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Metagenomic Sequencing -NGS
Metagenome ke pokello ea kakaretso ea liphatsa tsa lefutso tsa sehlopha se tsoakaneng sa lintho tse phelang, tse kang tikoloho le metagenome ea batho. E na le liphatsa tsa lefutso tsa likokoana-hloko tse lengoang le tse ke keng tsa lengoa. Shotgun metagenomic sequencing le NGS e nolofalletsa boithuto ba libaka tsena tse rarahaneng tsa genomic tse kentsoeng ka har'a lisampole tsa tikoloho ka ho fana ka lintlha tse fetang tsa taxonomic, ho fana ka leseli le hlakileng mabapi le mefuta-futa ea mefuta, matla a mangata le libopeho tse rarahaneng tsa baahi. Ntle le lithuto tsa taxonomic, shotgun metagenomics e boetse e fana ka pono e sebetsang ea genomics, e nolofalletsang ho hlahlojoa ha liphatsa tsa lefutso tse kentsoeng le likarolo tsa tsona tsa ho beha lintho lits'ebetsong tsa tikoloho. Qetellong, ho thehoa ha marang-rang a khokahano lipakeng tsa likarolo tsa lefutso le maemo a tikoloho ho kenya letsoho kutloisisong e akaretsang ea tšebelisano e rarahaneng lipakeng tsa lichaba tsa likokoana-hloko le semelo sa tsona sa tikoloho. Qetellong, tatellano ea metagenomic e eme e le sesebelisoa sa bohlokoa bakeng sa ho manolla merabe ea liphatsa tsa lefutso ea lichaba tse fapaneng tsa likokoana-hloko, e totobatsang likamano tse nang le likarolo tse ngata lipakeng tsa liphatsa tsa lefutso le ecology ka har'a tikoloho ena e rarahaneng.
Platforms: Illumina NovaSeq le DNBSEQ-T7
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Metagenomic Sequencing-TGS
Metagenome ke pokello ea liphatsa tsa lefutso tsa sehlopha se tsoakaneng sa lintho tse phelang, joalo ka tikoloho le metagenome ea batho. E na le liphatsa tsa lefutso tsa likokoana-hloko tse lengoang le tse ke keng tsa lengoa. Sequencing ea Metagenomic e nolofalletsa boithuto ba libaka tsena tse rarahaneng tsa genomic tse kentsoeng lisampoleng tsa tikoloho ka ho fana ka ho feta profiling ea taxonomic. E boetse e fana ka pono e sebetsang ea liphatsa tsa lefutso ka ho lekola liphatsa tsa lefutso tse kentsoeng le likarolo tsa tsona tsa ts'ebetso ea tikoloho. Le hoja lithunya tsa setso li atamela ka tatellano ea Illumina li 'nile tsa sebelisoa haholo lithutong tsa metagenomic, ho tla ha Nanopore le PacBio tatellano ea nako e telele ea ho bala e fetotse tšimo. Theknoloji ea Nanopore le PacBio e ntlafatsa litlhahlobo tse tlase tsa bioinformatic, haholoholo kopano ea metagenome, ho netefatsa likopano tse tsoelang pele. Litlaleho li bonts'a hore metagenomics e thehiloeng ho Nanopore le PacBio e atlehile ho hlahisa liphatsa tsa lefutso tsa baktheria tse felletseng le tse koetsoeng ho tsoa ho li-microbiomes tse rarahaneng (Moss, EL, et al., Nature Biotech, 2020). Ho kopanya ho bala ha Nanopore le ho bala ha Illumina ho fana ka mokhoa oa leano oa ho lokisa liphoso, ho fokotsa ho nepahala ha tlhaho ho tlase ha Nanopore. Motsoako ona oa synergistic o phahamisa matla a sethala se seng le se seng sa tatellano, se fana ka tharollo e matla ea ho hlola mefokolo e ka bang teng le ho ntšetsa pele ho nepahala le ho ts'epahala ha litlhahlobo tsa metagenomic.
Sethala: Nanopore PromethION 48, Illumia le PacBio Revio
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Whole genome bisulfite sequencing(WGBS)
Whole Genome Bisulfite Sequencing (WGBS) e eme e le mokhoa o tloaelehileng oa khauta bakeng sa ho fuputsa ka botebo DNA methylation, haholo-holo boemo ba bohlano ho cytosine (5-mC), molaoli oa bohlokoa oa polelo ea lefutso le tšebetso ea liselefouno. Molao-motheo o thehiloeng ho WGBS o kenyelletsa phekolo ea bisulfite, e susumetsang phetoho ea li-cytosine tse se nang methylated ho uracil (C ho ea ho U), ha li ntse li siea li-cytosine tsa methylated li sa fetohe. Mokhoa ona o fana ka tharollo ea motheo o le mong, o lumellang bafuputsi ho etsa lipatlisiso ka botlalo ka methylome le ho sibolla mekhoa e sa tloaelehang ea methylation e amanang le maemo a fapaneng, haholo mofets'e. Ka ho sebelisa WGBS, bo-ramahlale ba ka fumana leseli le ke keng la bapisoa mabapi le genome-wide methylation landscapes, ho fana ka kutloisiso e fapaneng ea mekhoa ea epigenetic e bakang lits'ebetso le mafu a fapaneng a tlhaho.
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Assay for Transposase-Accessible Chromatin with High Throughput Sequencing (ATAC-seq)
ATAC-seq ke mokhoa o phahameng oa tatellano o sebelisoang bakeng sa tlhahlobo ea phihlello ea chromatin ea genome. Tšebeliso ea eona e fana ka kutloisiso e tebileng ea mekhoa e rarahaneng ea taolo ea lefats'e ea epigenetic holim'a liphatsa tsa lefutso. Mokhoa ona o sebelisa hyperactive Tn5 transposase ho arola ka nako e le 'ngoe le ho tšoaea libaka tse bulehileng tsa chromatin ka ho kenya li-adapter tsa tatellano. Kholiso e latelang ea PCR e hlahisa laeborari ea tatellano, e lumellang ho tsebahatsa libaka tse bulehileng tsa chromatin tlas'a maemo a itseng a sebaka. ATAC-seq e fana ka pono e akaretsang ea libaka tse fihlellehang tsa chromatin, ho fapana le mekhoa e shebaneng feela le libaka tse tlamang tsa transcription factor kapa libaka tse itseng tse fetotsoeng tsa histone. Ka tatellano ea libaka tsena tse bulehileng tsa chromatin, ATAC-seq e senola libaka tse nang le monyetla oa ho ba le tatellano e sebetsang ea taolo le libaka tse tlamang tse ka bang teng, tse fanang ka leseli la bohlokoa mabapi le phetoho e matla ea polelo ea lefutso ho genome.
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16S/18S/ITS Amplicon Sequencing-PacBio
Liphatsa tsa lefutso tsa 16S le 18S rRNA, hammoho le sebaka sa Internal Transcribed Spacer (ITS), li sebetsa e le matšoao a bohlokoa a ho hatisa menoana ea molek'hule ka lebaka la motsoako oa tsona oa libaka tse baballetsoeng haholo le tse feto-fetohang, e leng se etsang hore e be lisebelisoa tsa bohlokoa bakeng sa ho khetholla likokoana-hloko tsa prokaryotic le eukaryotic. Ho holisa le tatellano ea libaka tsena ho fana ka mokhoa o se nang ho itšehla thajana bakeng sa ho batlisisa sebopeho sa likokoana-hloko le mefuta-futa ea tikoloho le tikoloho e fapaneng. Le hoja tatellano ea Illumina e atisa ho lebisa libaka tse khutšoanyane tsa hypervariable tse kang V3-V4 ea 16S le ITS1, ho bontšitsoe hore tlhaloso e phahameng ea taxonomic e ka finyelloa ka tatellano ea bolelele bo feletseng ba 16S, 18S, le ITS. Mokhoa ona o pharaletseng o fella ka liphesente tse phahameng tsa tatellano e hlophisitsoeng ka nepo, ho fihlela boemo ba tharollo bo fetelang ho khethoeng ha mefuta. Sethala sa tatellano sa PacBio's Single-Molecule Real-Time (SMRT) se hlahella ka ho fana ka lipalo tse telele tse nepahetseng haholo (HiFi) tse koahelang liamplicon tse bolelele bo felletseng, tse qothisanang lehlokoa le ho nepahala ha tatellano ea Illumina. Bokhoni bona bo lumella bafuputsi ho fumana monyetla o ke keng oa lekanngoa - pono e ntle ea sebopeho sa liphatsa tsa lefutso. Kakaretso e atolositsoeng e phahamisa tharollo haholo litlhalosong tsa mefuta, haholo-holo ka har'a lichaba tsa baktheria kapa fungal, e leng ho nolofalletsang kutloisiso e tebileng ea ho rarahana ha palo ea likokoana-hloko.
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16S/18S/ITS Amplicon Sequencing-NGS
Tatelano ea Amplicon le thekenoloji ea Illumina, e shebaneng ka ho khetheha le 16S, 18S, le matšoao a lefutso a ITS, ke mokhoa o matla oa ho manolla bongata ba phylogeny, taxonomy, le mefuta ka har'a lichaba tsa likokoana-hloko. Mokhoa ona o kenyelletsa tatellano ea libaka tse sa tsitsang tsa liphatsa tsa lefutso tsa ho boloka matlo. Qalong e hlahisitsoe e le monoana oa molek'hule kaWoeses et alka 1977, mokhoa ona o ntlafalitse profiling ea microbiome ka ho etsa hore litlhahlobo tse se nang ho itšehla thajana. Ka tatellano ea 16S (libaktheria), 18S (fungus), le Internal Transcribed Spacer (ITS, fungus), bafuputsi ba ke ke ba tseba feela mefuta e mengata empa hape le e sa tloaelehang le e sa tsejoeng. E amohetsoe haholo joalo ka sesebelisoa sa bohlokoa, tatellano ea amplicon e se e le sesebelisoa sa ho lemoha mefuta e fapaneng ea mefuta ea likokoana-hloko libakeng tse fapaneng, ho kenyeletsoa molomo oa motho, mala, setuloana le ho feta.
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Libaktheria le Fungus Whole Genome Re-Sequencing
Merero ea tatellano ea liphatsa tsa lefutso tsa baktheria le fungal kaofela ke ea bohlokoa bakeng sa ho ntšetsa pele liphatsa tsa lefutso tsa likokoana-hloko ka ho etsa hore ho phetheloe le ho bapisa liphatsa tsa lefutso tsa microbial. Sena se nolofatsa boenjiniere ba ho belisoa, ho ntlafatsa mekhoa ea indasteri, le ho hlahloba mekhoa ea bobeli ea metabolism. Ho feta moo, tatellano e ncha ea fungal le baktheria e bohlokoa bakeng sa ho utloisisa phetoho ea tikoloho, ho ntlafatsa mefuta, le ho senola matla a ho iphetola ha liphatsa tsa lefutso, ka litlamorao tse pharaletseng ho tsa bongaka, temo le mahlale a tikoloho.
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PacBio-Full-length 16S/18S/ITS Amplicon Sequencing
Sethala sa Amplicon (16S/18S/ITS) se entsoe ka boiphihlelo ba lilemo tsa tlhahlobo ea projeke ea mefuta-futa ea likokoana-hloko, e nang le tlhahlobo e tloaelehileng ea mantlha le tlhahlobo ea motho ka mong: tlhahlobo ea mantlha e akaretsa litaba tsa tlhahlobo e akaretsang ea lipatlisiso tsa hajoale tsa likokoana-hloko, litaba tsa tlhahlobo li ruile ebile li na le botlalo, le liphetho tsa tlhahlobo li hlahisoa ka mokhoa oa litlaleho tsa morero; Litaba tsa tlhahlobo ea motho ka mong li fapane. Mehlala e ka khethoa 'me liparamente li ka hlophisoa ka mokhoa o bonolo ho latela tlaleho ea tlhahlobo ea mantlha le sepheo sa lipatlisiso, ho fihlela litlhoko tsa motho ka mong. Sistimi ea ts'ebetso ea Windows, e bonolo ebile e potlakile.
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PacBio-Full-length Transcriptome (Non-Reference)
Ho nka data ea tatellano ea Isoform ea Pacific Bioscience (PacBio) joalo ka ho kenya, Sesebelisoa sena se khona ho tsebahatsa tatelano ea sengoloa sa bolelele bo felletseng (ntle le ho kopanya). Ka ho etsa 'mapa oa tatellano ea bolelele bo felletseng khahlano le genome ea litšupiso, lingoloa li ka ntlafatsoa ka liphatsa tsa lefutso tse tsebahalang, lingoliloeng, libaka tsa likhoutu, joalo-joalo. Tabeng ena, ho ka finyelloa ho tsebahatsa ka nepo haholoanyane meaho ea mRNA, joalo ka mefuta e meng ea ho kopanya, joalo-joalo. Tlhahlobo e kopaneng le data ea tatellano ea li-transcriptome ea NGS e etsa hore ho be le tlhaloso e felletseng le palo e nepahetseng haholoanyane ea polelo maemong a mongolo, e leng molemo haholo oa polelo e fapaneng le tlhahlobo ea ts'ebetso.
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Representation Bisulfite Sequencing (RRBS)
Reduced Representation Bisulfite Sequencing (RRBS) e hlahile e le mokhoa o sa sebetseng hantle le o sebetsang hantle oa Whole Genome Bisulfite Sequencing (WGBS) lipatlisisong tsa DNA methylation. Le ha WGBS e fana ka lintlha tse felletseng ka ho hlahloba genome eohle ka qeto e le 'ngoe ea motheo, litšenyehelo tsa eona tse phahameng e ka ba ntho e fokotsang. RRBS ka maano e fokotsa phephetso ena ka ho khetha karolo e emelang ea genome. Mokhoa ona o ipapisitse le ntlafatso ea libaka tse ruileng tsa lihlekehleke tsa CpG ke MspI cleavage e lateloang ke khetho ea boholo ba likhechana tsa 200-500/600 bps. Ka lebaka leo, ke libaka tse haufi le lihlekehleke tsa CpG feela tse latellanang, athe tse nang le lihlekehleke tse hole tsa CpG li sa kengoa tlhahlobong. Ts'ebetso ena, e kopantsoeng le tatellano ea bisulfite, e lumella ho lemoha ho phahameng ka ho fetisisa ha DNA methylation, le mokhoa oa ho latellana, PE150, o lebisa tlhokomelo ka ho khetheha lipheletsong tsa ho kenya ho e-na le bohareng, ho eketsa katleho ea profiling ea methylation. RRBS ke sesebelisoa sa bohlokoahali se nolofalletsang lipatlisiso tsa DNA methylation tse theko e tlaase le ho ntlafatsa tsebo ea mekhoa ea epigenetic.
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Prokaryotic RNA Sequencing
Tatelano ea RNA e matlafatsa profil e felletseng ea lingoliloeng tsohle tsa RNA ka har'a lisele tlas'a maemo a ikhethileng. Theknoloji ena e tsoetseng pele e sebetsa e le sesebelisoa se matla, se senolang liphatlalatso tse rarahaneng tsa liphatsa tsa lefutso, libopeho tsa liphatsa tsa lefutso, le mekhoa ea limolek'hule e amanang le mekhoa e fapaneng ea tlhaho. E amohetsoe haholo lipatlisisong tsa mantlha, tlhahlobo ea bongaka, le nts'etsopele ea lithethefatsi, tatellano ea RNA e fana ka leseli mabapi le ho rarahana ha matla a cellular le taolo ea liphatsa tsa lefutso. Ts'ebetso ea rona ea sampole ea prokaryotic RNA e etselitsoe li-transcriptomes tsa prokaryotic, tse kenyelletsang ho fokotseha ha rRNA le ho hlophisoa ha laeborari ea tataiso.
Sethala: Illumina NovaSeq
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Metatranscriptome Sequencing
Theknoloji ea tatellano ea tatellano ea Illumina, ts'ebeletso ea tatellano ea metaranscriptome ea BMKGENE e utolla polelo e matla ea mefuta e fapaneng ea likokoana-hloko, ho tloha ho li-eukaryote ho ea ho li-prokaryotes le likokoana-hloko, ka har'a libaka tsa tlhaho tse kang mobu, metsi, leoatle, setuloana le mala. Ts'ebeletso ea rona e felletseng e matlafatsa bafuputsi ho batlisisa ka har'a profaele e felletseng ea liphatsa tsa lefutso tsa lichaba. Ntle le tlhahlobo ea taxonomic, ts'ebeletso ea rona ea tatellano ea metatranscriptome e thusa ho lekola ntlafatso ea ts'ebetso, e fana ka leseli mabapi le liphatsa tsa lefutso tse hlahisitsoeng ka mokhoa o fapaneng le mesebetsi ea tsona. Fumana lintlha tse ngata tsa baeloji ha u ntse u sheba libaka tse rarahaneng tsa mofuta oa polelo, mefuta-futa ea taxonomic, le matla a sebetsang ka har'a libaka tsena tse fapaneng tsa tikoloho.
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Seboka sa Novo Fungal Genome
BMKGENE e fana ka litharollo tse fapaneng bakeng sa liphatsa tsa lefutso tsa fungal, ho fana ka litlhoko tse fapaneng tsa lipatlisiso le botlalo bo lakatsehang ba genome. Ho sebelisa tatellano e khuts'oane ea Illumina feela ho lumella ho hlahisa genome ea moralo. Tatelano ea ho bala nako e khuts'oane le ea nako e telele e sebelisang Nanopore kapa Pacbio e kopantsoe bakeng sa genome e ntlafalitsoeng ea fungal e nang le li-contig tse telele. Ho feta moo, ho kopanya tatellano ea Hi-C ho eketsa bokhoni le ho feta, ho nolofalletsa ho fihlella genome e felletseng ea boemo ba chromosome.
