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Biopsy ea metsi (LB) ke khopolo e ntseng e tuma ka potlako lefapheng la bongaka. Khopolo ena e thehiloe haholo-holo ho fumanoeng ha likaroloana tsa DNA e potolohang kantle ho sele (ccfDNA), tse lokolloang haholo-holo e le likaroloana tse nyane kamora lefu la sele liseleng tse fapaneng. Karolo e nyane ea likaroloana tsena e tsoa liseleng tse tsoang linaheng tse ling (tse tsoang linaheng tse ling) kapa linthong tse phelang. Mosebetsing oa hajoale, re sebelisitse khopolo ena ho li-mussel, mofuta oa selikalikoe o tsejoang ka bokhoni ba tsona bo phahameng ba ho sefa metsi a leoatle. Re sebelisa bokhoni ba li-mussel ho sebetsa e le li-filter tsa tlhaho ho hapa likaroloana tsa DNA tsa tikoloho ho tsoa mehloling e fapaneng ho fana ka tlhaiso-leseling mabapi le mefuta-futa ea lintho tse phelang lebopong la leoatle. Liphetho tsa rona li bontša hore hemolymph ea mussel e na le likaroloana tsa DNA tse fapaneng haholo ka boholo, ho tloha ho 1 ho isa ho 5 kb. Tatellano ea lithunya e bontšitse hore palo e kholo ea likaroloana tsa DNA e simolohile likokoana-hlokong tse tsoang linaheng tse ling. Har'a tsona, re fumane likaroloana tsa DNA tse tsoang libaktheria, archaea le livaerase, ho kenyeletsoa le livaerase tse tsejoang ka ho tšoaetsa mefuta e fapaneng ea batho ba lulang libakeng tse phelang lebopong la leoatle. Qetellong, phuputso ea rona e bontša hore khopolo ea LB e sebelisitsoeng ho li-mussels e emela mohloli o ruileng oa tsebo empa o e-so hlahlojoe mabapi le mefuta-futa ea likokoana-hloko tikolohong ea lebopong la leoatle.
Tšusumetso ea phetoho ea tlelaemete (CC) holim'a mefuta-futa ea lintho tse phelang metsing ke sebaka se ntseng se hola ka potlako sa lipatlisiso. Ho futhumala ha lefatše ha ho bake feela likhatello tsa bohlokoa tsa 'mele, empa hape ho sutumelletsa meeli ea ho iphetola ha lintho ea botsitso ba mocheso ba lintho tse phelang metsing, ho ama bolulo ba mefuta e mengata ea lintho tse phelang metsing, ho li susumelletsa ho batla maemo a matle haholoanyane [1, 2]. Ntle le ho ama mefuta-futa ea lintho tse phelang metsing, CC e sitisa tekano e bonolo ea likamano tsa moamoheli le likokoana-hloko. Dysbacteriosis ena ea likokoana-hloko e beha tšokelo e kholo ho lintho tse phelang metsing kaha e etsa hore lintho tse phelang metsing li be kotsing ea ho tšoaetsoa ke likokoana-hloko tse tšoaetsanoang [3, 4]. Ho lumeloa hore SS e bapala karolo ea bohlokoa lefung la bongata, e leng bothata bo boholo bakeng sa tsamaiso ea lintho tse phelang metsing lefatšeng ka bophara [5, 6]. Ena ke taba ea bohlokoa ka lebaka la litlamorao tsa moruo, tikoloho le phepo ea mefuta e mengata ea leoatle. Sena ke 'nete haholo-holo bakeng sa li-bivalve tse lulang libakeng tsa polar, moo liphello tsa CK li leng teng hang-hang le ka matla [6, 7]. Ha e le hantle, li-bivalve tse kang Mytilus spp. li sebelisoa haholo ho lekola liphello tsa CC ho lintho tse phelang metsing. Ha ho makatse hore ebe palo e kholo ea li-biomarker e ntlafalitsoe ho lekola bophelo ba tsona, hangata ho sebelisoa mokhoa oa mekhahlelo e 'meli o kenyeletsang li-biomarker tse sebetsang tse thehiloeng ts'ebetsong ea enzymatic kapa mesebetsi ea sele joalo ka ho phela ha sele le ts'ebetso ea phagocytic [8]. Mekhoa ena e boetse e kenyelletsa ho lekanya mahloriso a matšoao a itseng a khatello a bokellanang liseleng tse bonolo kamora ho monngoa ha metsi a mangata a leoatle. Leha ho le joalo, bokhoni bo phahameng ba ho sefa le sistimi ea potoloho ea mali e bulehileng hanyane ea li-bivalve li fana ka monyetla oa ho nts'etsapele li-biomarker tse ncha tsa hemolymph ho sebelisoa mohopolo oa biopsy ea metsi (LB), mokhoa o bonolo le o sa keneleng haholo oa tsamaiso ea bakuli. lisampole tsa mali [9, 10]. Le hoja mefuta e 'maloa ea limolek'hule tse potolohang e ka fumanoa ho LB ea motho, mohopolo ona o thehiloe haholo-holo tlhahlobong ea tatellano ea DNA ea likaroloana tsa DNA tse potolohang kantle ho sele (ccfDNA) plasma. Ha e le hantle, boteng ba DNA e potolohang plasma ea motho bo 'nile ba tsejoa ho tloha bohareng ba lekholo la bo20 la lilemo [11], empa ke lilemong tsa morao tjena feela moo ho fihla ha mekhoa ea tatellano ea phallo e phahameng ho lebisitseng tlhahlobong ea bongaka e thehiloeng ho ccfDNA. Boteng ba dikotwana tsena tsa DNA tse potolohang bo bakwa ke ho lokollwa ha DNA ya genomic (nyuklea le mitochondrial) ka mora lefu la sele. Ho batho ba phetseng hantle, mahloriso a ccfDNA hangata a tlase (<10 ng/mL) empa a ka eketseha ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa ba nang le khatello ea maikutlo, e leng se fellang ka tšenyo ea lisele. Ho batho ba phetseng hantle, mahloriso a ccfDNA hangata a tlase (<10 ng/mL) empa a ka eketseha ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa ba nang le khatello ea maikutlo, e leng se fellang ka tšenyo ea lisele. Те здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 раз у больныхй пилика подвергающихся стрессу, приводящему к повреждению тканей. Ho batho ba phetseng hantle, mahloriso a cccDNA hangata a tlase (<10 ng/mL), empa a ka eketseha ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa ba nang le khatello ea maikutlo e lebisang tšenyo ea 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悢1者倍，从而导致组织损伤。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5-10 倍 , 从而 组织。。。Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут быть увеличены в 5-10 раз у пациентоный стрессом, что приводит к повреждению тканей. Hangata mahloriso a ccfDNA a tlase (<10 ng/ml) ho batho ba phetseng hantle, empa a ka eketseha ka makhetlo a 5-10 ho bakuli ba nang le mafu a fapaneng kapa khatello ea maikutlo, e leng se fellang ka tšenyo ea lisele.Boholo ba dikotwana tsa ccfDNA bo fapana haholo, empa hangata bo tloha ho 150 ho isa ho 200 bp. [12]. Tlhahlobo ya ccfDNA e iphumanetseng, ke hore, ccfDNA ho tswa diseleng tse tlwaelehileng kapa tse fetotsweng, e ka sebediswa ho lemoha diphetoho tsa lefutso le tsa epigenetic tse teng ka hara genome ya nuclear le/kapa mitochondrial, ka hona e thusa dingaka ho kgetha mekhoa e ikgethang ya kalafo e shebaneng le dimolek'hule [13]. Leha ho le jwalo, ccfDNA e ka fumanwa mehloding e meng e kang ccfDNA ho tswa diseleng tsa lesea nakong ya boimana kapa ho tswa dithong tse kentsweng [14,15,16,17]. ccfDNA hape ke mohlodi wa bohlokwa wa tlhahisoleseding bakeng sa ho lemoha boteng ba nucleic acids ya moemedi ya tshwaetsanang (kantle ho naha), e leng se dumellang ho lemoha ditshwaetso tse atileng tse sa tsejweng ke ditso tsa madi, ho qoba biopsy e hlaselang ya dinama tse tshwaeditsweng [18]. Diphuputso tsa moraorao di bontshitse hore madi a motho a na le mohlodi o ruileng wa tlhahisoleseding o ka sebediswang ho hlwaya dikokwana-hloko tsa vaerase le baktheria, le hore hoo e ka bang 1% ya ccfDNA e fumanwang plasma ya motho e tswa kantle ho naha [19]. Lithuto tsena li bontša hore mefuta-futa ea likokoana-hloko tse potolohang tsa ntho e phelang e ka hlahlojoa ka ho sebelisa tlhahlobo ea ccfDNA. Leha ho le joalo, ho fihlela morao tjena, khopolo ena e ne e sebelisoa feela ho batho le, ka tekanyo e nyane, ho liphoofolo tse ling tse nang le lesapo la mokokotlo [20, 21].
Pampiring ena, re sebedisa bokgoni ba LB ho sekaseka ccfDNA ya Aulacomya atra, mofuta o ka borwa o atisang ho fumanwa Dihlekehlekeng tsa Kerguelen tse ka tlase ho antarctic, sehlopha sa dihlekehleke tse hodima lesabasaba le leholo le ileng la bopjwa dilemong tse dimilione tse 35 tse fetileng. Ho sebedisa sistimi ya teko ya in vitro, re fumane hore dikotwana tsa DNA metsing a lewatle di nkuwa kapele ke di-mussel mme di kena karolong ya hemolymph. Ho latellana ha dithunya ho bontshitse hore mussel hemolymph ccfDNA e na le dikotwana tsa DNA tsa yona le tse seng tsa yona, ho kenyeletswa le dibaktheria tse tshwanang le dikotwana tsa DNA tse tswang ho di-biomes tse tlwaelehileng tsa tikoloho e batang ya lewatle e nang le seretse se chesang. Hemolymph ccfDNA e boetse e na le dikotwana tsa vaerase tse tswang ho divaerase tse nang le mefuta e fapaneng ya moamoheli. Re boetse re fumane dikotwana tsa DNA tse tswang diphoofolong tse nang le disele tse ngata tse kang tlhapi ya masapo, anemone ya lewatle, bolele le dikokonyana. Qetellong, thuto ya rona e bontsha hore mohopolo wa LB o ka sebediswa ka katleho ho diphoofolo tse se nang lesapo la mokokotlo tsa lewatle ho hlahisa repertoire e ruileng ya genomic ditikolohong tsa lewatle.
Li-Mytilus platensis (M. platensis) tse kholo (bolelele ba 55-70 mm) le Aulacomya atra (A. atra) li ile tsa bokelloa lebopong le mafika la Port-au-France (049°21.235 S, 070°13.490 E .). Lihlekehleke tsa Kerguelen ka Tšitoe 2018. Li-mussel tse ling tse kholo tse putsoa (Mytilus spp.) li fumanoe ho mofani oa khoebo (PEI Mussel King Inc., Prince Edward Island, Canada) 'me tsa kenngoa ka tankeng e nang le moea o laoloang ke mocheso (4°C) e nang le 10-20 L ea metsi a letsoai a maiketsetso a 32‰. (letsoai la leoatle la maiketsetso Reef Crystal, Instant Ocean, Virginia, USA). Bakeng sa teko ka 'ngoe, bolelele le boima ba likhetla ka bonngoe li ile tsa lekanngoa.
Protokhole ya phihlello e bulehileng ya mahala bakeng sa lenaneo lena e fumaneha inthaneteng (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). Ka bokhutšoanyane, hemolymph ya LB e ile ya bokellwa ho tswa mesifeng ya abductor jwalo ka ha ho hlalositswe [22]. Hemolymph e ile ya hlakiswa ka ho centrifugation ho 1200×g metsotso e 3, supernatant e ile ya hoamiswa (-20°C) ho fihlela e sebediswa. Bakeng sa ho arola le ho hlwekisa cfDNA, disampole (1.5-2.0 ml) di ile tsa qhibilihiswa mme tsa sebetswa ho sebediswa NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ho ya ka ditaelo tsa moetsi. ccfDNA e ile ya bolokwa ho -80°C ho fihlela tlhahlobo e nngwe. Ditekong tse ding, ccfDNA e ile ya arolwa mme ya hlwekiswa ho sebediswa QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada). DNA e hlwekisitsweng e ile ya lekanngwa ho sebediswa PicoGreen assay e tloaelehileng. Kabo ea karoloana ea ccfDNA e arohaneng e hlahlobiloe ka capillary electrophoresis ho sebelisoa Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) ho sebelisoa High Sensitivity DNA Kit. Teko e entsoe ho sebelisoa 1 µl ea sampole ea ccfDNA ho latela litaelo tsa moetsi.
Bakeng sa ho hlophisa likaroloana tsa hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Canada) o lokisitse lilaebrari tsa lithunya a sebelisa kit ea Illumina DNA Mix ea kit ea Illumina MiSeq PE75. Ho sebelisitsoe adaptara e tloaelehileng (BioO). Lifaele tsa data tse tala lia fumaneha ho tsoa 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 sebelisitsoe bakeng sa ho khaola li-adapter le ho bala boleng bo bobe. Ho bala ha lithunya ka lipheletso tse kopantsoeng ho ile ha kopanngoa FLASH ho baleng nako e telele ka ho kopana bonyane ha 20 bp ho qoba ho se lumellane [25]. Dipalo tse kopantsweng di ile tsa ngolwa ka BLASTN ho sebediswa database ya bivalve NCBI Taxonomy (boleng ba e < 1e−3 le 90% homology), mme ho kwalwa ha ditatelano tse rarahaneng tse tlase ho ile ha etswa ho sebediswa DUST [26]. Dipalo tse kopantsweng di ile tsa ngolwa ka BLASTN ho sebediswa database ya bivalve NCBI Taxonomy (boleng ba e < 1e−3 le 90% homology), mme ho kwalwa ha ditatelano tse rarahaneng tse tlase ho ile ha etswa ho sebediswa DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN базы данных таксономии двустворчатых моллюсков NCBI (8) гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием LEROLE [26]. Dipalo tse kopaneng di ile tsa ngolwa ka BLASTN ho sebediswa database ya taxonomy ya NCBI bivalve (boleng ba e < 1e-3 le 90% homology), mme ho kwalwa ha tatellano e rarahaneng ho ile ha etswa ho sebediswa 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DUST [26]进行低复杂度序列的掩蔽.使用 双 壳类 ncbi 分类 (（（<1e-3 和 90% 同源） 用 用 注释 合并读数 ， 并 使光特片 [26] sОбъединенные чтения были аннотированы с помощью BLASTN беспользованием таксономической базы даных двустворчатых моллюсков-3% гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием LEROLE [26]. Dipalo tse kopaneng di ile tsa ngolwa ka BLASTN ho sebediswa database ya taxonomic ya NCBI bivalve taxonomic (boleng ba e <1e-3 le 90% homology), mme ho kwalwa ha tatellano e rarahaneng ho ile ha etswa ho sebediswa DUST [26].Dipalo di ne di arotswe ka dihlopha tse pedi: tse amanang le di-bivalve tatelano (mona di bitswa ho ipalla) le tse sa amaneng (tse sa ipaleng). Dihlopha tse pedi di ile tsa bokellwa ka thoko ho sebediswa MEGAHIT ho hlahisa di-contigs [27]. Ho sa le jwalo, kabo ya taxonomic ya dipalo tsa di-microbiome tse tswang kantle e ile ya arolwa ka ho sebedisa Kraken2 [28] mme ya emelwa ka setshwantsho ke chate ya pie ya Krona ho Galaxy [29, 30]. Di-kmers tse ntle ka ho fetisisa di ile tsa nkuwa e le di-kmers-59 ho tloha ditekong tsa rona tsa pele. Di-contig tse ikemetseng di ile tsa hlwauwa ka ho tsamaisana le BLASTN (database ya bivalve NCBI, boleng ba e < 1e−10 le 60% homology) bakeng sa tlhaloso ya ho qetela. Di-contig tse ikemetseng di ile tsa hlwauwa ka ho tsamaisana le BLASTN (database ya bivalve NCBI, boleng ba e < 1e−10 le 60% homology) bakeng sa tlhaloso ya ho qetela. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, значени10 e) для окончательной аннотации. Di-self-contig di ile tsa hlwauwa ka ho bapisa le BLASTN (database ya NCBI bivalve, boleng ba e <1e-10 le 60% homology) bakeng sa tlhaloso ya 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）对齐來识别自身重叠群以进行最终注释.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 (база данных NCBI ND значение e <1e-10 le гомология 60%). Di-self-contig di ile tsa hlwauwa bakeng sa tlhaloso ya ho qetela ka ho bapisa le BLASTN (database ya NCBI bivalve, boleng ba e <1e-10 le 60% homology). Ka nako e ts'oanang, lihlopha tsa sehlopha tse seng tsa boithati li ile tsa ngoloa ka BLASTN (database ea nt NCBI, boleng ba e < 1e−10 le homology ea 60%). Ka nako e ts'oanang, lihlopha tsa sehlopha tse seng tsa boithati li ile tsa ngoloa ka BLASTN (database ea nt NCBI, boleng ba e < 1e−10 le homology ea 60%). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 и гомология 60%). Ka nako e ts'oanang, lihlopha tsa lihlopha tsa kantle ho naha li ile tsa ngoloa ka BLASTN (database ea NT NCBI, boleng ba e <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 nako e ts'oanang, lihlopha tse sa itšehleng ka botsona li ile tsa ngoloa ka BLASTN (database ea nt NCBI, boleng ba e <1e-10 le 60% homology). BLASTX e boetse e entsoe ho li-nonself contigs ho sebelisoa li-database tsa protheine ea nr le RefSeq NCBI (boleng ba e < 1e−10 le 60% homology). BLASTX e boetse e entsoe ho li-nonself contigs ho sebelisoa li-database tsa protheine ea nr le RefSeq NCBI (boleng ba e < 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%). BLASTX e boetse e entsoe ho li-non-self contigs ho sebelisoa li-database tsa protheine ea nr le RefSeq NCBI (boleng ba e < 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 boetse e entsoe ho li-non-self contigs ho sebelisoa li-database tsa protheine ea nr le RefSeq NCBI (boleng ba e <1e-10 le 60% homology).Matamo a BLASTN le BLASTX a lintho tse sa itšetleheng ka tsona a emela likarolo tsa ho qetela (sheba faele e tlatsetsang).
Li-primer tse sebelisitsoeng bakeng sa PCR li thathamisitsoe ho Tafole ea S1. Taq DNA polymerase (Bio Basic Canada, Markham, ON) e sebelisitsoe ho matlafatsa liphatsa tsa lefutso tse reretsoeng tsa ccfDNA. Maemo a latelang a karabelo a sebelisitsoe: ho fetoha ha 95°C metsotso e 3, 95°C motsotso o le mong, ho beha mocheso oa annealing motsotso o le mong, ho lelefatsa ho 72°C motsotso o le mong, lipotoloho tse 35, 'me qetellong ho 72°C metsotsong e 10. . Lihlahisoa tsa PCR li ile tsa aroloa ka 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 ile tsa tloaeleha ka metsing a leoatle a nang le oksijene a 500 ml (32 PSU) ka lihora tse 24 mochesong oa 4°C. DNA ea plasmid e nang le insert e kenyang tatellano ea human galectin-7 cDNA (nomoro ea kamohelo ea NCBI L07769) e ile ea eketsoa ka har'a vial ka tekanyo ea ho qetela ea 190 μg/μl. Li-mussels tse neng li koaletsoe tlas'a maemo a tšoanang ntle le ho eketsoa ha DNA e ne e le tsona tse laolang. 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 ile tsa nkuoa ho tsoa tankeng ka 'ngoe ka nako e boletsoeng. Bakeng sa ho latela 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 phahameng la metsi a leoatle, li-aliquots li ile tsa hlapolotsoa ka metsing a boleng ba PCR (1:10) pele ho liteko tsohle tsa PCR.
PCR ea marotholi a dijithale (ddPCR) e entsoe ho sebelisoa protocol ea BioRad QX200 (Mississauga, Ontario, Canada). Sebelisa boemo ba mocheso ho fumana mocheso o motle ka ho fetisisa (Tafole ea S1). Marotholi a hlahisitsoe ho sebelisoa jenereithara ea marotholi ea QX200 (BioRad). ddPCR e entsoe ka tsela e latelang: 95°C metsotso e 5, lipotoloho tse 50 tsa 95°C metsotsoana e 30 le mocheso o fanoeng oa annealing metsotso e 1 le 72°C metsotsoana e 30, 4°C metsotso e 5 le 90°C metsotso e 5. Palo ea marotholi le likarabelo tse ntle (palo ea likopi/µl) li lekantsoe ho sebelisoa sebali sa marotholi sa QX200 (BioRad). Lisampole tse nang le marotholi a ka tlase ho 10,000 li ile tsa haneloa. Taolo ea lipaterone ha ea ka ea etsoa nako le nako ha ddPCR e sebelisoa.
qPCR e entsoe ho sebelisoa Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) le li-primer tse khethehileng tsa LGALS7. Li-PCR tsohle tsa bongata li entsoe ka 20 µl ho sebelisoa QuantiFast SYBR Green PCR Kit (QIAGEN). qPCR e qaliloe ka ho kenngoa ha mocheso oa metsotso e 15 mochesong oa 95°C ho lateloa ke lipotoloho tse 40 mochesong oa 95°C metsotsoana e 10 le mochesong oa 60°C metsotsoana e 60 ka pokello e le 'ngoe ea data. Li-curve tse qhibilihang li hlahisitsoe ho sebelisoa litekanyo tse latellanang ho 95°C metsotsoana e 5, 65°C metsotsoana e 60, 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 sekhahla sa tsona se phahameng sa ho sefa, re ile ra qala ka ho batlisisa hore na li ka sefa le ho boloka likaroloana tsa DNA tse teng metsing a leoatle. Re ne re boetse re thahasella hore na likaroloana tsena li bokellana tsamaisong ea tsona ea lymphatic e bulehileng hanyane. Re rarollotse bothata bona ka teko ka ho latela qetello ea likaroloana tsa DNA tse qhibilihang tse ekelitsoeng litankeng tsa mussel e putsoa. ​​Ho nolofatsa ho latela likaroloana tsa DNA, re sebelisitse DNA ea plasmid e tsoang linaheng tse ling (eseng ea motho ka boeena) e nang le gene ea human galectin-7. ddPCR e latela likaroloana tsa DNA tsa plasmid metsing a leoatle le li-mussels. Liphetho tsa rona li bontša hore haeba palo ea likaroloana tsa DNA metsing a leoatle e lula e tsitsitse ha nako e ntse e ea (ho fihlela matsatsi a 7) ha li-mussels li le sieo, joale ha li-mussels li le teng boemo bona bo batla bo nyamela ka ho feletseng nakong ea lihora tse 8 (Setšoantšo sa 1a,b). Likaroloana tsa DNA ea kantle li ile tsa fumanoa habonolo nakong ea metsotso e 15 ka har'a mokelikeli oa intravalvular le hemolymph (Setšoantšo sa 1c). Likaroloana tsena li ntse li ka fumanoa ho fihlela lihora tse 4 kamora ho pepesehela. Mosebetsi ona oa ho sefa mabapi le likaroloana tsa DNA o tšoana le mosebetsi oa ho sefa oa libaktheria le bolele [31]. Liphetho tsena li bontša hore li-mussels li ka sefa le ho bokella DNA ea linaheng tse ling likarolong tsa tsona tsa mokelikeli.
Maqhutsu a amanang a DNA ea plasmid ka metsing a leoatle boteng ba (A) kapa bosieo ba (B), bo lekanngoang ke ddPCR. Ho A, liphetho li hlahisoa e le liperesente, 'me meeli ea mabokose e emela liperesente tsa 75 le 25. Kou ea logarithmic e kentsoeng e bontšoa ka bofubelu, 'me sebaka se koahetsoeng ka bohlooho se emela nako ea kholiseho ea 95%. Ho B, mola o mofubelu o emela karolelano 'me mola o moputsoa o emela nako ea kholiseho ea 95% bakeng sa kholiseho. C Ho bokellana ha DNA ea plasmid ka har'a hemolymph le valvular fluid ea li-mussels ka linako tse fapaneng kamora ho eketsoa ha DNA ea plasmid. Liphetho li hlahisoa e le likopi tse felletseng tse fumanoeng/mL (±SE).
Ka mor'a moo, re ile ra batlisisa tšimoloho ea ccfDNA ka har'a li-mussel tse bokelletsoeng libetheng tsa li-mussel Lihlekehlekeng tsa Kerguelen, sehlopha se hōle sa lihlekehleke tse nang le tšusumetso e fokolang ea batho. Bakeng sa morero ona, cccDNA e tsoang ho li-hemolymph tsa mussel e ile ea aroloa le ho hloekisoa ka mekhoa e sebelisoang hangata ho hloekisa cccDNA ea batho [32, 33]. Re fumane hore karolelano ea mahloriso a ccfDNA a hemolymph ka har'a li-mussel e ka har'a li-microgram tse tlase ka ml ea hemolymph (sheba Tafole S2, Tlhahisoleseling e Tlatsetsang). Mefuta ena ea mahloriso e kholo haholo ho feta ho batho ba phetseng hantle (li-nanogram tse tlase ka milliliter), empa maemong a sa tloaelehang, ho bakuli ba kankere, boemo ba ccfDNA bo ka fihla ho li-microgram tse 'maloa ka milliliter [34, 35]. Tlhahlobo ea kabo ea boholo ba hemolymph ccfDNA e bontšitse hore likotoana 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 tšoanang li fumanoe ho sebelisoa QIAamp Investigator Kit e thehiloeng silica, e leng mokhoa o sebelisoang haholo saenseng ea forensic ho arola le ho hloekisa DNA ea genomic ka potlako ho tsoa lisampoleng tsa DNA tse nang le mahloriso a tlase, ho kenyeletsoa le ccfDNA [36].
Electrophoregram e emelang ccfDNA ea hemolymph ea mussel. E ntšoa ka NucleoSnap Plasma Kit (holimo) le QIAamp DNA Investigator Kit. Setšoantšo sa B Violin se bontšang kabo ea hemolymph mahloriso a ccfDNA (±SE) ka har'a mussels. Mela e metšo le e khubelu e emela li-quartile tse mahareng le tsa pele le tsa boraro, ka ho latellana.
Hoo e ka bang 1% ea ccfDNA ho batho le li-primate e na le mohloli o tsoang kantle ho naha [21, 37]. Ka lebaka la tsamaiso ea potoloho ea mali e bulehileng hanyane ea li-bivalve, metsi a leoatle a nang le likokoana-hloko tse ngata, le kabo ea boholo ba ccfDNA ea mussel, re ile ra nahana hore hemolymph ccfDNA ea mussel e ka 'na ea ba le letamo le ruileng le le fapaneng la DNA ea likokoana-hloko. Ho leka khopolo-taba ena, re ile ra hlophisa hemolymph ccfDNA ho tsoa lisampoleng tsa Aulacomya atra tse bokelletsoeng Lihlekehlekeng tsa Kerguelen, ra fana ka lipalo tse fetang limilione tse 10, tse 97.6% tsa tsona li fetile taolo ea boleng. Lipalo li ile tsa aroloa ho latela mehloli ea boinotši le e seng ea boinotši ho sebelisoa li-database tsa BLASTN le NCBI bivalve (Setšoantšo sa S1, Tlhahisoleseling e Tlatsetsang).
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 genomic ea nyutlelie ea li-mussels, kaha genome ea A. atra ha e so ka e hlophisoa kapa ea hlalosoa. Leha ho le joalo, re khonne ho khetholla likaroloana tse 'maloa tsa ccfDNA tsa tlhaho ea rona re sebelisa laeborari ea bivalve (Setšoantšo sa S2, Tlhahisoleseling e Tlatsetsang). Re boetse re tiisitse boteng ba likaroloana tsa DNA tsa tlhaho ea rona ka ho atolosoa ha PCR ka kotloloho ha liphatsa tseo tsa lefutso tsa A. atra tse ileng tsa hlophisoa (Setšoantšo sa 3). Ka mokhoa o ts'oanang, kaha genome ea mitochondrial ea A. atra e fumaneha li-database tsa sechaba, motho a ka fumana bopaki ba boteng ba likaroloana tsa ccfDNA tsa mitochondrial ka har'a hemolymph ea A. atra. Boteng ba likaroloana tsa DNA tsa mitochondrial bo tiisitsoe ke amplification ea PCR (Setšoantšo sa 3).
Liphatsa tsa lefutso tse fapaneng tsa mitochondrial li ne li le teng hemolymph ea A. atra (matheba a khubelu - nomoro ea setoko: SRX5705969) le M. platensis (matheba a maputsoa - nomoro ea setoko: SRX5705968) e ntlafalitsoeng ke PCR. Setšoantšo se fetotsoe ho tsoa ho Breton et al., 2011 B Ho atolosoa ha supernatant ea hemolymph ho tsoa ho A. atra E bolokiloe pampiring ea FTA. Sebelisa punch ea 3 mm ho eketsa ka kotloloho ho tube ea PCR e nang le motsoako oa PCR.
Ka lebaka la bongata ba dikokwana-hloko tse ka metsing a lewatle, qalong re ile ra tsepamisa maikutlo ho tlhalosong ya ditatelano tsa DNA tsa dikokwana-hloko ka hara hemolymph. Ho etsa sena, re sebedisa mawa a mabedi a fapaneng. Lewa la pele le sebedisitse Kraken2, lenaneo la ho arola tatellano le thehilweng ho algorithm le ka hlwayang ditatelano tsa dikokwana-hloko ka ho nepahala ho tshwanang le BLAST le disebediswa tse ding [28]. Dipalo tse fetang 6719 di ile tsa fumaneha hore di tswa baktheriang, ha tse 124 le 64 di ne di tswa ho archaea le dikokwana-hloko, ka ho latellana (Setshwantsho sa 4). Dikarolo tse ngata tsa DNA tsa baktheria e ne e le Firmicutes (46%), Proteobacteria (27%), le Bacteroidetes (17%) (Setshwantsho sa 4a). Kabo ena e tsamaellana le dithuto tsa pejana tsa marine blue mussel microbiome [39, 40]. Gammaproteobacteria e ne e le sehlopha se seholo sa Proteobacteria (44%), ho kenyeletswa le Vibrionales tse ngata (Setshwantsho sa 4b). Mokhoa oa ddPCR o tiisitse boteng ba likaroloana tsa DNA ea Vibrio ho ccfDNA ea A. atra hemolymph (Setšoantšo sa 4c) [41]. Ho fumana tlhaiso-leseling e batsi mabapi le tšimoloho ea baktheria ea ccfDNA, mokhoa o mong o ile oa nkuoa (Setšoantšo sa S2, Tlhahisoleseling e Tlatsetsang). Tabeng ena, dipalo tse kopaneng di ile tsa kopanngwa e le dipalo tse kopaneng mme tsa hlophiswa e le tsa motho ka mong (di-bivalve) kapa tse sa tsweng ho motho ka mong ho sebediswa BLASTN le boleng ba e ba 1e−3 le kgaotso e nang le homologi ya >90%. Tabeng ena, dipalo tse kopaneng di ile tsa kopanngwa e le dipalo tse kopaneng mme tsa hlophiswa e le tsa motho ka mong (di-bivalve) kapa tse sa tsweng ho motho ka mong ho sebediswa BLASTN le boleng ba e ba 1e−3 le kgaotso e nang le homologi ya >90%. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственый как собственыстиные чужие по происхождению с использованием BLASTN le значения e 1e-3 le отсечения с гомологией> 90%. Tabeng ena, ho bala ho kopaneng ho ile ha bokelloa e le ho bala ho fellang ka mahlakore a mabedi mme ha hlophisoa e le tsa tlhaho (bivalve) kapa tse seng tsa mantlha ho sebediswa boleng ba BLASTN le e ba 1e-3 mme ha kgaolwa ka ho tshwana ho >90%.在這种情况下，重叠的話数组装为配对末端读数，并使用BLASTN 和1e-3 tsa hao le>90%同源性的截止值分类為自身（双壳类）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 的 值% le 90%自身 （双 壳类） 非 自身。。。。。 В этом случае перекрывающиеся чтения были собраны как чтения парными концами и классифицированы как собственные несобственные по происхождению с использованием значений e BLASTN le 1e-3 le ho etsa гомологии> 90%. Tabeng ena, ho bala ho kopaneng ho ile ha bokelloa e le ho bala ho fellang ka mahlakore a mabedi mme ha hlophisoa e le tsa hao (di-bivalve) kapa tse seng tsa mantlha ho sebediswa boleng ba e BLASTN le 1e-3 le moedi wa homology >90%.Kaha genome ea A. atra ha e so ka e hlophisoa ka tatellano, re sebelisitse leano la kopano ea de novo la sehokelo sa MEGAHIT Next Generation Sequencing (NGS). Kakaretso ea li-contig tse 147,188 li fumanoe e le tse itšetlehileng ka (li-bivalve) tsa tšimoloho. Li-contig tsena li ile tsa phatloha ka boleng ba e-1e-10 ho sebelisoa BLASTN le BLASTX. Leano lena le re lumelletse ho khetholla li-fragment tse 482 tse seng tsa bivalve tse teng ho A. atra ccfDNA. Ho feta halofo (57%) ea li-fragment tsena tsa DNA li fumanoe ho tsoa libaktheria, haholo-holo ho tsoa ho li-symbionts tsa gill, ho kenyeletsoa le li-symbionts tsa sulfotrophic, le ho tsoa ho li-symbionts tsa gill Solemya velum (Setšoantšo sa 5).
Bongata bo lekanyelitsoeng boemong ba mofuta. B Mefuta e fapaneng ea likokoana-hloko tsa phyla tse peli tse kholo (Firmicutes le Proteobacteria). Keketseho e emelang ea ddPCR C Vibrio spp. A. Likaroloana tsa gene ea 16S rRNA (e putsoa) ho li-hemolymph tse tharo tsa atra.
Kakaretso ea li-contig tse bokelletsoeng tse 482 li ile tsa hlahlojoa. Boemo bo akaretsang ba kabo ea taxonomic ea litlhaloso tsa metagenomic contig (prokaryotes le eukaryotes). B Kabo e qaqileng ea likaroloana tsa DNA ea baktheria tse khethiloeng ke BLASTN le BLASTX.
Tlhahlobo ea Kraken2 e boetse e bontšitse hore mussel ccfDNA e ne e na le likaroloana tsa DNA tsa khale, ho kenyeletsoa likaroloana tsa DNA tsa Euryarchaeota (65%), Crenarchaeota (24%), le Thaurmarcheota (11%) (Setšoantšo sa 6a). Boteng ba likaroloana tsa DNA tse nkiloeng ho Euryarchaeota le Crenarchaeota, tse neng li fumanoa pele sechabeng sa likokoana-hloko tsa mussels tsa California, ha lia lokela ho makatsa [42]. Le hoja Euryarchaeota hangata e amahanngoa le maemo a feteletseng, joale hoa hlokomeleha hore Euryarchaeota le Crenarcheota ke tse ling tsa li-prokaryote tse tloaelehileng haholo tikolohong ea cryogenic ea leoatle [43, 44]. Boteng ba likokoana-hloko tsa methanogenic ka har'a mussels ha bo makatse, ha ho nahanoa ka litlaleho tsa morao tjena tsa ho lutla ho hoholo ha methane ho tsoa ho lutla ho tlase ho Kerguelen Plateau [45] le tlhahiso ea methane ea likokoana-hloko e ka bang teng e bonoang lebopong la Lihlekehleke tsa Kerguelen [46].
Tlhokomelo ea rona e ile ea fetela ho lipalo tse tsoang ho livaerase tsa DNA. Ho ea ka tsebo ea rona e ntle ka ho fetisisa, ena ke thuto ea pele e sa reroang ea litaba tsa vaerase ea mussels. Joalokaha ho ne ho lebelletsoe, re fumane likaroloana tsa DNA tsa bacteriophages (Caudovirales) (Setšoantšo sa 6b). Leha ho le joalo, DNA ea vaerase e tloaelehileng haholo e tsoa ho phylum ea nucleocytoviruses, e tsejoang hape e le vaerase ea nuclear cytoplasmic DNA e kholo (NCLDV), e nang le genome e kholo ka ho fetisisa ho feta vaerase efe kapa efe. Ka har'a phylum ena, boholo ba tatellano ea DNA ke ea malapa a Mimimidoviridae (58%) le Poxviridae (21%), ao beng ba 'ona ba tlhaho ba kenyelletsang liphoofolo tse nang le lesapo la mokokotlo le li-arthropod, ha karolo e nyane ea tatellano ena ea DNA e le ea algae e tsebahalang ea virological. E tšoaetsa algae ea leoatleng ea eukaryotic. Tatellano ena e boetse e fumanoe ho tsoa ho vaerase ea Pandora, vaerase e kholo e nang le genome e kholo ka ho fetisisa ea mofuta ofe kapa ofe oa vaerase o tsejoang. Hoa thahasellisa hore mefuta ea batho ba tsejoang ba tšoaelitsoeng ke kokoana-hloko ena, joalo ka ha ho khetholloa ke tatellano ea hemolymph ccfDNA, e ne e le kholo haholo (Setšoantšo sa S3, Tlhahisoleseling e Tlatsetsang). 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. Re boetse re fumane tatellano e tsamaellanang le genome ea Pithovirus sibericum. Li-Pitovirus (tse tsejoang hape e le "li-zombie virus") li ile tsa aroloa ka lekhetlo la pele ho permafrost ea lilemo tse 30,000 Siberia [47]. Kahoo, liphetho tsa rona li lumellana le litlaleho tsa pejana tse bontšang hore ha se mefuta eohle ea sejoale-joale ea likokoana-hloko tsena e felileng [48] le hore likokoana-hloko tsena li ka ba teng libakeng tse hōle tsa leoatle tse ka tlas'a Arctic.
Qetellong, re ile ra leka ho bona hore na re ka fumana likaroloana tsa DNA tse tsoang liphoofolong tse ling tse nang le lisele tse ngata. BLASTN le BLASTX li ile tsa khetholla likaroloana tse 482 tsa kantle ho naha ka lilaebrari tsa nt, nr le RefSeq (genomic le protein). Liphetho tsa rona li bontša hore har'a likaroloana tsa kantle ho naha tsa ccfDNA tsa liphoofolo tse nang le lisele tse ngata DNA ea masapo a masapo e atile (Setšoantšo sa 5). Likaroloana tsa DNA tse tsoang likokoanyana le mefuta e meng le tsona li fumanoe. Karolo e kholo ea likaroloana tsa DNA ha e so fumanoe, mohlomong ka lebaka la palo e fokolang ea mefuta e mengata ea leoatleng li-database tsa genomic ha e bapisoa le mefuta ea lefatše [49].
Pampiring ena ea hona joale, re sebelisa mohopolo oa LB ho li-mussel, re pheha khang ea hore hemolymph ccfDNA shot sequencing e ka fana ka temohisiso mabapi le sebopeho sa tikoloho ea lebopong la leoatle. Haholo-holo, re fumane hore 1) hemolymph ea mussel e na le maemo a holimo haholo (maemo a microgram) a likaroloana tsa DNA tse potolohang tse kholo (~1-5 kb); 2) likaroloana tsena tsa DNA li ikemetse ebile ha li itšetlehe ka tsona 3) Har'a mehloli ea kantle ho naha ea likaroloana tsena tsa DNA, re fumane DNA ea baktheria, ea khale le ea vaerase, hammoho le DNA ea liphoofolo tse ling tse ngata tse nang le lisele tse ngata; 4) Ho bokellana ha likaroloana tsena tsa ccfDNA tse tsoang kantle ho naha ho etsahala ka potlako 'me ho kenya letsoho mosebetsing oa ho sefa oa li-mussel ka hare. Qetellong, thuto ea rona e bontša hore mohopolo oa LB, oo ho fihlela joale o sebelisitsoeng haholo-holo lefapheng la biomedicine, o kenyelletsa mohloli o ruileng oa tsebo empa o sa hlahlojoa o ka sebelisoang ho utloisisa hamolemo tšebelisano-'moho lipakeng tsa mefuta ea sentinel le tikoloho ea eona.
Ntle le di-primates, ho aroloa ha ccfDNA ho tlalehiloe ho liphoofolo tse anyesang, ho kenyeletsoa litoeba, lintja, likatse le lipere [50, 51, 52]. Leha ho le joalo, ho ea ka tsebo ea rona, phuputso ea rona ke ea pele ho tlaleha ho fumanoa le ho latellana ha ccfDNA mefuteng ea leoatle e nang le sistimi e bulehileng ea potoloho ea mali. Tšobotsi ena ea anatomiki le bokhoni ba ho sefa ba li-mussels, bonyane ka karolo e itseng, li ka hlalosa litšobotsi tse fapaneng tsa boholo ba likaroloana tsa DNA tse potolohang ha li bapisoa le mefuta e meng. Ho batho, boholo ba likaroloana tsa DNA tse potolohang maling ke likaroloana tse nyane tse tlohang ho 150 ho isa ho 200 bp. ka tlhōrō e phahameng ea 167 bp [34, 53]. Karolo e nyane 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 kabo ena ea boholo ke hore mohloli o moholo oa ccfDNA plasma o hlaha ka lebaka la lefu la sele, ebang ke ka lebaka la lefu la sele kapa ka lebaka la ho bola ha lisele tsa hematopoietic tse potolohang ho batho ba phetseng hantle kapa ka lebaka la apoptosis ea lisele tsa hlahala ho bakuli ba kankere (e tsejoang e le DNA ea hlahala e potolohang). , ctDNA). Kabo ea boholo ba hemolymph ccfDNA eo re e fumaneng ho li-mussel e ne e le ho tloha ho 1000 ho isa ho 5000 bp, e leng se bontšang hore mussel ccfDNA e na le tšimoloho e fapaneng. Ena ke khopolo-taba e utloahalang, kaha li-mussel li na le tsamaiso ea methapo ea mali e bulehileng hanyane 'me li lula libakeng tsa metsing a leoatle tse nang le maemo a phahameng a DNA ea genomic ea microbial. Ha e le hantle, liteko tsa rona tsa laboratori tse sebelisang DNA ea kantle li bontšitse hore li-mussel li bokella likaroloana tsa DNA metsing a leoatle, bonyane ka mor'a lihora tse 'maloa lia senyeha ka mor'a ho monngoa ha sele le/kapa ho lokolloa le/kapa ho bolokoa mekhatlong e fapaneng. Ka lebaka la ho se fumanehe ha lisele (prokaryotic le eukaryotic), tšebeliso ea likarolo tsa intravalvular e tla fokotsa bongata ba ccfDNA ho tsoa mehloling ea eona hammoho le mehloling ea kantle ho naha. Ha re nahana ka bohlokoa ba tšireletso ea tlhaho ea bivalve le palo e kholo ea li-phagocyte tse potolohang, re ile ra nahana hape hore esita le ccfDNA ea kantle ho naha e ruisitsoe ke li-phagocyte tse potolohang tse bokellang DNA ea kantle ho naha ha li monngoa ke likokoana-hloko le/kapa lithōle tsa sele. Ha li kopanngoa, liphetho tsa rona li bontša hore bivalve hemolymph ccfDNA ke polokelo e ikhethang ea tlhahisoleseling ea limolek'hule 'me e matlafatsa boemo ba tsona e le mofuta oa sentinel.
Lintlha tsa rona li bontša hore tatellano le tlhahlobo ea likaroloana tsa hemolymph ccfDNA tse nkiloeng ke baktheria li ka fana ka tlhahisoleseling ea bohlokoa mabapi le limela tsa baktheria tse amohelang le libaktheria tse teng tikolohong e potolohileng ea leoatle. Mekhoa ea tatellano ea lithunya e senotse tatellano ea baktheria ea commensal A. atra gill e ka beng e fosahetse haeba mekhoa ea ho khetholla rRNA ea 16S e ne e sebelisitsoe, ka lebaka la leeme la laeborari ea litšupiso. Ha e le hantle, tšebeliso ea rona ea data ea LB e bokelletsoeng ho tsoa ho M. platensis moalong o tšoanang oa mussel ho Kerguelen e bontšitse hore sebopeho sa li-symbiont tsa baktheria tse amanang le gill se ne se tšoana bakeng sa mefuta ka bobeli ea mussel (Setšoantšo sa S4, Tlhahisoleseling e Tlatsetsang). Ho tšoana hona ha li-mussel tse peli tse fapaneng ka liphatsa tsa lefutso ho ka bonts'a sebopeho sa lichaba tsa baktheria libakeng tse batang, tse nang le sebabole le tse nang le seretse se chesang tsa Kerguelen [55, 56, 57, 58]. Maemo a holimo a likokoana-hloko tse fokotsang sebabole a hlalositsoe hantle ha ho kotuloa li-mussel libakeng tsa lebopong tse nang le bioturbated [59], joalo ka lebopong la Port-au-France. Monyetla o mong ke hore limela tsa mussel tsa commensal li ka angoa ke phetiso e otlolohileng [60, 61]. Ho hlokahala lipatlisiso tse ling ho fumana kamano pakeng tsa tikoloho ea leoatle, bokaholimo ba leoatle, le sebopeho sa libaktheria tse sebelisanang le li-mussel. Lithuto tsena li ntse li tsoela pele hona joale.
Bolelele le mahloriso a hemolymph ccfDNA, bonolo ba yona ba ho hlwekisa, le boleng bo hodimo ho dumella tatellano e potlakileng ya shotgun ke tse ding tsa melemo e mengata ya ho sebedisa mussel ccfDNA ho lekola mefutafuta ya diphedi tikolohong ya lewatle. Mokgwa ona o sebetsa haholoholo bakeng sa ho hlalosa ditjhaba tsa dikokwana-hloko (viromes) tikolohong e itseng [62, 63]. Ho fapana le dibaktheria, archaea, le eukaryotes, di-genome tsa dikokwana-hloko ha di na dijini tse bolokilweng ka phylogenetically jwalo ka di-sequence tsa 16S. Diphetho tsa rona di bontsha hore di-biopsies tsa metsi tse tswang mefuteng ya ditshupiso tse kang di-mussel di ka sebediswa ho hlwaya palo e kgolo ya dikokwana-hloko tsa ccfDNA tse tsejwang ho tshwaetsa beng ba tsona ba atisang ho dula tikolohong ya lewatle ya lewatle. Sena se kenyeletsa dikokwana-hloko tse tsejwang ho tshwaetsa di-protozoa, di-arthropod, dikokonyana, dimela le dikokwana-hloko tsa baktheria (mohlala, bacteriophages). Kabo e tshwanang e fumanwe ha re hlahloba hemolymph ccfDNA virome ya di-mussel tse putswa (M. platensis) tse bokelletsweng lera le le leng la mussel ho Kerguelen (Tafole ya S2, Tlhahisoleseding e Tlatsetsang). Ho latellana ha lithunya tsa ccfDNA ka sethunya ke mokhoa o mocha o ntseng o eketseha thutong ea virome ea batho kapa mefuta e meng [21, 37, 64]. Mokhoa ona o molemo haholo bakeng sa ho ithuta likokoana-hloko tsa DNA tse nang le marapo a mabeli, kaha ha ho na jini e le 'ngoe e bolokiloeng har'a likokoana-hloko tsohle tsa DNA tse nang le marapo a mabeli, tse emelang sehlopha sa likokoana-hloko tse fapaneng ka ho fetisisa le tse pharaletseng Baltimore [65]. Leha boholo ba likokoana-hloko tsena li ntse li sa aroloa 'me li ka kenyelletsa likokoana-hloko tse tsoang karolong e sa tsejoeng ka ho feletseng ea lefats'e la vaerase [66], re fumane hore li-virome le mefuta ea moamoheli oa li-mussels A. atra le M. platensis li oela lipakeng tsa mefuta ena e 'meli. ka ho tšoanang (sheba setšoantšo S3, tlhaiso-leseling e eketsehileng). Ho tšoana hona ha ho makatse, kaha ho ka 'na ha bontša ho hloka khetho ho nkeng DNA e teng tikolohong. Lithuto tsa nakong e tlang tse sebelisang RNA e hloekisitsoeng lia hlokahala hona joale ho hlalosa RNA virome.
Thutong ea rona, re sebelisitse phaephe e thata haholo e nkiloeng mosebetsing oa Kowarski le basebetsi-'moho le eena [37], ba sebelisitseng ho hlakola ka mehato e 'meli li-reads tse kopantsoeng le li-contig pele le ka mor'a ho bokella ccfDNA ea tlhaho, e leng se ileng sa fella ka karolo e kholo ea li-reads tse sa kang tsa etsoa 'mapeng. Ka hona, re ke ke ra qhelela ka thoko hore tse ling tsa li-reads tsena tse sa kang tsa etsoa 'mapeng li ntse li e-na le tšimoloho ea tsona, haholo-holo hobane ha re na genome ea litšupiso bakeng sa mofuta ona oa mussel. Re boetse re sebelisitse phaephe ena hobane re ne re tšoenyehile ka li-chimera pakeng tsa li-reads tsa motho ka mong le tse seng tsa motho ka mong le bolelele ba ho bala bo hlahisoang ke Illumina MiSeq PE75. Lebaka le leng la boholo ba li-reads tse sa kang tsa etsoa ke hore boholo ba likokoana-hloko tsa leoatleng, haholo-holo libakeng tse hōle tse kang Kerguelen, ha lia ngoloa. Re sebelisitse Illumina MiSeq PE75, re nka hore bolelele ba likaroloana tsa ccfDNA bo tšoana le ccfDNA ea motho. Bakeng sa lithuto tsa nakong e tlang, ka lebaka la liphetho tsa rona tse bontšang hore hemolymph ccfDNA e na le nako e telele ea ho bala ho feta batho le/kapa liphoofolo tse anyesang, re khothaletsa ho sebelisa sethala sa tatellano se loketseng haholoanyane likaroloana tse telele tsa ccfDNA. Mokhoa ona o tla etsa hore ho be bonolo haholo ho khetholla matšoao a mangata bakeng sa tlhahlobo e tebileng. Ho fumana tatellano e felletseng ea genome ea nyutlelie ea A. atra e sa fumaneheng hona joale ho tla boela ho nolofatse haholo khethollo ea ccfDNA ho tsoa mehloling ea motho ka mong le e seng ea motho ka mong. Kaha lipatlisiso tsa rona li shebane le monyetla oa ho sebelisa mohopolo oa biopsy ea metsi ho li-mussels, re tšepa hore ha mohopolo ona o sebelisoa lipatlisisong tsa nakong e 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.
Jwalo ka biomarker ya kliniki e sa hlaseleng, maemo a hodimo a ccfDNA plasma ya motho a amana le mafu a fapaneng, tshenyo ya dinama, le maemo a kgatello [67,68,69]. Keketseho ena e amana le ho lokollwa ha dikotwana tsa DNA tsa tlhaho ya tsona kamora tshenyo ya dinama. Re ile ra sebetsana le bothata bona ka ho sebedisa kgatello e matla ya mocheso, moo dinama di ileng tsa pepesetswa mocheso wa 30 °C ka nako e kgutshwane. Re entse tlhahlobo ena mefuteng e meraro e fapaneng ya dinama tse nyane ditekong tse tharo tse ikemetseng. Leha ho le jwalo, ha re a ka ra fumana phetoho maemong a ccfDNA kamora kgatello e matla ya mocheso (sheba Setshwantsho S5, tlhahisoleseding e eketsehileng). Tshibollo ena e ka hlalosa, bonyane ka karolo e itseng, taba ya hore dinama tse nyane di na le tsamaiso ya potoloho e bulehileng hanyane mme di bokella bongata bo boholo ba DNA ya kantle ho naha ka lebaka la tshebetso ya tsona e phahameng ya ho sefa. Ka lehlakoreng le leng, dinama tse nyane, jwalo ka diphoofolo tse ngata tse se nang lesapo la mokokotlo, di ka nna tsa hanela tshenyo e bakwang ke kgatello ya dinama tse nyane, ka hona tsa fokotsa ho lokollwa ha ccfDNA hemolymph ya tsona [70, 71].
Ho fihlela jwale, tlhahlobo ya DNA ya mefutafuta ya diphedi tikolohong ya metsing e shebane haholo le metabarcoding ya DNA ya tikoloho (eDNA). Leha ho le jwalo, mokgwa ona hangata o lekanyeditswe tlhahlobong ya mefutafuta ya diphedi ha ho sebediswa di-primer. Tshebediso ya tatellano ya dithunya e thibela meedi ya PCR le kgetho e leeme ya di-primer sets. Kahoo, ka tsela e itseng, mokgwa wa rona o haufi le mokgwa o sa tswa sebediswa wa ho latellana ha dithunya tsa eDNA tse nang le bokgoni bo phahameng, o kgonang ho hlophisa DNA e arohaneng ka ho toba le ho sekaseka hoo e ka bang diphedi tsohle [72, 73]. Leha ho le jwalo, ho na le dintlha tse mmalwa tsa motheo tse kgethollang LB ho mekgwa e tloaelehileng ya eDNA. Ehlile, phapang e kgolo pakeng tsa eDNA le LB ke tshebediso ya di-filter host tsa tlhaho. Tshebediso ya mefuta ya lewatle e kang di-sponge le di-bivalve (Dresseina spp.) e le sefe sa tlhaho bakeng sa ho ithuta eDNA e tlalehilwe [74, 75]. Leha ho le jwalo, phuputso ya Dreissena e sebedisitse di-biopsies tsa dinama tseo DNA e ntshitsweng ho tsona. Tlhahlobo ea ccfDNA ho tsoa ho LB ha e hloke biopsy ea lisele, lisebelisoa tse ikhethileng le tse theko e boima tse amanang le eDNA kapa biopsy ea lisele. Ha e le hantle, re sa tsoa tlaleha hore ccfDNA ho tsoa ho LB e ka bolokoa le ho hlahlojoa ka tšehetso ea FTA ntle le ho boloka ketane e batang, e leng phephetso e kholo bakeng sa lipatlisiso libakeng tse hole [76]. Ho ntšoa ha ccfDNA ho tsoa ho li-biopsies tsa metsi le hona ho bonolo 'me ho fana ka DNA ea boleng bo holimo bakeng sa tatellano ea lithunya le tlhahlobo ea PCR. Ena ke monyetla o moholo ha ho nahanoa ka meeli e meng ea tekheniki e amanang le tlhahlobo ea eDNA [77]. Bonolo le litšenyehelo tse tlase tsa mokhoa oa ho nka sampole le tsona li loketse haholo mananeo a tlhokomelo ea nako e telele. Ntle le bokhoni ba tsona bo phahameng ba ho sefa, tšobotsi e 'ngoe e tsebahalang ea li-bivalve ke motsoako oa lik'hemik'hale oa mucopolysaccharide oa mucus ea tsona, e khothalletsang ho monngoa ha livaerase [78, 79]. Sena se etsa hore li-bivalve e be sefe se loketseng sa tlhaho bakeng sa ho khetholla mefuta-futa ea lintho tse phelang le tšusumetso ea phetoho ea tlelaemete tikolohong e fanoeng ea metsing. Leha boteng ba dikotwana tsa DNA tse tswang ho moamoheli bo ka bonwa e le moedi wa mokgwa ha o bapiswa le eDNA, ditjeo tse amanang le ho ba le ccfDNA ya tlhaho jwalo ha e bapiswa le eDNA di utlwahala ka nako e le nngwe bakeng sa tlhahisoleseding e ngata e fumanehang bakeng sa dithuto tsa bophelo bo botle. Sena se kenyeletsa boteng ba di-sequence tsa vaerase tse kentsweng ho genome ya moamoheli wa moamoheli. Sena se bohlokwa haholo bakeng sa di-mussels, ka lebaka la boteng ba di-retrovirus tse fetisetswang ka ho lebanya ho di-bivalve [80, 81]. Molemo o mong wa LB ho feta eDNA ke hore e sebedisa ts'ebetso ya phagocytic ya disele tsa madi tse potolohang ka hara hemolymph, e tshwasang dikokwana-hloko (le di-genome tsa tsona). Phagocytosis ke mosebetsi o ka sehloohong wa disele tsa madi ka hara di-bivalve [82]. Qetellong, mokgwa ona o sebedisa bokgoni bo phahameng ba ho sefa ba di-mussels (karolelano ya 1.5 l/h ya metsi a lewatle) le potoloho ya matsatsi a mabedi, e eketsang ho kopanngwa ha dikarolo tse fapaneng tsa metsi a lewatle, e leng se dumellang ho tshwarwa ha heterologous eDNA. [83, 84]. Ka hona, tlhahlobo ea mussel ccfDNA ke mokhoa o khahlisang ka lebaka la litlamorao tsa phepo e nepahetseng, moruo le tikoloho tsa mussel. Ho tšoana 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 moamoheli ho arabela linthong tse kantle. Mohlala, mahlale a tatellano ea moloko oa boraro a ka nahanoa ho etsa tlhahlobo ea methylation ea genome-wide ho ccfDNA ea tlhaho ho sebelisoa tatellano ea nanopore. Ts'ebetso ena e lokela ho nolofalloa ke taba ea hore bolelele ba likotoana tsa mussel ccfDNA bo lumellana hantle le li-platform tsa tatellano tse baloang nako e telele tse lumellang tlhahlobo ea methylation ea DNA ea genome-wide ho tloha ho tatellano e le 'ngoe ntle le tlhoko ea liphetoho tsa lik'hemik'hale.85,86] Ena ke monyetla o khahlisang, kaha ho bontšitsoe hore mekhoa ea methylation ea DNA e bonts'a karabelo khatellong ea tikoloho 'me e tsoela pele ho feta meloko e mengata. Ka hona, e ka fana ka temohisiso ea bohlokoa mabapi le mekhoa ea motheo e laolang karabelo kamora ho pepesehela phetoho ea tlelaemete kapa litšila [87]. Leha ho le joalo, ts'ebeliso ea LB ha e na meeli. Ha ho hlokahale hore ho boleloe, sena se hloka boteng ba mefuta ea lipontšo tikolohong. Jwalo ka ha ho boletswe ka hodimo, ho sebedisa LB ho lekola mefutafuta ya diphedi tsa tikoloho e itseng ho boetse ho hloka mothapo o matla wa bioinformatics o nahanang ka boteng ba dikotwana tsa DNA tse tswang mohloding. Bothata bo bong bo boholo ke ho fumaneha ha di-genome tsa referense bakeng sa mefuta ya lewatle. Ho tshepilwe hore merero e kang Morero wa Di-Genome tsa Mammal tsa Marine le morero o sa tswa thehwa wa Fish10k [88] e tla nolofatsa tlhahlobo e jwalo nakong e tlang. Tshebediso ya mohopolo wa LB ho diphedi tse fepang di-filter tsa lewatle e boetse e tsamaisana le kgatelopele ya moraorao ya theknoloji ya tatellano, e leng se etsang hore e tshwanelehe hantle bakeng sa ntshetsopele ya di-biomarker tse ngata tsa ohm ho fana ka tlhahisoleseding ya bohlokwa mabapi le bophelo bo botle ba dibaka tsa lewatle ho arabela kgatellong ya tikoloho.
Lintlha tsa tatellano ea liphatsa tsa lefutso li kentsoe ho NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 tlas'a Bioprojects SRR8924808.
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