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Ulandelelwano lweMetagenomic -NGS
I-metagenome yingqokelela yemathiriyeli yemfuzo iyonke yoluntu oluxubeneyo lwezinto eziphilayo, ezifana nokusingqongileyo kunye neemetagenomes zabantu. Iqulethe ii-genomes zombini ii-microorganisms ezinokulinywa kunye nezingenakulinywa. I-Shotgun metagenomic sequencing kunye ne-NGS yenza ukuba kuphononongwe le mihlaba intsonkothileyo ye-genomic emiliselwe kwiisampulu zokusingqongileyo ngokubonelela ngokungaphaya kweprofayili ye-taxonomic, inika ulwazi oluphangaleleyo kwiyantlukwano yeentlobo, ukuguquguquka kobuninzi, kunye nezakhiwo ezintsonkothileyo zabemi. Ngaphandle kwezifundo ze-taxonomic, i-shotgun metagenomics ikwabonelela ngembono esebenzayo ye-genomics, eyenza ukuphononongwa kofuzo olufakwe kwikhowudi kunye neendima zabo zokubeka kwiinkqubo zendalo. Okokugqibela, ukusekwa kothungelwano lothungelwano phakathi kwezinto zofuzo kunye nemiba yokusingqongileyo inegalelo ekuqondeni okupheleleyo konxibelelwano oluntsonkothileyo phakathi koluntu lwemicrobial kunye nemvelaphi yazo yendalo. Ukuqukumbela, ulandelelwano lwe-metagenomic limi njengesixhobo esibalulekileyo sokutyhila ubugocigoci be-genomic boluntu olwahlukeneyo lwe-microbial, ikhanyisela ubudlelwane obuninzi phakathi kofuzo kunye ne-ikholoji ngaphakathi kwezi zixokelelwano zendalo ezintsonkothileyo.
Iiplatforms: Illumina NovaSeq kunye ne-DNBSEQ-T7
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Ulandelelwano lweMetagenomic-TGS
I-metagenome yingqokelela yemathiriyeli yemfuzo yoluntu oluxubeneyo lwezinto eziphilayo, ezifana nokusingqongileyo kunye neemetagenomes zabantu. Iqulethe ii-genomes zombini ii-microorganisms ezinokulinywa kunye nezingenakulinywa. Ulandelelwano lwe-Metagenomic lwenza uphononongo lwezi ndawo zintsonkothileyo ze-genomic ezizinziswe kwiisampulu ze-ikholoji ngokubonelela ngokungaphezulu kweprofayili ye-taxonomic. Ikwabonelela ngembono esebenzayo ye-genomics ngokuphonononga ijene ekhowudiweyo kunye neendima zabo zokubeka kwiinkqubo zokusingqongileyo. Ngelixa i-shotgun yendabuko isondela kunye nokulandelelana kwe-Illumina isetyenziswe ngokubanzi kwizifundo ze-metagenomic, ukufika kwe-Nanopore kunye ne-PacBio yokulandelelana kwexesha elide iguqule intsimi. Itekhnoloji yeNanopore kunye nePacBio iphucula uhlalutyo lwe-bioinformatic esezantsi, ngakumbi indibano yemetagenome, iqinisekisa iindibano eziqhubekayo. Iingxelo zibonisa ukuba i-Nanopore-based and PacBio-based metagenomics ivelise ngempumelelo i-genomes yebhakteria epheleleyo kunye nevaliweyo ukusuka kwi-microbiomes eyinkimbinkimbi (Moss, EL, et al., Nature Biotech, 2020). Ukudibanisa i-Nanopore ifundeka kunye nokufundwa kwe-Illumina kunika indlela yobuchule yokulungiswa kweempazamo, ukunciphisa ukuchaneka okuphantsi kweNanopore. Le ndibaniselwano ye-synergistic iphakamisa amandla eqonga ngalinye lokulandelelana, linikezela ngesisombululo esomeleleyo sokunqoba imida enokubakho kunye nokuqhubela phambili ukuchaneka kunye nokuthembeka kohlalutyo lwe-metagenomic.
Iqonga: iNanopore PromethION 48, Illumia kunye nePacBio Revio
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I-Whole genome bisulfite sequencing(WGBS)
I-Whole Genome Bisulfite Sequencing (WGBS) ime njengendlela yomgangatho wegolide wokuhlola nzulu kwe-DNA methylation, ngokukodwa indawo yesihlanu kwi-cytosine (5-mC), umlawuli obalulekileyo we-gene expression kunye nomsebenzi weselula. Umgaqo ophantsi kwe-WGBS ubandakanya unyango lwe-bisulfite, ukuguqula ukuguqulwa kwe-cytosines engaxutywanga kwi-uracil (C ukuya ku-U), ngelixa ishiya i-cytosine ye-methylated ingatshintshi. Obu buchule bubonelela ngesisombululo esisisiseko esinye, sivumela abaphandi ukuba baphande ngokubanzi i-methylome kwaye bafumanise iipateni ze-methylation ezingaqhelekanga ezinxulumene neemeko ezahlukeneyo, ngakumbi umhlaza. Ngokusebenzisa i-WGBS, izazinzulu zinokufumana ulwazi olungenakuthelekiswa nanto kwi-genome-wide methylation landscapes, ukunika ukuqonda okucacileyo kweendlela ze-epigenetic eziphantsi kweenkqubo ezahlukeneyo zebhayoloji kunye nezifo.
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I-Assay ye-Chromatin eFumanekayo ye-Transposase ene-High throughput Sequencing (ATAC-seq)
I-ATAC-seq yindlela yolandelelwano oluphezulu olusetyenziselwa uhlalutyo lokufikeleleka kwechromatin yegenome ngokubanzi. Ukusetyenziswa kunika ukuqonda okunzulu kwiindlela ezintsonkothileyo zolawulo lwe-epigenetic lwehlabathi malunga nokuchazwa kofuzo. Indlela isebenzisa i-hyperactive Tn5 transposase ukuqhekeza ngaxeshanye kwaye ithegi imimandla yechromatin evulekileyo ngokufaka iiadaptha zolandelelwano. Ukwandiswa okulandelayo kwePCR iziphumo ekudalweni kwethala leencwadi elilandelelanayo, elivumela ukuchongwa ngokubanzi kwemimandla yechromatin evulekileyo phantsi kweemeko ezithile zexesha lesithuba. I-ATAC-seq ibonelela ngombono obanzi wokufikeleleka kwe-chromatin landscapes, ngokungafaniyo neendlela ezigxile kuphela kwiindawo ezibophelelayo ze-transcription factor okanye imimandla ethile ye-histone-modified. Ngokulandelelanisa le mimandla yechromatin ivulekileyo, i-ATAC-seq ityhila imimandla ekunokwenzeka ukuba ibe nolandelelwano lolawulo olusebenzayo kunye neendawo ezinokubakho zokukhuphela izinto ezibophelelayo, inikezela ngezimvo ezixabisekileyo kumodareyithwa oguqukayo wokubonakaliswa kofuzo kuyo yonke igenome.
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16S/18S/ITS Amplicon Sequencing-PacBio
Iijini ze-rRNA ze-16S kunye ne-18S, kunye nengingqi ye-Internal Transcribed Spacer (ITS), isebenza njengeyona nto ibalulekileyo yokuphawula iminwe yemolekyuli ngenxa yokudityaniswa kwemimandla egcinwe kakhulu kunye ne-hyper-variable, izenza izixhobo ezixabiseke kakhulu zokubonisa iimpawu zeprokaryotic kunye ne-eukaryotic. Ukwandiswa kunye nolandelelwano lwale mimandla lubonelela ngendlela engenakwahlulwa yokuphanda ubume bemicrobial kunye neyantlukwano kuzo zonke ii-ecosystems. Ngelixa ulandelelwano lwe-Illumina ngokuqhelekileyo lujolise kwimimandla emifutshane ye-hypervariable efana ne-V3-V4 ye-16S kunye ne-ITS1, kubonakaliswe ukuba i-taxonomic annotation ephezulu ifikeleleke ngokulandelelana ubude obupheleleyo be-16S, 18S, kunye ne-ITS. Le ndlela ibanzi iphumela kwiipesenti eziphezulu zolandelelwano oluhlelwe ngokuchanekileyo, ukufikelela kwinqanaba lesisombululo esinabela ekuchongweni kweentlobo. I-PacBio ye-Single-Molecule ye-Real-Time (SMRT) iqonga lokulandelelanisa iqonga livelele ngokubonelela ngofundo oluchanekileyo olude (i-HiFi) olugubungela i-amplicon yobude obupheleleyo, ikhuphisana nokuchaneka kokulandelelana kwe-Illumina. Esi sikhundla sivumela abaphandi ukuba bafumane inzuzo engenakulinganiswa - imbono ye-panoramic ye-genetic landscape. Ugutyulo olwandisiweyo lusiphakamisa kakhulu isisombululo kwingcaciso yeentlobo, ngakumbi phakathi kweebhaktiriya okanye kuluntu lomngundo, okwenza ukuba kuqondwe nzulu ukuntsonkotha kobuninzi bentsholongwane.
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16S/18S/ITS Amplicon Sequencing-NGS
Ukulandelelana kwe-Amplicon kunye neteknoloji ye-Illumina, ejolise ngokukodwa kwi-16S, i-18S, kunye neempawu ze-ITS zofuzo, yindlela enamandla yokutyhila i-phylogeny, i-taxonomy, kunye nobuninzi beentlobo ngaphakathi kwe-microbial community. Le ndlela ibandakanya ukulandelelana kwemimandla eguquguqukayo yogcino lweempawu zofuzo. Yaziswa ekuqaleni njengophawu lweminwe yemolekyuli nguWoeses et alngo-1977, obu buchule buguqule iprofayili ye-microbiome ngokwenza uhlalutyo olungakhethiyo. Ngokulandelelana kwe-16S (iibhaktheriya), i-18S (i-fungi), kunye ne-Internal Transcribed Spacer (ITS, i-fungi), abaphandi abakwazi ukuchonga kuphela iintlobo ezininzi kodwa kunye nezinqabileyo nezingaziwa. Yamkelwa ngokubanzi njengesixhobo esibalulekileyo, ulandelelwano lwe-amplicon lube sisixhobo ekuqondeni ukuqanjwa kwe-microbial eyahlukileyo kwiindawo ezahlukeneyo, kubandakanya umlomo womntu, amathumbu, isitulo, nangaphaya.
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IBacterial kunye neFungal Whole Genome Re-Sequencing
Iiprojekthi zebhaktiriya kunye nokungunda kokulandelelana kwakhona kwegenome zibalulekile ekuqhubeleni phambili i-microbial genomics ngokwenza ukuba kugqitywe kwaye kuthelekiswe i-microbial genomes. Oku kuququzelela ubunjineli bokubiliswa, ukulungelelaniswa kweenkqubo zoshishino, kunye nokuphononongwa kweendlela zemetabolism yesibini. Ngaphaya koko, ulandelelwano lokungunda kunye nebhaktiriya lubalulekile ekuqondeni ulungelelwaniso lwendalo esingqongileyo, ukukhulisa ubunzima, kunye nokutyhila ukuguquguquka kofuzo, kunye nefuthe elibanzi kwezamayeza, ezolimo, kunye nesayensi yokusingqongileyo.
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PacBio-Full-length 16S/18S/ITS Amplicon Sequencing
Iqonga le-Amplicon (16S / 18S / ITS) liphuhliswa kunye neminyaka yamava kuhlalutyo lweprojekthi ye-microbial diversity, equlethe uhlalutyo olusisiseko olusemgangathweni kunye nohlalutyo lomntu siqu: uhlalutyo olusisiseko lubandakanya umxholo wohlalutyo oluqhelekileyo lophando lwangoku lwe-microbial, umxholo wokuhlalutya ucebile kwaye ubanzi, kunye neziphumo zohlalutyo zinikezelwe ngohlobo lweengxelo zeprojekthi; Umxholo wohlalutyo lobuqu uhlukile. Iisampulu zinokukhethwa kwaye iiparameters zinokumiselwa ngokuguquguqukayo ngokwengxelo yohlalutyo olusisiseko kunye nenjongo yophando, ukufezekisa iimfuno zomntu. Inkqubo yokusebenza yeWindows, ilula kwaye iyakhawuleza.
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I-PacBio-ubude obugcweleyo be-Transcriptome (Non-Reference)
Ukuthatha i-Pacific Biosciences (i-PacBio) ye-Isoform yokulandelelanisa idatha njengegalelo, le App iyakwazi ukuchonga ulandelelwano olupheleleyo lwe-transcript (ngaphandle kwendibano). Ngokwenza imephu yolandelelwano lobude obupheleleyo ngokuchasene ne-genome yereferensi, imibhalo inokuthi iphuculwe ngejene eyaziwayo, imibhalo ebhaliweyo, imimandla yekhowudi, njl.njl. Kule meko, ukuchongwa okuchaneke ngakumbi kwezakhiwo ze-mRNA, njengokunye ukudityaniswa, njl njl, kunokufezekiswa. Uhlalutyo oludityanisiweyo kunye nedatha ye-NGS yolandelelwano lwe-transcriptome yenza ukuba inkcazo ebanzi ngakumbi kunye nobungakanani obuchanekileyo ekubonakalisweni kwinqanaba le-transcript, elizuza kakhulu kwintetho ephantsi yolwahlulo kunye nohlalutyo olusebenzayo.
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Umekelo oluNcitshisiweyo lweBisulfite Sequencing (RRBS)
I-Reduced Representation Bisulfite Sequencing (RRBS) iye yavela njengendlela engabizi kwaye esebenzayo kwi-Whole Genome Bisulfite Sequencing (WGBS) kuphando lwe-DNA methylation. Ngelixa i-WGBS ibonelela ngemibono ebanzi ngokuphonononga yonke i-genome kwisisombululo sesiseko esinye, iindleko zayo eziphezulu zinokuba ngumba osisithintelo. I-RRBS ngobuchule iwunciphisa lo mngeni ngokuhlalutya ngokukhetha inxalenye emele yegenome. Le ndlela yokusebenza ixhomekeke ekutyebiseni imimandla etyebileyo yeziqithi ze-CpG ngokucolwa kwe-MspI kulandele ukukhethwa kobungakanani bamaqhekeza angama-200-500/600 e-bps. Ngenxa yoko, kuphela imimandla esondele kwiziqithi ze-CpG ezilandelelanayo, ngelixa ezo zineziqithi ze-CpG ezikude zingabandakanywa kuhlalutyo. Le nkqubo, idibene ne-bisulfite sequencing, ivumela ukufunyanwa kwe-high-resolution ye-DNA methylation, kunye nendlela yokulandelelana, i-PE150, igxininise ngokukodwa kwiiphelo zokufaka esikhundleni se-middle, ukwandisa ukusebenza kakuhle kwe-methylation profiling. I-RRBS sisixhobo esixabisekileyo esenza ukuba uphando lwe-DNA methylation lungabizi kakhulu kwaye luqhubele phambili ulwazi lweendlela ze-epigenetic.
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Prokaryotic RNA Ulandelelwano
Ulandelelwano lwe-RNA luxhobisa iprofayili ebanzi yazo zonke iikopi ze-RNA ngaphakathi kweeseli phantsi kweemeko ezithile. Le teknoloji ye-cutting-edge isebenza njengesixhobo esinamandla, ityhila iiprofayili zegenesis ezintsonkothileyo, ulwakhiwo lwemfuza, kunye neendlela zemolekyuli ezinxulumene neenkqubo ezahlukeneyo zebhayoloji. Yamkelwa ngokubanzi kuphando olusisiseko, uxilongo lweklinikhi, kunye nophuhliso lwamachiza, ulandelelwano lwe-RNA lubonelela ngokuqonda kubuntsonkothe bokutshintshatshintsha kweeseli kunye nolawulo lwemfuzo. Inkqubo yethu yesampulu yeprokaryotic ye-RNA ilungiselelwe i-prokaryotic transcriptomes, ebandakanya ukuncipha kwe-rRNA kunye nokulungiswa kwethala leencwadi.
Iqonga: Illumina NovaSeq
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Ulandelelwano lweMetatranscriptome
Itekhnoloji yokulandelelanisa i-Illumina, inkonzo yolandelelwano ye-metaranscriptome ye-BMKGENE ityhila ukubonakaliswa kofuzo oluguquguqukayo lweentlobo ngeentlobo zeentsholongwane, ezithatha ii-eukaryotes ukuya kwiiprokaryotes kunye neentsholongwane, ngaphakathi kwendawo yendalo efana nomhlaba, amanzi, ulwandle, isitulo, kunye namathumbu. Inkonzo yethu ebanzi ixhobisa abaphandi ukuba baphengulule kwiiprofayile ezipheleleyo zegenesis yegenesis yasekuhlaleni. Ngaphandle kohlalutyo lwe-taxonomic, inkonzo yethu yolandelelwano ye-metatranscriptome iququzelela ukuphononongwa ekutyebiseni okusebenzayo, ikhanyisela iigenes ezichazwe ngokwahlukileyo kunye neendima zabo. Fumanisa ubutyebi bengqiqo yebhayoloji njengoko ujonga kumhlaba ontsonkothileyo wokuchazwa kofuzo, iyantlukwano yetaxonomic, kunye nokuguquguquka okusebenzayo ngaphakathi kwezi niches zahlukeneyo zokusingqongileyo.
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De novo Fungal Genome Assembly
I-BMKGENE ibonelela ngezisombululo eziguquguqukayo kwiigenomes zokungunda, ukulungiselela iimfuno zophando ezahlukeneyo kunye nokugqibelela kwegenome enqwenelekayo. Ukusebenzisa ukulandelelana kwe-Illumina okufutshane kuphela kuvumela ukuveliswa kwe-genome eyidrafti. Ukufundwa okufutshane kunye nokulandelelana kwexesha elide usebenzisa i-Nanopore okanye i-Pacbio zidityanisiwe kwi-genome yefungal esulungekileyo kunye ne-contigs emide. Ngaphezu koko, ukudibanisa ulandelelwano lwe-Hi-C kuphucula ngakumbi amandla, okwenza ukuba kufunyanwe igenome yomgangatho wechromosome epheleleyo.
