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Liquid biopsy (LB) ke mohopolo o ntseng o tsebahala ka potlako lefapheng la biomedical.Khopolo ena e thehiloe haholo-holo ho sibolloeng ha likhechana tsa DNA ea extracellular (ccfDNA), eo haholo-holo e lokolloang e le likhechana tse nyenyane ka mor'a lefu la sele liseleng tse sa tšoaneng.Karolo e nyenyane ea likaroloana tsena e tsoa ho lisele tse tsoang linaheng tse ling (lisele) kapa lintho tse phelang.Mosebetsing oa hajoale, re sebelisitse mohopolo ona ho li-mussels, mofuta oa molebeli o tsebahalang ka bokhoni ba ona bo phahameng ba ho sefa metsi a leoatle.Re sebelisa bokhoni ba li-mussels ho sebetsa e le lihloela tsa tlhaho ho hapa likaroloana tsa DNA tsa tikoloho ho tsoa mehloling e fapaneng ho fana ka leseli mabapi le mefuta-futa ea tikoloho ea maoatle a leoatle.Liphetho tsa rona li bontša hore mussel hemolymph e na le likaroloana tsa DNA tse fapaneng haholo ka boholo, ho tloha ho 1 ho isa ho 5 kb.Tatelano ea lithunya e bontšitse hore palo e kholo ea likhechana tsa DNA li tsoa linaheng tse ling.Har'a tsona, re fumane likhechana tsa DNA ho tsoa ho libaktheria, archaea, le livaerase, ho kenyeletsoa likokoana-hloko tse tsejoang hore li tšoaetsa batho ba bangata ba atisang ho fumanoa tikolohong ea leoatle.Qetellong, boithuto ba rona bo bonts'a hore mohopolo oa LB o sebelisitsoeng ho li-mussels o emela mohloli o ruileng empa o e-so ka o batlisisoa ka mefuta-futa ea likokoana-hloko tikolohong ea maoatleng a leoatle.
Tšusumetso ea phetoho ea maemo a leholimo (CC) ho mefuta-futa ea tikoloho ea leoatle ke sebaka sa lipatlisiso se ntseng se hola ka potlako.Ho futhumala ha lefatše ha ho bake likhatello tsa 'mele feela, empa hape ho sutumelletsa meeli ea ho iphetola ha lintho tsa ho tsitsa ha mocheso oa lintho tse phelang metsing, e amang bolulo ba mefuta e mengata, e ba susumelletsang ho batla maemo a molemo haholoanyane [1,2].Ntle le ho ama mefuta-futa ea lihloliloeng tsa metazoan, CC e sitisa ho leka-lekana ha litšebelisano tsa likokoana-hloko tse ling.Dysbacteriosis ena ea likokoana-hloko e baka tšokelo e tebileng ho tikoloho ea leoatle kaha e etsa hore likokoana-hloko tsa leoatleng li hlaseloe habonolo ke likokoana-hloko tse tšoaetsanoang [3, 4].Ho lumeloa hore SS e bapala karolo ea bohlokoa lefung la batho ba bangata, e leng bothata bo tebileng ba tsamaiso ea tikoloho ea leoatleng [5, 6].Ena ke taba ea bohlokoa ho latela litlamorao tsa moruo, tikoloho le phepo ea mefuta e mengata ea leoatle.Sena ke 'nete ka ho khetheha bakeng sa li-bivalves tse lulang libakeng tsa polar, moo liphello tsa CK li leng teng hang-hang le tse matla haholoanyane [6, 7].Ha e le hantle, li-bivalves tse kang Mytilus spp.li sebelisoa haholo ho beha leihlo litlamorao tsa CC tikolohong ea maoatle.Ha ho makatse hore ebe palo e kholo ea li-biomarker e entsoe ho lekola bophelo ba bona, hangata ba sebelisa mokhoa oa mekhahlelo e 'meli o kenyelletsang li-biomarker tse sebetsang tse ipapisitseng le ts'ebetso ea enzymatic kapa mesebetsi ea cellular joalo ka ts'ebetso ea lisele le phagocytic [8].Mekhoa ena e boetse e kenyelletsa tekanyo ea khatello ea likhatello tse khethehileng tse bokellang liseleng tse bonolo ka mor'a ho kenngoa ha metsi a mangata a leoatle.Leha ho le joalo, matla a phahameng a ho sefa le mokhoa o sa buleheng oa potoloho ea li-bivalves o fana ka monyetla oa ho hlahisa li-biomarker tse ncha tsa hemolymph ho sebelisa khopolo ea "liquid biopsy" (LB), mokhoa o bonolo le o fokolang oa tsamaiso ea mokuli.disampole tsa madi [9, 10].Leha mefuta e 'maloa ea limolek'hule tse potolohang e ka fumanoa ho LB ea motho, mohopolo ona o ipapisitse le tlhahlobo ea tatellano ea DNA ea ho potoloha likhechana tsa extracellular DNA (ccfDNA) ho plasma.Ha e le hantle, ho ba teng ha DNA e potolohang ka har'a plasma ea motho ho 'nile ha tsejoa ho tloha bohareng ba lekholo la bo20 la lilemo [11], empa ke lilemong tsa morao tjena moo ho fihla ha mekhoa e phahameng ea ho latellana ho lebisitseng ho hlahlojoa ke lingaka ho latela ccfDNA.Ho ba teng ha likhechana tsena tsa DNA tse potolohang ho bakoa ke karolo e itseng ea ho lokolloa ha DNA ea genomic (nuclear le mitochondrial) ka mor'a lefu la sele. Ho batho ba phetseng hantle, bongata ba ccfDNA hangata bo tlase (<10 ng/mL) empa bo ka eketsoa ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa ba tlas'a khatello ea maikutlo, e leng se bakang tšenyo ea lisele. Ho batho ba phetseng hantle, bongata ba ccfDNA hangata bo tlase (<10 ng/mL) empa bo ka eketsoa ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa ba tlas'a khatello ea maikutlo, e leng se bakang tšenyo ea lisele. Те здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 раз у больных песние стрессу, приводящему к повреждению тканей. Ho batho ba phetseng hantle, khatello ea cccDNA hangata e tlase (<10 ng / mL), empa e ka eketseha ka makhetlo a 5-10 ho bakuli ba nang le mafu a sa tšoaneng kapa tlas'a khatello ea kelello e lebisang ho senyeha ha lisele.在健康个体中，ccfDNA的浓度通常较低（<10 ng/mL），但在患有各种病理或承受压力的患者力的患者力的患10致组织损伤。在 健康 个体 中 , ccfdna 的 浓度 较 低 （<10 ng/ml） 但 在 各 种病理 或 承叅 压 承叅 压 1倍 , 从而 组织。。。 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут быть увеличены в 5-10 что приводит к повреждению тканей. ccfDNA concentrations hangata e tlase (<10 ng/ml) ho batho ba phetseng hantle, empa e ka eketsoa ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa khatello ea maikutlo, e bakang tšenyo ea lisele.Boholo ba likhechana tsa ccfDNA bo fapana haholo, empa hangata bo fapana ho tloha ho 150 ho isa ho 200 bp.[12].Tlhahlobo ea ccfDNA e entsoeng ka boeona, ke hore, ccfDNA ho tloha liseleng tse tloaelehileng kapa tse fetotsoeng tse amohelang batho, e ka sebelisoa ho lemoha liphetoho tsa lefutso le tsa epigenetic tse teng ho genome ea nyutlelie le / kapa mitochondrial, kahoo ho thusa baoki ho khetha mekhoa e itseng ea phekolo e lebisitsoeng ho molek'hule [13] .Leha ho le joalo, ccfDNA e ka fumanoa mehloling e tsoang linaheng tse ling e kang ccfDNA ho tloha liseleng tsa lesea nakong ea bokhachane kapa lithong tse fetisitsoeng [14,15,16,17].ccfDNA hape ke mohloli oa bohlokoa oa tlhaiso-leseling bakeng sa ho bona boteng ba li-nucleic acid tsa moemeli ea tšoaetsanoang (osele), e lumellang ho sibolloa ho sa hlaseheng ha tšoaetso e atileng e sa tsejoeng ke litloaelo tsa mali, ho qoba biopsy e hlaselang ea lisele tse nang le tšoaetso [18].Liphuputso tsa morao-rao li bontšitse hore mali a motho a na le mohloli o mongata oa tlhahisoleseding e ka sebelisoang ho khetholla likokoana-hloko tsa kokoana-hloko le baktheria, le hore hoo e ka bang 1% ea ccfDNA e fumanoang ho plasma ea motho e tsoa linaheng tse ling [19].Liphuputso tsena li bonts'a hore mefuta-futa ea likokoana-hloko ea likokoana-hloko e potolohang e ka hlahlojoa ho sebelisoa tlhahlobo ea ccfDNA.Leha ho le joalo, ho fihlela morao tjena, khopolo ena e ne e sebelisoa feela ho batho, 'me, ho isa bohōleng bo itseng, ho liphoofolo tse ling tse nang le lesapo la mokokotlo [20, 21].
Pampiring ea hona joale, re sebelisa monyetla oa LB ho hlahloba ccfDNA ea Aulacomya atra, mofuta o ka boroa o atisang ho fumanoa Lihlekehlekeng tsa Kerguelen tse ka tlaase ho antarctic, sehlopha sa lihlekehleke tse holim'a sehlaba se seholo se ileng sa thehoa lilemong tse limilione tse 35 tse fetileng.ho foqoha ha seretse se chesang.Re sebelisa mokhoa oa ho etsa liteko oa in vitro, re fumane hore likhechana tsa DNA metsing a leoatle li nkoa ka potlako ke li-mussels ebe li kena ka phaposing ea hemolymph.Tatelano ea Shotgun e bonts'itse hore mussel hemolymph ccfDNA e na le likhechana tsa DNA tsa eona le tseo e seng tsa eona, ho kenyeletsoa libaktheria tsa symbiotic le likaroloana tsa DNA tse tsoang ho biomes tse tloaelehileng tsa tikoloho e batang ea leoatle ea seretse se chesang.Hemolymph ccfDNA e boetse e na le tatelano ea vaerase e nkiloeng ho livaerase tse nang le mefuta e fapaneng ea moamoheli.Re boetse re fumane likhechana tsa DNA ho tsoa liphoofolong tse ngata tse nang le lisele tse ngata tse kang tlhapi ea masapo, anemone ea leoatleng, algae le likokoanyana.Qetellong, boithuto ba rona bo bonts'a hore mohopolo oa LB o ka sebelisoa ka katleho ho li-invertebrates tsa leoatleng ho hlahisa li-genomic repertoire e ruileng tikolohong ea maoatle.
Batho ba baholo (55-70 limilimithara bolelele) Mytilus platensis (M. platensis) le Aulacomya atra (A. atra) li ile tsa bokelloa ho tloha mabōpong a mafika a intertidal a Port-au-France (049°21.235 S, 070°13.490 E.).Lihlekehleke tsa Kerguelen ka December 2018. Li-mussels tse ling tsa batho ba baholo ba buluu (Mytilus spp.) li ile tsa fumanoa ho mofani oa khoebo (PEI Mussel King Inc., Prince Edward Island, Canada) 'me tsa kenngoa ka mocheso o laoloang (4 ° C) tanka ea aerated e nang le 10-20 L ea 32‰ brine ea maiketsetso.(letsoai la maiketsetso la leoatle Reef Crystal, Instant Ocean, Virginia, USA).Bakeng sa teko ka 'ngoe, bolelele le boima ba likhetla ka bomong li ne li lekanngoa.
Protocol ea mahala ea phihlello ea lenaneo lena e fumaneha inthaneteng (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Ka bokhutšoanyane, LB hemolymph e ile ea bokelloa ho tloha mesifa ea abductor joalokaha ho hlalositsoe [22].Hemolymph e ile ea hlakisoa ka centrifugation ho 1200×g bakeng sa metsotso ea 3, supernatant e ne e le leqhoa (-20 ° C) ho fihlela e sebelisoa.Bakeng sa ho itšehla thajana le ho hloekisa cfDNA, lisampole (1.5-2.0 ml) li ile tsa qhibilihisoa le ho sebetsoa ho sebelisoa NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ho latela litaelo tsa moetsi.ccfDNA e ne e bolokoa ho -80 ° C ho fihlela tlhahlobo e 'ngoe.Litekong tse ling, ccfDNA e ile ea aroloa le ho hloekisoa ho sebelisoa QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada).DNA e hloekisitsoeng e ile ea lekanyetsoa ho sebelisoa tlhahlobo e tloaelehileng ea PicoGreen.Kabo ea sekhechana sa ccfDNA e ka thoko e ile ea hlahlojoa ke capillary electrophoresis ho sebelisa Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) ho sebelisa High Sensitivity DNA Kit.Teko e entsoe ho sebelisoa 1 µl ea sampole ea ccfDNA ho latela litaelo tsa moetsi.
Bakeng sa tatellano ea likhechana tsa hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Canada) o ile a lokisa lilaebrari tsa lithunya a sebelisa Illumina DNA Mix kit ea Illumina MiSeq PE75 kit.Ho ile ha sebelisoa adaptara e tloaelehileng (BioO).Lifaele tsa data tse tala li fumaneha ho NCBI Sequence Read Archive (SRR8924808 le SRR8924809).Boleng ba motheo ba ho bala bo ile ba hlahlojoa ho sebelisoa FastQC [23].Trimmomatic [24] e 'nile ea sebelisoa bakeng sa li-adapter tsa ho khaola le ho bala ha boleng bo tlaase.Lithunya tsa Shotgun tse nang le lipheletso tse kopantsoeng li ile tsa kopanngoa hore FLASH e kopanngoe ho bala nako e telele ka ho kopana ha 20 bp ho qoba ho se lumellane [25]. Libali tse kopantsoeng li ne li hlalositsoe ka BLASTN ho sebelisoa database ea bivalve NCBI Taxonomy (e value <1e-3 le 90% homology), le masking ea tatellano e sa rarahanang e ne e etsoa ho sebelisoa DUST [26]. Libali tse kopantsoeng li ne li hlalositsoe ka BLASTN ho sebelisoa database ea bivalve NCBI Taxonomy (e value <1e-3 le 90% homology), le masking ea tatellano e sa rarahanang e ne e etsoa ho sebelisoa DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN базы данных таксономии двустворчатых моллюсков NCBI (8) маскирование последовательностей низкой сложности было выполнено с использованием LEROLE [26]. Lipalo tse kopaneng li ile tsa hlakisoa ka BLASTN ho sebelisoa database ea NCBI bivalve taxonomy (e value <1e-3 le 90% homology), le masking a tatellano a rarahaneng a tlase a entsoe ka DUST [26].使用双壳类NCBI 分类数据库（e 值< 1e-3 和90% 同源性）用BLASTN 注释合并的读数，并使强特强 [26]掩蔽.使用 双 壳类 ncbi 分类 (（（<1e-3 和 90% 同源） 用 用 注释 合并 读数 , 并 读数 。掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽Объединенные чтения были аннотированы с помощью BLASTN беспользованием таксономической базы даных двустворчатых молюсков-NCBI а маскирование последовательностей низкой сложности было выполнено с использованием LEROLE [26]. Lipalo tse kopaneng li ile tsa hlakisoa ka BLASTN ho sebelisoa database ea NCBI bivalve taxonomic (e value <1e-3 le 90% homology), le masking a tatellano a rarahaneng a tlase a entsoe ka DUST [26].Ho bala ho ne ho arotsoe ka lihlopha tse peli: tse amanang le tatellano ea li-bivalve (mona li bitsoa ho ipalla) le tse sa amaneng (tse sa ipallang).Lihlopha tse peli li ile tsa bokelloa ka thoko ho sebelisa MEGAHIT ho hlahisa li-contigs [27].Ho sa le joalo, kabo ea taxonomic ea li-microbiome tsa kantle ho naha e ne e arotsoe ho sebelisoa Kraken2 [28] mme e emeloa ka mokhoa o hlakileng ke chate ea phae ea Krona ho Galaxy [29, 30].Li-kmers tse nepahetseng li ne li ikemiselitse ho ba kmers-59 ho tsoa litekong tsa rona tsa pele. Li-self contigs li ile tsa khetholloa ka ho lumellana le BLASTN (database ea bivalve NCBI, e bohlokoa <1e-10 le 60% homology) bakeng sa tlhaloso ea ho qetela. Li-self contigs li ile tsa khetholloa ka ho lumellana le BLASTN (database ea bivalve NCBI, e bohlokoa <1e-10 le 60% homology) bakeng sa tlhaloso ea ho qetela. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, зная-010) нчательной аннотации. Li-self-contigs li ile tsa khetholloa ka ho bapisa le BLASTN (NCBI bivalve database, e value <1e-10 le 60% homology) bakeng sa tlhaloso ea ho qetela.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% 同源性)然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% Затем были идентифицированы собственные контиги для окончательной аннотации путем сопоставления с BLASTN е e <1e-10 le гомология 60%). Li-self-contigs li ile tsa khetholloa bakeng sa tlhaloso ea ho qetela ka ho bapisa le BLASTN (NCBI bivalve database, e value <1e-10 le 60% homology). Ka mokhoa o ts'oanang, li-contigs tsa sehlopha tseo e seng tsa sehlopha li ile tsa hlakisoa ka BLASTN (nt NCBI database, e value <1e-10 le 60% homology). Ka mokhoa o ts'oanang, li-contigs tsa sehlopha tseo e seng tsa sehlopha li ile tsa hlakisoa ka BLASTN (nt NCBI database, e value <1e-10 le 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 и гомология 60%). Ka mokhoa o ts'oanang, li-contigs tsa lihlopha tsa kantle ho naha li ile tsa hlakisoa ka BLASTN (database ea NT NCBI, boleng ba <1e-10 le 60% homology).平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。 Параллельно контиги, не относящиеся к собственной группе, были аннотированы с помощью BLASTN (база данных nt NCBI, значение e и 1-1%). Ka mokhoa o ts'oanang, li-contigs tse seng tsa sehlopha li ile tsa hlalosoa ka BLASTN (nt NCBI database, e value <1e-10 le 60% homology). BLASTX e ile ea boela ea etsoa ho li-contigs tse se nang boithati ho sebelisoa li-database tsa nr le RefSeq protein NCBI (e value <1e−10 le 60% homology). BLASTX e ile ea boela ea etsoa ho li-contigs tse se nang boithati ho sebelisoa li-database tsa nr le RefSeq protein NCBI (e value <1e−10 le 60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 6%). BLATX e ne e boetse e etsoa ho li-contig tseo e seng tsa boithati tse sebelisang marang-rang a protheine ea nr le RefSeq NCBI (e value <1e-10 le 60% homology).还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 同源性）.还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 同源性）. BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 6мог%). BLASTX e ne e boetse e etsoa ho li-contig tseo e seng tsa boithati tse sebelisang li-protein tsa nr le RefSeq NCBI (e value <1e-10 le 60% homology).Matamo a BLASTN le a BLASTX a batho bao e seng batho ba ikemetseng a emela li-contigs tsa ho qetela (bona faele ea Tlatsetso).
Lintlha tse sebelisoang bakeng sa PCR li thathamisitsoe ho Lethathamo la S1.Taq DNA polymerase (Bio Basic Canada, Markham, ON) e ne e sebelisetsoa ho holisa liphatsa tsa lefutso tsa ccfDNA.Maemo a latelang a karabelo a sebelisitsoe: denaturation ho 95 ° C bakeng sa metsotso e 3, 95 ° C bakeng sa motsotso o le mong, beha mocheso oa annealing bakeng sa motsotso o le mong, elongation ho 72 ° C motsotso o le mong, lipotoloho tse 35, 'me qetellong 72 ° C nakong ea metsotso e 10..Lihlahisoa tsa PCR li ne li arotsoe ke electrophoresis ka li-gel tsa agarose (1.5%) tse nang le SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Canada) ho 95 V.
Li-mussels (Mytilus spp.) li ne li tloaelehile metsing a leoatle a nang le oksijene ea 500 ml (32 PSU) ka lihora tse 24 ho 4 ° C.Plasmid DNA e nang le kenyelletso e kenyelletsang tatellano ea motho oa galectin-7 cDNA (nomoro ea ho kena ea NCBI L07769) e kentsoe ka har'a vial ka har'a khatello ea ho qetela ea 190 μg/μl.Li-mussels tse kentsoeng tlas'a maemo a tšoanang ntle le tlatsetso ea DNA e ne e le taolo.Tanka ea boraro ea taolo e ne e e-na le DNA e se nang li-mussels.Ho beha leihlo boleng ba DNA metsing a leoatle, lisampole tsa metsi a leoatle (20 μl; makhetlo a mararo) li nkiloe tanka ka 'ngoe ka nako e bontšitsoeng.Bakeng sa ho lateloa ha DNA ea plasmid, li-mussels tsa LB li ile tsa kotuloa ka linako tse bontšitsoeng 'me tsa hlahlojoa ke qPCR le ddPCR.Ka lebaka la letsoai le lengata la metsi a leoatle, li-aliquots li ile tsa hlapolloa ka metsi a boleng ba PCR (1:10) pele ho liteko tsohle tsa PCR.
Digital droplet PCR (ddPCR) e ile ea etsoa ho sebelisoa protocol ea BioRad QX200 (Mississauga, Ontario, Canada).Sebelisa profaele ea mocheso ho fumana mocheso o nepahetseng (Letlapa la S1).Marotholi a ile a hlahisoa ho sebelisoa jenereithara ea lerotholi ea QX200 (BioRad).ddPCR e entsoe ka tsela e latelang: 95 ° C bakeng sa 5 min, 50 cycles ea 95 ° C bakeng sa 30 s le mocheso o fanoeng oa annealing bakeng sa 1 min le 72 ° C bakeng sa 30 s, 4 ° C bakeng sa metsotso e 5 le 90 ° C nakong ea metsotso e 5.Palo ea marotholi le karabelo e ntle (palo ea likopi/µl) e ne e lekantsoe ho sebelisoa sebali sa lerotholi sa QX200 (BioRad).Mehlala e nang le marotholi a ka tlase ho 10,000 e ile ea hanoa.Taolo ea mohlala e ne e sa etsoa nako le nako ha ddPCR e tsamaisoa.
qPCR e entsoe ho sebelisoa Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) le li-primers tse khethehileng tsa LGALS7.Li-PCR tsohle tse ngata li entsoe ka 20 µl ho sebelisoa QuantiFast SYBR Green PCR Kit (QIAGEN).qPCR e ile ea qalisoa ka 15 min incubation ho 95 ° C e lateloa ke li-cycle tse 40 ho 95 ° C bakeng sa metsotsoana e 10 le ho 60 ° C bakeng sa metsotsoana e 60 ka pokello e le 'ngoe ea data.Li-curve tse qhibilihang li ile tsa hlahisoa ho sebelisoa litekanyo tse latellanang ho 95 ° C bakeng sa 5 s, 65 ° C bakeng sa 60 s, le 97 ° C qetellong ea qPCR.QPCR ka 'ngoe e entsoe ka makhetlo a mararo, ntle le lisampole tsa taolo.
Kaha li-mussels li tsebahala ka lebelo la tsona le holimo la ho sefa, re ile ra etsa lipatlisiso pele hore na li ka sefa le ho boloka likhechana tsa DNA tse teng metsing a leoatle.Re ne re boetse re thahasella ho tseba hore na likaroloana tsena li bokellana tsamaisong ea tsona ea lymphatic e semi-open.Re rarolle bothata bona ka liteko ka ho batlisisa qetello ea likhechana tsa DNA tse qhibilihang tse kentsoeng litankang tsa mussel tse putsoa.Ho thusa ho lateloa ha likhechana tsa DNA, re sebelisitse DNA ea kantle ho naha (eseng ea rona) e nang le mofuta oa galectin-7 oa motho.ddPCR e lata likhechana tsa plasmid tsa DNA metsing a leoatle le li-mussels.Liphetho tsa rona li bontša hore haeba palo ea likhechana tsa DNA metsing a leoatle a lula a le teng ka nako e telele (ho fihlela ho matsatsi a 7) ha ho se na li-mussels, joale ka pel'a li-mussels boemo bona bo batla bo nyamela ka ho feletseng ka hare ho lihora tse 8 (setšoantšo sa 1a, b).Likaroloana tsa DNA exogenous li ile tsa fumanoa habonolo ka hare ho metsotso ea 15 ka intravalvular fluid le hemolymph (setšoantšo sa 1c).Likaroloana tsena li ntse li ka bonoa ho fihlela lihora tse 4 kamora ho pepeseha.Ts'ebetso ena ea ho sefa mabapi le likhechana tsa DNA e bapisoa le ts'ebetso ea ho sefa ea libaktheria le algae [31].Liphetho tsena li fana ka maikutlo a hore li-mussels li ka sefa le ho bokella DNA ea kantle ho likarolo tsa mokelikeli oa tsona.
Likarolo tse amanang tsa plasmid DNA metsing a leoatle ka boteng (A) kapa bosieo (B) ba li-mussels, tse lekantsoeng ka ddPCR.Ho A, liphetho li hlahisoa e le liperesente, 'me meeli ea mabokose e emetse liperesente tsa 75 le 25.Mokokotlo o kentsoeng oa logarithmic o bontšitsoe ka bofubelu, 'me sebaka se nang le moriti o moputsoa se emela 95% ea nako ea ho itšepa.Ho B, mola o mofubelu o emela moelelo 'me mola o moputsoa o emela nako ea ho itšepa ea 95% bakeng sa mahloriso.C Ho bokellana ha plasmid DNA ho hemolymph le valvular fluid ea li-mussels ka linako tse fapaneng ka mor'a ho eketsoa ha plasmid DNA.Liphetho li hlahisoa e le likopi tse felletseng tse fumanoeng/mL (±SE).
Ka mor'a moo, re ile ra batlisisa ka tšimoloho ea ccfDNA ka li-mussels tse bokelitsoeng libetheng tsa li-mussel Lihlekehlekeng tsa Kerguelen, sehlopha se hōle sa lihlekehleke tse nang le tšusumetso e fokolang ea anthropogenic.Bakeng sa morero ona, cccDNA e tsoang ho mussel hemolymphs e ne e arotsoe le ho hloekisoa ka mekhoa e tloaelehileng e sebelisoang ho hloekisa cccDNA ea batho [32, 33].Re fumane hore karolelano ea likhakanyo tsa hemolymph ccfDNA ka har'a li-mussels li ka har'a li-micrograms ka ml ea hemolymph range (sheba Letlapa la S2, Litaba tsa Tlatsetso).Lethathamo lena la likhakanyo li kholo ho feta ho batho ba phetseng hantle (li-nanograms tse tlase ka milliliter), empa maemong a sa tloaelehang, ho bakuli ba mofetše, boemo ba ccfDNA bo ka fihla ho li-micrograms tse 'maloa ka milliliter [34, 35].Tlhahlobo ea boholo ba kabo ea hemolymph ccfDNA e bontšitse hore likhechana tsena li fapana haholo ka boholo, ho tloha ho 1000 bp ho isa ho 1000 bp.ho fihlela ho 5000 bp (setšoantšo sa 2).Liphetho tse ts'oanang li ile tsa fumanoa ho sebelisoa silika-based QIAamp Investigator Kit, mokhoa o atisang ho sebelisoa ho saense ea forensic ho arola le ho hloekisa DNA ea genomic ho tloha lisampole tse tlaase tsa DNA, ho akarelletsa le ccfDNA [36].
Moemeli oa ccfDNA electrophoregram ea mussel hemolymph.E ntšitsoe ka NucleoSnap Plasma Kit (holimo) le QIAamp DNA Investigator Kit.B morero oa violin o bontšang kabo ea li-mussels tsa hemolymph ccfDNA (±SE) ka har'a li-mussels.Mela e ntšo le e khubelu e emela bohareng le quartiles ea pele le ea boraro, ka ho latellana.
Hoo e ka bang 1% ea ccfDNA ho batho le litšoene e na le mohloli o tsoang kantle ho naha [21, 37].Ka lebaka la mokhoa o bulehileng oa potoloho ea li-bivalves, metsi a leoatle a nang le likokoana-hloko tse ngata, le kabo ea boholo ba mussel ccfDNA, re hakantse hore mussel hemolymph ccfDNA e kanna ea ba le letamo le ruileng le le fapaneng la DNA ea likokoana-hloko.E le ho hlahloba khopolo-taba ena, re ile ra tatellana hemolymph ccfDNA ho tloha lisampole tsa Aulacomya atra tse bokelitsoeng ho tloha Lihlekehlekeng tsa Kerguelen, tsa fana ka lipalo tse fetang limilione tse 10, 97.6% ea tsona li fetisitse taolo ea boleng.Ka nako eo ho baloa ho ile ha aroloa ho ea ka mehloli ea boithati le eo e seng ea boithati ho sebelisa BLASTN le NCBI bivalve databases (Fig. S1, Supplementary Information).
Ho batho, DNA ea nyutlelie le ea mitochondrial e ka lokolloa maling [38].Leha ho le joalo, thutong ea hona joale, ho ne ho sa khonehe ho hlalosa ka botlalo DNA ea nyutlelie ea li-mussels, kaha A. atra genome ha e e-s'o be le tatellano kapa ho hlalosoa.Leha ho le joalo, re khonne ho tseba likarolo tse 'maloa tsa ccfDNA tsa tšimoloho ea rona ka ho sebelisa laebrari ea bivalve (Fig. S2, Supplementary Information).Re boetse re netefalitse boteng ba likhechana tsa DNA tsa tšimoloho ea rona ka ho holisa PCR e laetsoeng ea liphatsa tsa lefutso tsa A. atra tse neng li hlophisitsoe (setšoantšo sa 3).Ka mokhoa o ts'oanang, ha ho nahanoa hore genome ea mitochondrial ea A. atra e fumaneha lits'ebetsong tsa sechaba, motho a ka fumana bopaki ba boteng ba likhechana tsa mitochondrial ccfDNA ho hemolymph ea A. atra.Ho ba teng ha likhechana tsa DNA tsa mitochondrial ho netefalitsoe ke amplification ea PCR (setšoantšo sa 3).
Mefuta e fapa-fapaneng ea liphatsa tsa lefutso tsa mitochondrial e ne e le teng hemolymph ea A. atra (matheba a khubelu - nomoro ea thepa: SRX5705969) le M. platensis (matheba a maputsoa - nomoro ea thepa: SRX5705968) e matlafalitsoeng ke PCR.Setšoantšo se nkiloeng ho tsoa ho Breton et al., 2011 B Amplification ea hemolymph supernatant ho tloha A. atra E bolokiloe pampiring ea FTA.Sebelisa punch ea 3 mm ho kenya ka ho toba tube ea PCR e nang le motsoako oa PCR.
Ka lebaka la litaba tsa likokoana-hloko tse ngata metsing a leoatle, qalong re ile ra shebana le sebopeho sa tatellano ea likokoana-hloko tsa DNA ho hemolymph.Ho etsa sena, re sebelisa mekhoa e 'meli e fapaneng.Leano la pele le sebelisitsoeng Kraken2, lenaneo la ho arola ka tatellano ea algorithm le ka khethollang tatellano ea likokoana-hloko ka ho nepahala ho bapisoang le BLAST le lisebelisoa tse ling [28].Ho feta ho bala ha 6719 ho ne ho ikemiseditse ho ba baktheria, ha 124 le 64 li ne li tsoa ho archaea le likokoana-hloko, ka ho latellana (setšoantšo sa 4).Likaroloana tse ngata ka ho fetisisa tsa baktheria ea DNA e ne e le Firmicutes (46%), Proteobacteria (27%), le Bacteroidetes (17%) (Fir. 4a).Kabo ena e lumellana le lithuto tse fetileng tsa marine blue mussel microbiome [39, 40].Gammaproteobacteria e ne e le sehlopha se seholo sa Proteobacteria (44%), ho akarelletsa le Vibrionales tse ngata (setšoantšo sa 4b).Mokhoa oa ddPCR o netefalitse ho ba teng ha likaroloana tsa Vibrio DNA ho ccfDNA ea A. atra hemolymph (Fig. 4c) [41].Ho fumana boitsebiso bo eketsehileng mabapi le tšimoloho ea baktheria ea ccfDNA, ho ile ha nkoa mokhoa o eketsehileng (Fig. S2, Tlhahisoleseding ea Tlatsetso). Tabeng ena, ho baloa hore lintho tse fetelletseng li ile tsa kopanngoa e le tse baloang ka bobeli 'me li ile tsa aroloa e le tsa motho ka mong (bivalves) kapa li sa tsoa hlaha ho sebelisoa BLASTN le boleng ba 1e−3 le khaollo e nang le >90% homology. Tabeng ena, ho baloa hore lintho tse fetelletseng li ile tsa kopanngoa e le tse baloang ka bobeli 'me li ile tsa aroloa e le tsa motho ka mong (bivalves) kapa li sa tsoa hlaha ho sebelisoa BLASTN le boleng ba 1e−3 le khaollo e nang le >90% homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственные песни происхождению с использованием BLASTN le значения e 1e-3 le отсечения с гомологией> 90%. Tabeng ena, lipalo tse fetelletseng li ile tsa bokelloa e le tse baloang ka bobeli 'me tsa hlalosoa e le tsa tlhaho (bivalve) kapa tseo e seng tsa mantlha ho sebelisoa BLASTN le boleng ba 1e-3 le cutoff ka >90% homology.在這种情况下，重叠的話数组装为配对末端读数，并使用BLASTN le1e-3 tsa hao le>90% mohoeletsi类）或非自身來源.在 這 种 情况 下 , 重叠 读数 组装 為 配 末端 读数 , 使用 使用 blastn le 1e-3 mohopo oa mohono le 90% （双 壳类） 非 自身。。。。。. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицированы как собственные происхождению с использованием значений e BLASTN le 1e-3 and порога гомологии> 90%. Tabeng ena, ho bala ho fetelletseng ho ile ha bokelloa e le lipalo tse phethiloeng ka bobeli 'me tsa khetholloa e le tsa tsona (bivalves) kapa tseo e seng tsa mantlha ho sebelisoa e BLASTN le 1e-3 le moeli oa homology> 90%.Kaha A. atra genome ha e e-s'o be le tatellano, re sebelisitse leano la kopano ea de novo ea sekopanyi sa MEGAHIT Next Generation Sequencing (NGS).Kakaretso ea li-contigs tse 147,188 li khethiloe e le tse itšetlehileng ka (bivalves) tsa tlhaho.Li-contigs tsena li ile tsa phatloha ka litekanyetso tsa e-1e-10 tse sebelisang BLASTN le BLASTX.Leano lena le ile la re lumella ho khetholla likaroloana tse 482 tse seng tsa bivalve tse teng ho A. atra ccfDNA.Ho feta halofo (57%) ea likaroloana tsena tsa DNA li ile tsa fumanoa ho tsoa ho libaktheria, haholo-holo ho tloha ho li-symbionts tsa gill, ho akarelletsa le sulfotrophic symbionts, le ho tloha ho li-gill symbionts Solemya velum (setšoan. 5).
Bongata bo amanang le boemo ba mofuta.B Phapang ea likokoana-hloko tsa li-phyla tse peli tse kholo (Firmicutes le Proteobacteria).Ntlafatso e emelang ddPCR C Vibrio spp.A. Likaroloana tsa lefutso la 16S rRNA (putsoa) ka li-atra hemolymphs tse tharo.
Kakaretso ea li-contigs tse 482 tse bokeletsoeng li ile tsa hlahlojoa.Boemo bo akaretsang ba kabo ea taxonomic ea litlhaloso tsa metagenomic contig (prokaryotes le eukaryotes).B Kabo e felletseng ea likhechana tsa DNA tsa baktheria tse khethiloeng ke BLASTN le BLASTX.
Tlhahlobo ea Kraken2 e boetse e bontšitse hore mussel ccfDNA e na le likaroloana tsa DNA tsa khale, ho kenyelletsa le likaroloana tsa DNA tsa Euryarchaeota (65%), Crenarchaeota (24%), le Thaurmarcheota (11%) (Fig. 6a).Ho ba teng ha likhechana tsa DNA tse nkiloeng ho Euryarchaeota le Crenarchaeota, tseo pele li neng li fumanoa tikolohong ea li-mussels tsa California, ha lia lokela ho makatsa [42].Le hoja Euryarchaeota e atisa ho amahanngoa le maemo a feteletseng, hona joale hoa hlokomeloa hore Euryarchaeota le Crenarcheota ke tse ling tsa li-prokaryote tse tloaelehileng ka ho fetisisa tikolohong ea marine cryogenic [43, 44].Ho ba teng ha likokoana-hloko tsa methanogenic ka har'a li-mussels ha ho makatse, ha ho nahanoa ka litlaleho tsa morao-rao tsa ho lutla ho hoholo ha methane ho tsoa ho lutla ho tlase Sehlabeng sa Kerguelen [45] le tlhahiso ea methane e ka bang teng e bonoang lebopong la Lihlekehleke tsa Kerguelen [46].
Joale maikutlo a rona a ile a fetela ho baleng ho tsoa ho livaerase tsa DNA.Ho ea ka tsebo ea rona, ena ke thuto ea pele e sa lebelloang ea litaba tsa vaerase ea li-mussels.Joalokaha ho lebeletsoe, re fumane likaroloana tsa DNA tsa bacteriophages (Caudovirales) (setšoantšo sa 6b).Leha ho le joalo, DNA ea kokoana-hloko e tloaelehileng ka ho fetisisa e tsoa ho phylum of nucleocytoviruses, e tsejoang hape e le nuclear cytoplasmic big DNA virus (NCLDV), e nang le genome e kholo ka ho fetisisa ea kokoana leha e le efe.Ka har'a phylum ena, boholo ba tatellano ea DNA ke ba malapa a Mimimidoviridae (58%) le Poxviridae (21%), ao likokoana-hloko tsa tlhaho li kenyelletsang likokoana-hloko le li-arthropods, ha karolo e nyenyane ea tatellano ena ea DNA e le ea algae e tsebahalang ea virological.E tšoaetsa algae ea marine eukaryotic.Tatelano e ile ea boela ea fumanoa ho kokoana-hloko ea Pandora, kokoana-hloko e kholo ka ho fetisisa ea genome ea mofuta ofe kapa ofe o tsejoang oa kokoana-hloko.Hoa thahasellisa hore mefuta e mengata ea mabotho a tsejoang hore a tšoaelitsoe ke kokoana-hloko, joalo ka ha ho laetsoe ke hemolymph ccfDNA sequencing, e ne e batla e le khōlō (Figure S3, Supplementary Information).E kenyelletsa likokoana-hloko tse tšoaetsang likokoanyana tse kang Baculoviridae le Iridoviridae, hammoho le likokoana-hloko tse tšoaetsang amoeba, algae le liphoofolo tse nang le lesapo la mokokotlo.Hape re fumane tatellano e tsamaellanang le genome ea Pithovirus sibericum.Li-pitoviruses (tse tsejoang hape e le "livaerase tsa zombie") li qalile ho aroloa ho tloha ho permafrost ea lilemo tse 30,000 Siberia [47].Kahoo, liphetho tsa rona li lumellana le litlaleho tse fetileng tse bontšang hore ha se mefuta eohle ea morao-rao ea likokoana-hloko tsena e felileng [48] le hore likokoana-hloko tsena li ka 'na tsa e-ba teng libakeng tse hōle tsa leoatle tse ka tlas'a lefatše.
Qetellong, re ile ra etsa liteko ho bona hore na re ka fumana likhechana tsa DNA liphoofolong tse ling tse nang le lisele tse ngata.Kakaretso ea li-contigs tsa kantle tse 482 li ile tsa khetholloa ke BLASTN le BLASTX e nang le lilaebrari tsa nt, nr le RefSeq (genomic le protheine).Liphello tsa rona li bontša hore har'a likaroloana tsa linaheng tse ling tsa ccfDNA ea liphoofolo tse ngata tsa DNA ea masapo a masapo a mangata (setšoantšo sa 5).Ho fumanoe likaroloana tsa DNA tse tsoang likokoanyana le mefuta e meng.Karolo e batlang e le kholo ea likaroloana tsa DNA ha e e-s'o tsejoe, mohlomong ka lebaka la ho fokotseha ha palo e kholo ea mefuta ea leoatleng ka har'a boitsebiso ba genomic ha e bapisoa le mefuta ea lefatše [49].
Leqepheng la hona joale, re sebelisa mohopolo oa LB ho li-mussels, re pheha khang ea hore tatellano ea thunya ea hemolymph ccfDNA e ka fana ka temohisiso mabapi le sebopeho sa tikoloho ea maoatleng a leoatle.Haholo-holo, re fumane hore 1) mussel hemolymph e na le likhahla tse batlang li phahame (maemo a microgram) a batlang a le maholo (~ 1-5 kb) a potolohang likaroloana tsa DNA;2) likhechana tsena tsa DNA li ikemetse ebile ha li na boikemelo 3) Har'a mehloli e tsoang linaheng tse ling ea likhechana tsena tsa DNA, re fumane DNA ea baktheria, ea khale le ea kokoana-hloko, hammoho le DNA ea liphoofolo tse ling tse ngata;4) Ho bokellana ha likhechana tsena tsa ccfDNA tsa kantle ho hemolymph ho etsahala ka potlako mme ho kenya letsoho tšebetsong ea ka hare ea ho sefa ea li-mussels.Qetellong, thuto ea rona e bonts'a hore mohopolo oa LB, oo ho fihlela joale o sebelisitsoeng haholo-holo lefapheng la biomedicine, o kenyelletsa mohloli o ruileng empa o sa lekoa oa tsebo o ka sebelisoang ho utloisisa hamolemo tšebelisano pakeng tsa mefuta ea balebeli le tikoloho ea bona.
Ntle le litšoene, ho itšehla thajana ha ccfDNA ho tlalehiloe liphoofolong tse anyesang, ho kenyeletsoa litoeba, lintja, likatse le lipere [50, 51, 52].Leha ho le joalo, ho ea ka tsebo ea rona, thuto ea rona ke ea pele ho tlaleha ho fumanoa le tatellano ea ccfDNA mefuteng ea leoatle ka mokhoa o bulehileng oa ho potoloha.Tšobotsi ena ea sebōpeho le bokhoni ba ho sefa ba li-mussels, bonyane ka karolo e itseng, e ka hlalosa litšobotsi tse fapaneng tsa boholo ba likhechana tsa DNA tse potolohang ha li bapisoa le mefuta e meng.Ho batho, likaroloana tse ngata tsa DNA tse potolohang maling ke likhechana tse nyane ho tloha ho 150 ho isa ho 200 bp.ka tlhōrō e phahameng ea 167 bp [34, 53].Karolo e nyenyane empa e le ea bohlokoa ea likaroloana tsa DNA e pakeng tsa 300 le 500 bp ka boholo, 'me hoo e ka bang 5% e telele ho feta 900 bp.[54].Lebaka la ho ajoa ha boholo bona ke hore mohloli o ka sehloohong oa ccfDNA ho plasma o hlaha ka lebaka la lefu la sele, ebang ke ka lebaka la lefu la sele kapa ka lebaka la necrosis ea lisele tsa hematopoietic tse potolohang ho batho ba phetseng hantle kapa ka lebaka la apoptosis ea lisele tsa hlahala ho bakuli ba mofets'e (e tsejoang ka hore ke DNA e potolohang ea hlahala)., ctDNA).Kabo ea boholo ba hemolymph ccfDNA eo re e fumaneng ho li-mussels e tloha ho 1000 ho isa ho 5000 bp, ho fana ka maikutlo a hore mussel ccfDNA e na le tšimoloho e fapaneng.Ena ke khopolo-taba e utloahalang, kaha li-mussels li na le tsamaiso ea methapo e bulehileng ka halofo 'me li phela libakeng tse phelang metsing tse nang le likarolo tse phahameng tsa DNA ea likokoana-hloko.Ha e le hantle, liteko tsa rona tsa laboratori tse sebelisang DNA e sa tloaelehang li bontšitse hore li-mussels li bokella likhechana tsa DNA metsing a leoatle, bonyane ka mor'a lihora tse 'maloa li senyeha ka mor'a ho kenngoa ha lisele le / kapa ho lokolloa le / kapa ho bolokoa mekhatlong e sa tšoaneng.Ka lebaka la ho haella ha lisele (ka bobeli prokaryotic le eukaryotic), tšebeliso ea likaroloana tsa intravalvular e tla fokotsa bongata ba ccfDNA ho tsoa mehloling ea boinotšing hammoho le mehloling ea kantle ho naha.Ha re nahana ka bohlokoa ba boits'ireletso ba tlhaho ba bivalve le palo e kholo ea li-phagocyte tse potolohang, re ile ra tsoela pele ho nahana hore esita le ccfDNA e tsoang linaheng tse ling e ruisitsoe ka phagocyte e potolohang e bokellang DNA e tsoang linaheng tse ling ha e kenngoa ka likokoana-hloko le / kapa litšila tsa cellular.Ha li kopane, liphetho tsa rona li bonts'a hore bivalve hemolymph ccfDNA ke polokelo e ikhethang ea tlhaiso-leseling ea limolek'hule mme e tiisa boemo ba bona joalo ka mofuta oa molebeli.
Lintlha tsa rona li bonts'a hore tatellano le tlhahlobo ea likhechana tsa hemolymph ccfDNA tse tsoang ho baktheria li ka fana ka leseli la bohlokoa mabapi le limela tsa baktheria le libaktheria tse teng tikolohong e haufi ea leoatle.Mekhoa ea tatellano ea lithunya e senotse tatelano ea libaktheria tsa commensal A. atra gill tse ka beng li fositse haeba mekhoa e tloaelehileng ea ho tsebahatsa ea 16S rRNA e ne e sebelisitsoe, ka lebaka la karolo e 'ngoe ea leeme la laeborari ea litšupiso.Ha e le hantle, tšebeliso ea rona ea lintlha tsa LB tse bokelitsoeng ho M. platensis sebakeng se tšoanang sa mussel ho Kerguelen se bontšitse hore ho hlophisoa ha likokoana-hloko tse amanang le gill ho ne ho tšoana le mefuta e 'meli ea li-mussel (Fig. S4, Tlhahisoleseding ea Tlatsetso).Ho tšoana hona ha li-mussels tse peli tse fapaneng ka liphatsa tsa lefutso ho ka bonts'a sebopeho sa libaka tsa baktheria sebakeng se batang, sebabole le seretse se chesang sa Kerguelen [55, 56, 57, 58].Maemo a phahameng a likokoana-hloko tse fokotsang sebabole a hlalositsoe hantle ha ho kotuloa li-mussels libakeng tse nang le bioturbated lebōpong la leoatle [59], joalo ka lebopo la Port-au-France.Monyetla o mong ke hore limela tsa commensal mussel li ka angoa ke phetisetso e otlolohileng [60, 61].Ho hlokahala lipatlisiso tse ling ho fumana kamano pakeng tsa tikoloho ea leoatle, bokaholimo ba leoatle, le sebopeho sa libaktheria tsa symbiotic li-mussels.Lithuto tsena li ntse li tsoela pele.
Bolelele le mahloriso a hemolymph ccfDNA, ho hloekisoa ha eona habonolo, le boleng bo phahameng ba ho lumella tatellano ea lithunya tse potlakileng ke e meng ea melemo e mengata ea ho sebelisa li-mussel ccfDNA ho lekola mefuta-futa ea lintho tse phelang tikolohong ea maoatleng a leoatle.Mokhoa ona o sebetsa ka ho khetheha bakeng sa ho khetholla lichaba tsa likokoana-hloko (viromes) sebakeng se itseng sa tikoloho [62, 63].Ho fapana le libaktheria, archaea, le eukaryotes, liphatsa tsa lefutso tsa kokoana-hloko ha li na liphatsa tsa lefutso tse bolokiloeng ka phylogenetically tse kang tatellano ea 16S.Liphetho tsa rona li bonts'a hore li-biopsies tsa metsi tse tsoang mefuteng e bonts'ang joalo ka li-mussels li ka sebelisoa ho tseba palo e kholo ea likhechana tsa vaerase ea ccfDNA tse tsejoang ho tšoaetsa batho ba lulang libakeng tse lebōpong la leoatle.Sena se kenyelletsa likokoana-hloko tse tsejoang ka ho tšoaetsa protozoa, arthropods, likokoanyana, limela le likokoana-hloko tsa baktheria (mohlala, bacteriophages).Kabo e tšoanang e ile ea fumanoa ha re hlahloba hemolymph ccfDNA virome ea li-mussels tse putsoa (M. platensis) tse bokelletsoeng ka lera le tšoanang la mussel Kerguelen (Letlapa la S2, Boitsebiso bo Tlatselletsang).Ho latellana ha lithunya tsa ccfDNA ke mokhoa o mocha o ntseng o eketseha thutong ea virome ea batho kapa mefuta e meng [21, 37, 64].Mokhoa ona o bohlokoa haholo ho ithuteng livaerase tsa DNA tse nang le mela e 'meli, kaha ha ho na mofuta o le mong o bolokiloeng har'a livaerase tsa DNA tse nang le mela e 'meli, tse emelang sehlopha sa livaerase tse fapaneng ka ho fetisisa Baltimore [65].Leha boholo ba likokoana-hloko tsena li lula li sa aroloa 'me li ka kenyelletsa likokoana-hloko tse tsoang karolong e sa tsejoeng ka ho feletseng ea lefats'e la kokoana-hloko [66], re fumane hore li-viromes le mefuta e mengata ea li-mussels A. atra le M. platensis li oela pakeng tsa mefuta e 'meli.ka mokhoa o ts'oanang (sheba setšoantšo sa S3, lintlha tse ling).Ho tšoana hona ha ho makatse, kaha ho ka 'na ha bontša ho hloka boikhethelo ho nkeng DNA e teng tikolohong.Lithuto tsa nako e tlang tse sebelisang RNA e hloekisitsoeng lia hlokahala hajoale ho tšoaea RNA virome.
Thutong ea rona, re sebelisitse liphaephe tse matla haholo tse ikamahanyang le mosebetsi oa Kowarski le basebetsi-'moho le bona [37], ba sebelisitseng ho hlakola ka mehato e 'meli ea ho bala le li-contigs pele le ka morao ho kopano ea matsoalloa a ccfDNA, e leng se ileng sa fella ka karolo e phahameng ea lipalo tse sa ngolisoang.Ka hona, re ke ke ra hanela hore tse ling tsa limmapa tsena li ntse li e-na le tšimoloho ea tsona, haholo-holo hobane ha re na genome ea litšupiso bakeng sa mofuta ona oa li-mussel.Re boetse re sebelisitse pipeline ena hobane re ne re amehile ka li-chimeras pakeng tsa batho ba ikemetseng le ba sa ipallang le bolelele ba ho bala bo hlahisoang ke Illumina MiSeq PE75.Lebaka le leng la lipalo tse ngata tse sa hlalosoang ke hore boholo ba likokoana-hloko tsa leoatleng, haholo-holo libakeng tse ka thōko tse kang Kerguelen, ha li so hlalosoe.Re sebelisitse Illumina MiSeq PE75, re nka bolelele ba sekhechana sa ccfDNA bo ts'oana le ccfDNA ea motho.Bakeng sa lithuto tsa nakong e tlang, ho latela liphetho tsa rona tse bonts'ang hore hemolymph ccfDNA e bala nako e telele ho feta batho le/kapa liphoofolo tse anyesang, re khothaletsa ho sebelisa sethala sa tatellano se loketseng likaroloana tse telele tsa ccfDNA.Mokhoa ona o tla etsa hore ho be bonolo haholo ho tseba matšoao a mangata bakeng sa tlhahlobo e tebileng.Ho fumana tatelano e felletseng ea jenome ea nyutlelie ea A. atra e sa fumaneheng hona joale ho ka thusa haholo khethollo ea ccfDNA ho tsoa mehloling ea hau le eo e seng ea hau.Ka lebaka la hore lipatlisiso tsa rona li shebane le monyetla oa ho sebelisa mohopolo oa biopsy ea metsi ho li-mussels, re tšepa hore ha khopolo ena e sebelisoa liphuputsong tse tlang, lisebelisoa tse ncha le liphaephe li tla ntlafatsoa ho eketsa bokhoni ba mokhoa ona oa ho ithuta mefuta-futa ea likokoana-hloko tsa li-mussels.tikoloho ea leoatle.
Joaloka biomarker ea kliniki e sa hlaseleng, maemo a phahameng a plasma ea ccfDNA a amahanngoa le mafu a sa tšoaneng, tšenyo ea lisele le maemo a khatello ea kelello [67,68,69].Keketseho ena e amahanngoa le ho lokolloa ha likhechana tsa DNA tsa tšimoloho ea eona ka mor'a tšenyo ea lisele.Re ile ra rarolla bothata bona re sebelisa khatello e matla ea mocheso, moo li-mussels li ileng tsa pepesetsoa mocheso oa 30 ° C nakoana.Re entse tlhahlobo ena mefuteng e meraro e fapaneng ea li-mussels litekong tse tharo tse ikemetseng.Leha ho le joalo, ha rea ​​ka ra fumana phetoho leha e le efe maemong a ccfDNA ka mor'a khatello e matla ea mocheso (bona Setšoantšo sa S5, boitsebiso bo eketsehileng).Ho sibolloa hona ho ka hlalosa, bonyane ka karolo e 'ngoe, taba ea hore li-mussels li na le mokhoa o bulehileng oa ho potoloha ha mali 'me li bokella bongata bo boholo ba DNA ea kantle ho naha ka lebaka la ts'ebetso ea tsona e phahameng ea ho sefa.Ka lehlakoreng le leng, li-mussels, joalo ka liphoofolo tse ngata tse se nang lesapo la mokokotlo, li ka 'na tsa hanana le tšenyo ea lisele tse bakoang ke khatello ea maikutlo, kahoo li fokotsa ho lokolloa ha ccfDNA ho hemolymph [70, 71].
Ho fihlela joale, tlhahlobo ea DNA ea mefuta-futa ea lintho tse phelang metsing e shebane haholo le metabarcoding ea DNA (eDNA) ea tikoloho.Leha ho le joalo, mokhoa ona hangata o lekanyelitsoe tlhahlobong ea mefuta-futa ea lihloliloeng ha ho sebelisoa li-primers.Tšebeliso ea tatellano ea lithunya e qoba meeli ea PCR le khetho e leeme ea li-primer sets.Ka hona, ka tsela e itseng, mokhoa oa rona o haufi le mokhoa oa morao-rao o sebelisoang haholo oa eDNA Shotgun tatellano, o khonang ho tatellana ka ho toba DNA e arohaneng le ho hlahloba hoo e batlang e le lintho tsohle tse phelang [72, 73].Leha ho le joalo, ho na le lintlha tse 'maloa tsa bohlokoa tse khethollang LB ho mekhoa e tloaelehileng ea eDNA.Ehlile, phapang e kholo lipakeng tsa eDNA le LB ke ts'ebeliso ea li-host host tsa tlhaho.Tšebeliso ea mefuta ea leoatleng e kang liponche le li-bivalves (Dresseina spp.) e le sefe ea tlhaho ea ho ithuta eDNA e tlalehiloe [74, 75].Leha ho le joalo, phuputso ea Dreissena e sebelisitse li-biopsies tsa lisele tseo DNA e ileng ea ntšoa ho tsona.Tlhahlobo ea ccfDNA ho tsoa ho LB ha e hloke biopsy ea lisele, lisebelisoa tse khethehileng 'me ka linako tse ling li turu le lisebelisoa tse amanang le eDNA kapa biopsy ea lisele.Ha e le hantle, re sa tsoa tlaleha hore ccfDNA e tsoang LB e ka bolokoa le ho hlahlojoa ka tšehetso ea FTA ntle le ho boloka ketane e batang, e leng phephetso e kholo ea lipatlisiso libakeng tse hōle [76].Ho ntšoa ha ccfDNA ho li-biopsies tsa metsi ho boetse ho bonolo ebile ho fana ka DNA ea boleng bo holimo bakeng sa tatellano ea lithunya le tlhahlobo ea PCR.Ona ke monyetla o moholo ho fanoe ka tse ling tsa mefokolo ea tekheniki e amanang le tlhahlobo ea eDNA [77].Bonolo le litšenyehelo tse tlase tsa mokhoa oa ho etsa lisampole le tsona li loketse ka ho khetheha bakeng sa mananeo a nako e telele a ho beha leihlo.Ntle le bokhoni ba bona bo phahameng ba ho sefa, tšobotsi e 'ngoe e tsebahalang ea li-bivalves ke motsoako oa lik'hemik'hale oa mucopolysaccharide oa mucus oa bona, o khothalletsang ho monya ha livaerase [78, 79].Sena se etsa hore bivalves e be sefe se loketseng sa tlhaho bakeng sa ho khetholla mefuta-futa ea lihloliloeng le phello ea phetoho ea maemo a leholimo tikolohong e itseng ea metsing.Le hoja boteng ba likhechana tsa DNA tse nkiloeng ho moamoheli bo ka bonoa e le moeli oa mokhoa o bapisoang le eDNA, litšenyehelo tse amanang le ho ba le ccfDNA e joalo ea tlhaho ha e bapisoa le eDNA e utloahala ka nako e le 'ngoe bakeng sa boitsebiso bo bongata bo fumanehang bakeng sa lithuto tsa bophelo bo botle.offset host.Sena se kenyelletsa boteng ba tatellano ea vaerase e kopantsoeng le genome ea moamoheli.Sena se bohlokoa haholo bakeng sa li-mussels, ho latela boteng ba li-leukemic retroviruses tse fetisetsoang ka holimo ho li-bivalves [80, 81].Molemo o mong oa LB ho feta eDNA ke hore e sebelisa ts'ebetso ea phagocytic ea ho potoloha lisele tsa mali ho hemolymph, e kenyang likokoana-hloko (le liphatsa tsa lefutso tsa tsona).Phagocytosis ke mosebetsi o ka sehloohong oa lisele tsa mali ka bivalves [82].Qetellong, mokhoa ona o nka monyetla oa matla a phahameng a ho sefa ha li-mussels (karolelano ea 1.5 l/h ea metsi a leoatle) le potoloho ea matsatsi a mabeli, e leng ho eketsang ho tsoakana ha likarolo tse fapaneng tsa metsi a leoatle, ho lumella ho tšoaroa ha eDNA ea heterologous.[83,84].Kahoo, tlhahlobo ea li-mussel ccfDNA ke mokhoa o khahlisang ho latela litlamorao tsa phepo, moruo le tikoloho ea li-mussels.Ho ts'oana le tlhahlobo ea LB e bokelletsoeng ho batho, mokhoa ona o boetse o bula monyetla oa ho lekanya liphetoho tsa liphatsa tsa lefutso le tsa epigenetic ho DNA ea moeti ho arabela lintho tse sa tloaelehang.Mohlala, ho ka nahanoa ka theknoloji ea tatellano ea moloko oa boraro ho etsa tlhahlobo ea genome-wide methylation ho ccfDNA ea matsoalloa ho sebelisa nanopore sequencing.Ts'ebetso ena e lokela ho nolofatsoa ke taba ea hore bolelele ba likhechana tsa ccfDNA tsa mussel bo lumellana hantle le liforomo tsa tatellano tsa nako e telele tse lumellang genome-wide DNA methylation analysis ho tloha ho sequencing e le 'ngoe ntle le tlhokahalo ea liphetoho tsa lik'hemik'hale.Ka hona, e ka fana ka temohisiso ea bohlokoa mabapi le mekhoa ea motheo e laolang karabelo ka mor'a ho pepesehela phetoho ea boemo ba leholimo kapa litšila [87].Leha ho le joalo, tšebeliso ea LB ha e na meeli.Ha ho pelaelo hore sena se hloka hore ho be le mefuta e bontšang lintho tse phelang tikolohong.Joalokaha ho boletsoe ka holimo, ho sebelisa LB ho lekola mefuta-futa ea tikoloho ea tikoloho e fanoeng ho boetse ho hloka phala e matla ea bioinformatics e nahanelang boteng ba likhechana tsa DNA ho tsoa mohloling.Bothata bo bong bo boholo ke ho fumaneha ha liphatsa tsa lefutso tsa mefuta ea metsing.Ho na le tšepo ea hore mehato e kang ea Marine Mammal Genomes Project le morero o sa tsoa thehoa oa Fish10k [88] o tla thusa tlhahlobo e joalo nakong e tlang.Tšebeliso ea khopolo ea LB ho lintho tse phelang 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