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Ukulandelana kwe-Metagenomic -NGS
I-metagenome iqoqo lengqikithi yezakhi zofuzo zomphakathi oxubile wezinto eziphilayo, ezifana nemvelo kanye ne-metagenomes yabantu. Iqukethe ama-genomes womabili ama-microorganisms alinywayo nangenakulinywa. Ukulandelana kwe-Shotgun metagenomic ne-NGS kuvumela ukucwaninga kwalezi zindawo eziyinkimbinkimbi ze-genomic ezishumekwe kumasampuli emvelo ngokunikeza okungaphezu kokwenza iphrofayili ye-taxonomic, kunikeze nemininingwane eyimbudumbudu ngokuhlukahluka kwezinhlobo zezilwane, ukuguquguquka kobuningi, kanye nezakhiwo eziyinkimbinkimbi zabantu. Ngaphandle kwezifundo ze-taxonomic, i-shotgun metagenomics iphinde inikeze umbono osebenzayo we-genomics, ovumela ukuhlolwa kwezakhi zofuzo ezifakwe ikhodi kanye nezindima zazo zokubeka ezinqubweni zemvelo. Okokugcina, ukusungulwa kwamanethiwekhi okuxhumana phakathi kwezakhi zofuzo kanye nezici zemvelo kunomthelela ekuqondeni okuphelele kokuhlangana okuyinkimbinkimbi phakathi kwemiphakathi yezilwanyana ezincane kanye nesizinda sayo semvelo. Sengiphetha, ukulandelana kwe-metagenomic kumi njengethuluzi elibalulekile lokwembula ubunkimbinkimbi bofuzo lwemiphakathi ehlukahlukene yamagciwane, okukhanyisa ubudlelwano obunezinhlangothi eziningi phakathi kofuzo kanye ne-ecology ngaphakathi kwalezi zinhlelo zemvelo eziyinkimbinkimbi.
Amapulatifomu: Illumina NovaSeq kanye ne-DNBSEQ-T7
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I-Metagenomic Sequencing-TGS
I-metagenome iqoqo lezakhi zofuzo zomphakathi oxubile wezinto eziphilayo, ezifana nemvelo kanye ne-metagenomes yabantu. Iqukethe ama-genomes womabili ama-microorganisms alinywayo nangenakulinywa. Ukulandelana kwe-Metagenomic kunika amandla ukucwaninga kwalezi zindawo eziyinkimbinkimbi ze-genomic ezishumekwe kumasampula emvelo ngokunikeza okungaphezu kokuphrofayili we-taxonomic. Iphinde inikeze umbono osebenzayo we-genomics ngokuhlola izakhi zofuzo ezifakwe ikhodi kanye nezindima zazo zokubeka ezinqubweni zemvelo. Ngenkathi isibhamu sendabuko sisondela ngokulandelana kwe-Illumina sisetshenziswe kakhulu ezifundweni ze-metagenomic, ukuvela kwe-Nanopore ne-PacBio ukulandelana okufundwe isikhathi eside kuye kwashintsha insimu. Ubuchwepheshe be-Nanopore ne-PacBio buthuthukisa ukuhlaziya kwe-bioinformatic engezansi, ikakhulukazi ukuhlangana kwe-metagenome, okuqinisekisa ukuhlangana okuqhubekayo. Imibiko ikhombisa ukuthi i-Nanopore-based and PacBio-based metagenomics ikhiqize ngempumelelo ama-genome ebhaktheriya aphelele navaliwe avela kuma-microbiomes ayinkimbinkimbi (Moss, EL, et al., Nature Biotech, 2020). Ukuhlanganisa ukufundwa kwe-Nanopore nokufundwa kwe-Illumina kunikeza indlela yamasu yokulungisa amaphutha, kunciphisa ukunemba okuphansi kwe-Nanopore okungokwemvelo. Le nhlanganisela ye-synergistic isebenzisa amandla enkundla ngayinye yokulandelana, inikeza isixazululo esiqinile sokunqoba imikhawulo engaba khona futhi ithuthukise ukunemba nokuthembeka kokuhlaziywa kwe-metagenomic.
I-Platform: Nanopore PromethION 48, Illumia ne-PacBio Revio
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I-Whole genome bisulfite sequencing(WGBS)
I-Whole Genome Bisulfite Sequencing (WGBS) ime njengendlela yegolide esezingeni eliphezulu yokuhlola ngokujulile i-DNA methylation, ikakhulukazi indawo yesihlanu ku-cytosine (5-mC), umlawuli obalulekile wokuvezwa kofuzo kanye nomsebenzi wamaselula. Umgomo oyisisekelo we-WGBS uhilela ukwelashwa kwe-bisulfite, okwenza ukuguqulwa kwama-cytosine angenayo i-methylated abe yi-uracil (C kuya ku-U), kuyilapho kushiya ama-cytosine e-methylated engashintshiwe. Le nqubo inikezela ngesixazululo esisisekelo esisodwa, okuvumela abacwaningi ukuthi baphenye kabanzi nge-methylome futhi bathole amaphethini e-methylation angajwayelekile ahlobene nezimo ezahlukahlukene, ikakhulukazi umdlavuza. Ngokusebenzisa i-WGBS, ososayensi bangathola ukuqonda okungenakuqhathaniswa ku-genome-wide methylation landscapes, banikeze ukuqonda okufingqiwe kwezinqubo ze-epigenetic ezingaphansi kwezinqubo nezifo ezihlukahlukene zebhayoloji.
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I-Assay ye-Chromatin Efinyelelekayo Ye-Transposase Enokulandelanisa Okuphezulu Kokudlula (i-ATAC-seq)
I-ATAC-seq iyindlela yokulandelanisa ephezulu esetshenziselwa ukuhlaziywa kokufinyeleleka kwe-chromatin yofuzo olubanzi. Ukusetshenziswa kunikeza ukuqonda okujulile kwezinqubo eziyinkimbinkimbi zokulawula i-epigenetic emhlabeni jikelele phezu kokuchazwa kwezakhi zofuzo. Indlela isebenzisa i-hyperactive Tn5 transposase ukuze ngesikhathi esisodwa ihlukanise futhi ithege izifunda ze-chromatin ezivulekile ngokufaka ama-adaptha alandelanayo. Ukukhulisa i-PCR okulandelayo kuphumela ekudalweni kwelabhulali elandelanayo, evumela ukuhlonza okuphelele kwezifunda ze-chromatin evulekile ngaphansi kwezimo ezithile zesikhathi sesikhala. I-ATAC-seq inikeza umbono ophelele wezindawo ze-chromatin ezifinyelelekayo, ngokungafani nezindlela ezigxila kuphela kumasayithi abophezela isici sokuloba noma izifunda ezithile ezilungiswe i-histone. Ngokulandelanisa lezi zifunda ze-chromatin evulekile, i-ATAC-seq iveza izifunda okungenzeka kakhulu ukuthi zilandele ukulandelana kokulawula okusebenzayo kanye namasayithi angaba khona abopha isici sokuloba, enikeza imininingwane ebalulekile ekuguquguqukeni okuguquguqukayo kwesisho sofuzo kuyo yonke igenome.
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16S/18S/ITS Amplicon Sequencing-PacBio
Izakhi zofuzo ze-16S kanye ne-18S rRNA, kanye nesifunda se-Internal Transcribed Spacer (ITS), zisebenza njengezigxivizo zeminwe zemolekyuli ngenxa yenhlanganisela yazo yezifunda ezilondolozwe kakhulu neziguquguqukayo, okuzenza zibe amathuluzi abalulekile okubonisa izinto eziphilayo ze-prokaryotic ne-eukaryotic. Ukukhulisa nokulandelana kwalezi zifunda kunikeza indlela yokungahlukaniswa yodwa yokuphenya ukwakheka kwama-microbial kanye nokwehlukahlukana ezindaweni ezihlukahlukene zemvelo. Nakuba ukulandelana kwe-Illumina ngokuvamile kuqondise izifunda ezimfushane ezingaguquguquki njenge-V3-V4 ye-16S ne-ITS1, kuboniswe ukuthi isichasiselo se-taxonomic esiphezulu sifinyeleleka ngokulandelanisa ubude obugcwele be-16S, 18S, ne-ITS. Le ndlela ebanzi iphumela kumaphesenti aphezulu okulandelana okuhlukaniswe ngokunembile, kufinyelele izinga lokuxazulula elidlulela ekuhlonzweni kwezinhlobo zezilwane. Inkundla yokulandelana ye-Single-Molecule Real-Time (SMRT) ye-PacBio igqama ngokunikeza ukufundwa okude okunembe kakhulu (i-HiFi) okumboza ama-amplicon anobude obugcwele, ephikisana nokunemba kokulandelana kwe-Illumina. Leli khono livumela abacwaningi ukuthi bathole inzuzo engenakuqhathaniswa - umbono we-panoramic we-genetic landscape. Ukufakwa okunwetshiwe kuphakamisa kakhulu ukulungiswa kwesichasiselo sezinhlobo zezilwane, ikakhulukazi phakathi kwemiphakathi yamagciwane noma isikhunta, okuvumela ukuqonda okujulile kobunzima bezibalo zezinambuzane.
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16S/18S/ITS Amplicon Sequencing-NGS
Ukulandelana kwe-Amplicon ngobuchwepheshe be-Illumina, eqondise ngokukhethekile omaka bezakhi zofuzo be-16S, 18S, kanye ne-ITS, kuyindlela enamandla yokwembula ukuchichima kwe-phylogeny, taxonomy, kanye nezinhlobo zezilwane ngaphakathi kwemiphakathi encane. Le ndlela ibandakanya ukulandelana kwezifunda ezingaguquguquki zezimpawu zofuzo zokugcina izindlu. Ekuqaleni yethulwe njengezigxivizo zeminwe ze-molecular byWoeses et alngo-1977, le ndlela iguqule iphrofayili ye-microbiome ngokuvumela ukuhlaziya okungenakho ukuhlukaniswa. Ngokulandelana kwe-16S (ibhaktheriya), i-18S (isikhunta), kanye ne-Internal Transcribed Spacer (ITS, isikhunta), abacwaningi abakwazi ukuhlonza kuphela izinhlobo zezilwane kodwa nezingavamile nezingaziwa. Njengoba yamukelwe kabanzi njengethuluzi elibalulekile, ukulandelana kwe-amplicon sekube wusizo ekuhlukaniseni ukwakheka kwama-microbial ahlukene ezindaweni ezihlukahlukene, okuhlanganisa umlomo womuntu, amathumbu, indle, nangale kwalokho.
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Ukulandelana Kabusha Kwe-Genome Yamagciwane kanye Nesikhunta
Amaphrojekthi okulandelana kabusha kwe-bacterial and fungal whole-genome abalulekile ekuthuthukiseni i-microbial genomics ngokunika amandla ukuqedwa nokuqhathaniswa kwama-microbial genomes. Lokhu kusiza ubunjiniyela bokuvutshelwa, ukwenziwa kahle kwezinqubo zezimboni, kanye nokuhlolwa kwemizila yesibili yemetabolism. Ngaphezu kwalokho, ukulandelana kabusha kwesikhunta namagciwane kubalulekile ekuqondeni ukujwayela kwemvelo, ukukhulisa izinhlobo, nokwembula ukuguquguquka kofuzo, okunomthelela obanzi kwezokwelapha, ezolimo, kanye nesayensi yezemvelo.
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I-PacBio-Full-length 16S/18S/ITS Amplicon Sequencing
Iplathifomu ye-Amplicon (16S/18S/ITS) yathuthukiswa yaba neminyaka yokuhlangenwe nakho ekuhlaziyweni kwephrojekthi ye-microbial diversity, equkethe ukuhlaziya okuyisisekelo okujwayelekile kanye nokuhlaziya komuntu siqu: ukuhlaziya okuyisisekelo kuhlanganisa okuqukethwe kokuhlaziya okujwayelekile kocwaningo lwamanje lwe-microbial, okuqukethwe kokuhlaziya kucebile futhi kubanzi, futhi imiphumela yokuhlaziya yethulwa ngendlela yemibiko yephrojekthi; Okuqukethwe kokuhlaziywa komuntu siqu kuhlukahlukene. Amasampuli angakhethwa futhi amapharamitha angasethwa ngendlela evumelana nezimo ngokuvumelana nombiko wokuhlaziya oyisisekelo kanye nenjongo yocwaningo, ukuze kufezeke izidingo eziqondene nawe. Isistimu yokusebenza yeWindows, elula futhi esheshayo.
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I-PacBio-Full-length Transcriptome (Non-Reference)
Ithatha idatha yokulandelanisa ye-Pacific Biosciences (PacBio) Isoform njengokufakwayo, lolu hlelo lokusebenza luyakwazi ukubona ukulandelana kokulotshiweyo kobude obugcwele (ngaphandle kokuhlanganisa). Ngokwenza imephu ukulandelana kobude obugcwele ngokumelene ne-reference genome, okulotshiweyo kungenziwa ngcono ngezakhi zofuzo ezaziwayo, imibhalo, izifunda zekhodi, njll. Kulokhu, ukuhlonza okunembe kakhudlwana kwezakhiwo ze-mRNA, njengokunye ukuhlanganisa, njll, kungafinyelelwa. Ukuhlaziywa okuhlangene nedatha yokulandelanisa ye-NGS transcriptome kunika amandla isichasiselo esibanzi kanye nokulinganisa okunembe kakhudlwana ekukhulumeni ezingeni lokuloba, okuzuzisa kakhulu ukuvezwa komehluko ongezansi nokuhlaziywa kokusebenza.
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I-Reduced Representation Bisulfite Sequencing (RRBS)
I-Reduced Representation Bisulfite Sequencing (RRBS) ivele njengendlela engabizi futhi ephumelelayo ye-Whole Genome Bisulfite Sequencing (WGBS) ocwaningweni lwe-DNA methylation. Ngenkathi i-WGBS ihlinzeka ngemininingwane ebanzi ngokuhlola yonke i-genome ekuxazululeni kwesisekelo esisodwa, izindleko zayo eziphezulu zingaba yisici esikhawulelayo. I-RRBS inciphisa ngokuhlelekile le nselele ngokukhetha ingxenye emele yofuzo. Le ndlela yokwenza incike ekuthuthukisweni kwezifunda ezicebile eziqhingini ze-CpG nge-MspI cleavage elandelwa ukukhethwa kosayizi wezingcezu ze-bps ezingu-200-500/600. Ngokulandelayo, yizifunda kuphela eziseduze neziqhingi ze-CpG ezilandelanayo, kuyilapho lezo ezineziqhingi ezikude ze-CpG azifakwanga ekuhlaziyweni. Le nqubo, ehlangene nokulandelana kwe-bisulfite, ivumela ukutholakala kwe-high-resolution ye-DNA methylation, kanye nendlela yokulandelana, i-PE150, igxile ikakhulukazi emaphethelweni okufaka esikhundleni esiphakathi, okwandisa ukusebenza kahle kwephrofayili ye-methylation. I-RRBS iyithuluzi eliyigugu elivumela ucwaningo lwe-DNA methylation olungabizi futhi lithuthukise ulwazi lwezinqubo ze-epigenetic.
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Ukulandelana kwe-Prokaryotic RNA
Ukulandelana kwe-RNA kunika amandla ukuphrofayili okuphelele kwakho konke okulotshiwe kwe-RNA ngaphakathi kwamaseli ngaphansi kwezimo ezithile. Lobu buchwepheshe obusezingeni eliphezulu busebenza njengethuluzi elinamandla, lembula amaphrofayli ayinkimbinkimbi enkulumo yofuzo, izakhiwo zofuzo, nezindlela zamangqamuzana ezihambisana nezinqubo ezihlukahlukene zebhayoloji. Yamukelwe kabanzi ocwaningweni oluyisisekelo, ekuxilongweni komtholampilo, nasekuthuthukisweni kwezidakamizwa, ukulandelana kwe-RNA kunikeza ukuqonda ngobunkimbinkimbi bokuguquguquka kwamaselula kanye nokulawulwa kofuzo. Ukucubungula kwethu kwesampula ye-prokaryotic RNA yakhelwe ukubhalwa kwe-prokaryotic, okubandakanya ukuncipha kwe-rRNA kanye nokulungiswa komtapo wolwazi.
Ipulatifomu: Illumina NovaSeq
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Ukulandelana kwe-Metatranscriptome
Ubuchwepheshe bokulandelana kwe-Illumina, isevisi yokulandelana kwe-metaranscriptome ye-BMKGENE yembula ukubonakaliswa kofuzo okuhlukahlukene kwezinhlobonhlobo zamagciwane, ahlanganisa ama-eukaryote kuya kuma-prokaryotes nama-virus, endaweni yemvelo efana nomhlabathi, amanzi, ulwandle, indle, namathumbu. Isevisi yethu ebanzi inika amandla abacwaningi ukuthi bahlole amaphrofayli aphelele ezinhlobo zofuzo zemiphakathi eyinkimbinkimbi. Ngaphandle kokuhlaziywa kwe-taxonomic, isevisi yethu yokulandelanisa i-metatranscriptome isiza ukuhlola ekuthuthukisweni kokusebenza, okukhanyisa izakhi zofuzo ezivezwe ngokuhlukile kanye nezindima zazo. Yembula ingcebo yemininingwane yebhayoloji njengoba uzulazula ezindaweni eziyinkimbinkimbi zokuchazwa kofuzo, ukwehlukahlukana kwe-taxonomic, namandla okusebenza ngaphakathi kwalezi zindawo ezihlukahlukene zemvelo.
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I-Novo Fungal Genome Assembly
I-BMKGENE inikeza izixazululo eziguquguqukayo zezakhi zofuzo zesikhunta, ihlinzekela izidingo zocwaningo ezihlukahlukene kanye nokuphelela kofuzo olufiswayo. Ukusebenzisa ukulandelana kwe-Illumina yokufunda okufushane kuphela kuvumela ukukhiqizwa kwe-genome esalungiswa. Ukufunda okufushane nokufunda isikhathi eside kusetshenziswa i-Nanopore noma i-Pacbio kuhlanganiswe ukuze kube nofuzo lwesikhunta olucwengisiswe kakhulu olunama-contig amade. Ngaphezu kwalokho, ukuhlanganisa ukulandelana kwe-Hi-C kuthuthukisa futhi amandla, kuvumela ukutholwa kwegenome yeleveli ye-chromosome ephelele.
