarista aasaasiga ah, baarista bukaan-socodka, iyo horumarinta daroogada, taxanaha RNA wuxuu bixiyaa aragtiyo ku saabsan qallafsanaanta dhaqdhaqaaqa gacanta iyo habaynta hiddaha. Habaynta muunada prokaryotic RNA eeyada waxa loo habeeyey qoraalada prokaryotic, ee ku lug leh dhimista rRNA iyo diyaarinta maktabada jihaynta.
Platform: Illumina NovaSeq
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Taxanaha Metatranscriptome
Ka faa'iidaysiga tignoolajiyada isku xigxiga ee Illumina, adeegga isku xigxiga ee metatranscriptome ee BMKGENE ayaa daaha ka qaadaya muujinta firfircoonida hidda-wadaha ee noocyo kala duwan oo microbes ah, oo u dhexeeya eukaryotes ilaa prokaryotes iyo fayrasyada, gudaha deegaannada dabiiciga ah sida ciidda, biyaha, badda, saxarada, iyo xiidmaha. Adeegeena dhamaystiran waxa uu awooda saarayaa cilmi-baarayaasha in ay dhexgalaan bulshooyinka microbial-ka adag ee muuqaalada muujinta hidda-wadaha. Marka laga soo tago falanqaynta taxonomic, adeegeena isku xigxiga ee metatranscriptome waxa uu fududeeyaa sahaminta kobcinta shaqaynta, iftiiminta hiddo-wadaha si kala duwan loo muujiyay iyo doorkooda. Soo hel fikrado badan oo bayooloji ah markaad dhex socoto muuqaalada kakan ee muujinta hidde-sidaha, kala duwanaanta taxonomiga, iyo firfircoonida shaqaynaysa gudaha meelahan kala duwan ee deegaanka.
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De novo Fungal Genome Assembly
BMKGENE waxay bixisaa xalal kala duwan oo loogu talagalay genome-ka fangas-ka, oo daboolaya baahiyaha cilmi-baarista ee kala duwan iyo dhammaystirka genome-ga la doonayo. Isticmaalka akhrinta gaaban ee Illumina kaligaa waxay ogolaataa abuuritaanka genome qabyo ah. Akhris-gaaban iyo isku xigxiga-akhrinta-dheer iyadoo la adeegsanayo Nanopore ama Pacbio ayaa lagu daraa genome fangas oo aad loo sifeeyay oo leh cidhifyo dhaadheer. Intaa waxaa dheer, isku-darka taxanaha Hi-C waxay sii kordhisaa awoodaha, taasoo awood u siinaysa helitaanka genome-heer-koromosoom oo dhammaystiran.
