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I-Liquid biopsy (LB) umqondo othola ukuduma ngokushesha emkhakheni we-biomedical.Lo mqondo usekelwe kakhulu ekutholweni kwezingcezu ze-DNA engaphandle kweseli (ccfDNA), ekhishwa ikakhulukazi njengezingcezu ezincane ngemva kokufa kwamangqamuzana ezicutshini ezihlukahlukene.Ingxenye encane yalezi zingcezu ivela ezicutshini zangaphandle (zangaphandle) noma izinto eziphilayo.Emsebenzini wamanje, sisebenzise lo mqondo ezimbazeni, uhlobo lwezimbaza ezaziwa ngamandla azo aphezulu okuhlunga amanzi olwandle.Sisebenzisa ikhono lezimbaza ukuze zisebenze njengezihlungi zemvelo ukuze sithwebule izingcezu ze-DNA zemvelo ezivela emithonjeni ehlukahlukene ukuze sinikeze ulwazi mayelana nokuhlukahluka kwemvelo yezinto eziphilayo zasogwini.Imiphumela yethu ibonisa ukuthi i-mussel hemolymph iqukethe izingcezu ze-DNA ezihluka kakhulu ngosayizi, ukusuka ku-1 ukuya ku-5 kb.Ukulandelana kwesibhamu kubonise ukuthi inqwaba yezingcezu ze-DNA imvelaphi yamagciwane angaphandle.Phakathi kwazo, sithole izingcezwana ze-DNA ezivela kubhaktheriya, i-archaea, namagciwane, okuhlanganisa namagciwane aziwa ngokuthelela ababungazi abahlukahlukene abavame ukutholakala ezindaweni eziphilayo zasogwini.Sengiphetha, ucwaningo lwethu lubonisa ukuthi umqondo we-LB osetshenziswa ezimbazeni umelela umthombo ocebile kodwa ongakahlolisiswa wolwazi mayelana nokuhlukahluka kwamagciwane ezindaweni eziphila ogwini zasogwini.
Umthelela wokuguquka kwesimo sezulu (CC) ezinhlobonhlobo zezinto eziphilayo zasolwandle uyindawo ekhula ngokushesha yocwaningo.Ukufudumala kwembulunga yonke akudali nje kuphela ukucindezeleka okubalulekile ngokomzimba, kodwa futhi kucindezela imingcele yokuziphendukela kwemvelo kokuzinza okushisayo kwezinto eziphilayo zasolwandle, okuthinta indawo yokuhlala yezinhlobo eziningi zezilwane, okuzishukumisela ukuba zifune izimo ezingcono kakhulu [1, 2].Ngaphezu kokuthinta izinhlobonhlobo zezinto eziphilayo ze-metazoan, i-CC iphazamisa ibhalansi ethambile yokusebenzisana kwe-host-microbial.Le dysbacteriosis ye-microbial ibeka usongo olukhulu kuzinto eziphilayo zasolwandle njengoba yenza izinto eziphilayo zasolwandle zibe sengozini yokuthola izifo ezithathelwanayo [3, 4].Kukholakala ukuthi i-SS idlala indima ebalulekile ekufeni kwabantu abaningi, okuyinkinga enkulu yokuphathwa kwemvelo yasolwandle yomhlaba wonke [5, 6].Lolu wudaba olubalulekile uma kubhekwa imithelela yezomnotho, yemvelo kanye nokudla okunomsoco kwezinhlobo eziningi zasolwandle.Lokhu kuyiqiniso ikakhulukazi kuma-bivalves ahlala ezindaweni ezipholile, lapho imiphumela ye-CK ishesha kakhulu futhi inzima [6, 7].Eqinisweni, ama-bivalves afana ne-Mytilus spp.zisetshenziswa kakhulu ukuqapha imiphumela ye-CC ku-ecosystem yasolwandle.Akumangalisi ukuthi inani elikhulu kakhulu lama-biomarker athuthukisiwe ukuze aqaphe impilo yawo, ngokuvamile kusetshenziswa indlela yezigaba ezimbili ehlanganisa ama-biomarker asebenzayo asekelwe kumsebenzi we-enzymatic noma imisebenzi yeselula efana nokusebenza kwamaseli kanye nomsebenzi we-phagocytic [8].Lezi zindlela zihlanganisa nokukalwa kokuhlushwa kwezinkomba ezithile zokucindezela eziqoqana ezicutshini ezithambile ngemva kokumuncwa kwamanzi amaningi olwandle.Kodwa-ke, umthamo omkhulu wokuhlunga kanye nohlelo lokujikeleza kwegazi oluvulekile kancane lwe-bivalves lunikeza ithuba lokuthuthukisa ama-biomarker amasha e-hemolymph kusetshenziswa umqondo we-liquid biopsy (LB), indlela elula nengena kancane ekuphathweni kwesiguli.amasampula egazi [9, 10].Nakuba izinhlobo ezimbalwa zama-molecule ajikelezayo zingatholakala ku-LB yomuntu, lo mqondo usekelwe ngokuyinhloko ekuhlaziyweni kokulandelana kwe-DNA kwezingcezu ezijikelezayo ze-extracellular DNA (ccfDNA) ku-plasma.Eqinisweni, ukuba khona kwe-DNA ejikelezayo ku-plasma yomuntu kwaziwa kusukela maphakathi nekhulu lama-20 [11], kodwa kuseminyakeni yamuva nje lapho ukuvela kwezindlela zokulandelana kwemiphumela ephezulu kuye kwaholela ekuxilongweni komtholampilo okusekelwe ku-ccfDNA.Ukuba khona kwalezi zingcezu ze-DNA ezijikelezayo kungenxa yengxenye yokukhululwa kwe-genomic DNA (i-nuclear ne-mitochondrial) ngemva kokufa kweseli. Kubantu abanempilo, ukuhlushwa kwe-ccfDNA ngokuvamile kuphansi (<10 ng/mL) kodwa kungandiswa izikhathi ezingu-5-10 ezigulini ezihlushwa izifo ezihlukahlukene noma ezingaphansi kwengcindezi, okuholela ekulimaleni kwezicubu. Kubantu abanempilo, ukuhlushwa kwe-ccfDNA ngokuvamile kuphansi (<10 ng/mL) kodwa kungandiswa izikhathi ezingu-5-10 ezigulini ezihlushwa izifo ezihlukahlukene noma ezingaphansi kwengcindezi, okuholela ekulimaleni kwezicubu. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться ku 5–10 раз у больных у больных писангей правдия стрессу, приводящему к повреждению тканей. Kubantu abanempilo, ukuhlushwa kwe-cccDNA ngokuvamile kuphansi (<10 ng/mL), kodwa kungakhula izikhathi ezingu-5-10 ezigulini ezinama-pathologies ahlukahlukene noma ngaphansi kokucindezeleka okuholela ekulimaleni kwezicubu.在健康个体中，ccfDNA的浓度通常较低（<10 ng/mL)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损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损Концентрации ccfDNA обычно низкие (<10 нг/мл) у здоровых людей, но могут быть увеличены ku 5-10 что приводит к повреждению тканей. Ukugxila kwe-ccfDNA ngokuvamile kuphansi (<10 ng/ml) kubantu abanempilo, kodwa kungandiswa izikhathi ezingu-5-10 ezigulini ezinama-pathologies ahlukahlukene noma ukucindezeleka, okuholela ekulimaleni kwezicubu.Ubukhulu bezingcezu ze-ccfDNA buyahlukahluka kakhulu, kodwa ngokuvamile busukela ku-150 kuye ku-200 bp.[12].Ukuhlaziywa kwe-ccfDNA eyenziwe ngokwayo, okungukuthi, i-ccfDNA evela kumaseli abamba avamile noma aguquliwe, ingasetshenziswa ukuthola izinguquko zofuzo kanye ne-epigenetic ezikhona ku-genome yenukliya kanye/noma ye-mitochondrial, ngaleyo ndlela kusiza odokotela ukuba bakhethe izindlela zokwelapha eziqondiswe kuma-molecular [13].Kodwa-ke, i-ccfDNA ingatholakala emithonjeni yangaphandle njenge-ccfDNA kumaseli engane ngesikhathi sokukhulelwa noma ezithweni ezitshaliwe [14,15,16,17].I-ccfDNA futhi ingumthombo obalulekile wolwazi wokuthola ubukhona be-nucleic acid ye-ejenti ethelelanayo (yangaphandle), evumela ukutholwa okungahlanyisi kwezifo ezisabalele ezingabonwa ngamasiko egazi, ukugwema i-biopsy ehlaselayo yezicubu ezithelelekile [18].Ucwaningo lwakamuva lubonise ngempela ukuthi igazi lomuntu liqukethe umthombo ocebile wolwazi ongasetshenziswa ukuhlonza amagciwane egciwane kanye namagciwane, nokuthi cishe i-1% ye-ccfDNA etholakala ku-plasma yomuntu imvelaphi yangaphandle [19].Lolu cwaningo lukhombisa ukuthi ukuhlukahluka kwemvelo kwe-microbiome ejikelezayo yento ephilayo kungahlolwa kusetshenziswa ukuhlaziywa kwe-ccfDNA.Kodwa-ke, kuze kube muva nje, lo mqondo wawusetshenziswa kuphela kubantu futhi, ngokwezinga elincane, kwezinye izilwane ezinomgogodla [20, 21].
Ephepheni lamanje, sisebenzisa amandla e-LB ukuhlaziya i-ccfDNA ye-Aulacomya atra, uhlobo oluseningizimu oluvame ukutholakala e-subantarctic Kerguelen Islands, iqoqo leziqhingi eziphezu kwethafa elikhulu elakheka eminyakeni eyizigidi ezingu-35 edlule.ukuqhuma kwentaba-mlilo.Sisebenzisa uhlelo lokuhlola lwe-in vitro, sithole ukuthi izingcezu ze-DNA emanzini olwandle zithathwa ngokushesha yizimbaza bese zingena egunjini le-hemolymph.Ukulandelana kwesibhamu sesibhamu kubonise ukuthi i-mussel hemolymph ccfDNA iqukethe izingcezu ze-DNA ezisuka kuyo nengezona uqobo, okuhlanganisa amagciwane e-symbiotic kanye nezingcezu ze-DNA ezivela ku-biomes ejwayelekile yemvelo ebandayo yasogwini lwentabamlilo.I-Hemolymph ccfDNA futhi iqukethe ukulandelana kwegciwane elitholakala kumagciwane anezigaba ezihlukene zokusingatha.Siphinde sathola izingcezu ze-DNA ezivela ezilwaneni ezinamangqamuzana amaningi njengezinhlanzi ezinamathambo, ama-anemones olwandle, ulwelwe nezinambuzane.Sengiphetha, ucwaningo lwethu lubonisa ukuthi umqondo we-LB ungasetshenziswa ngempumelelo kuma-invertebrates olwandle ukuze ukhiqize i-genomic repertoire ecebile ku-ecosystems yasolwandle.
Abantu abadala (55-70 mm ubude) I-Mytilus platensis (M. platensis) kanye ne-Aulacomya atra (A. atra) baqoqwa ogwini olunamadwala aphakathi kwamatshe e-Port-au-France (049°21.235 S, 070°13.490 E.).I-Kerguelen Islands ngo-December 2018. Ezinye izimbaza ezindala eziluhlaza okwesibhakabhaka (Mytilus spp.) zitholwe kumthengisi wezohwebo (PEI Mussel King Inc., Prince Edward Island, Canada) futhi zafakwa ethangini elilawulwayo lokushisa (4 ° C) eline-aerated eliqukethe u-10-20 L we-32‰ brine yokufakelwa.(usawoti wasolwandle wokwenziwa iReef Crystal, i-Instant Ocean, eVirginia, e-USA).Ngokuhlolwa ngakunye, ubude nesisindo samagobolondo ngamanye kukalwa.
Iphrothokholi yokufinyelela evulekile yamahhala yalolu hlelo iyatholakala ku-inthanethi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Kafushane, i-LB hemolymph yaqoqwa emisipha ye-abductor njengoba kuchaziwe [22].I-hemolymph yacaciswa nge-centrifugation ku-1200×g imizuzu engu-3, ​​i-supernatant yayiqhwa (-20°C) ize isetshenziswe.Ukuze ahlukaniselwe futhi ahlanzwe i-cfDNA, amasampula (1.5-2.0 ml) ancibilika futhi acutshungulwa kusetshenziswa i-NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ngokuya ngeziyalezo zomenzi.I-ccfDNA yagcinwa ku -80°C kuze kube yilapho kuhlaziywa okwengeziwe.Kokunye ukuhlola, i-ccfDNA yahlukaniswa futhi yahlanzwa kusetshenziswa i-QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada).I-Purified DNA yalinganiswa kusetshenziswa i-assay evamile ye-PicoGreen.Ukusatshalaliswa kweziqephu ze-ccfDNA ehlukanisiwe kwahlaziywa yi-capillary electrophoresis kusetshenziswa i-Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) kusetshenziswa i-High Sensitivity DNA Kit.Ukuhlolwa kwenziwe kusetshenziswa u-1 µl wesampula ye-ccfDNA ngokuya ngemiyalelo yomkhiqizi.
Ngokulandelana kwezingcezu ze-hemolymph ccfDNA, u-Génome Québec (Montreal, Quebec, Canada) ulungise imitapo yolwazi yesibhamu esebenzisa ikhithi ye-Illumina DNA Mix yekhithi ye-Illumina MiSeq PE75.Kusetshenziswe i-adaptha ejwayelekile (BioO).Amafayela edatha eluhlaza ayatholakala kokuthi NCBI Sequence Read Archive (SRR8924808 kanye ne-SRR8924809).Ikhwalithi yokufunda eyisisekelo yahlolwa kusetshenziswa i-FastQC [23].I-Trimmomatic [24] isetshenziselwe ukunamathisela ama-adaptha kanye nokufundwa kwekhwalithi embi.I-Shotgun ifundeka ngeziphetho ezibhanqiwe i-FLASH ihlanganiswe ekufundeni okude okukodwa nokugqagqana okuncane okungu-20 bp ukuze kugwenywe ukungafani [25]. Ukufundwa okuhlanganisiwe kwachasiselwa nge-BLASTN kusetshenziswa isizindalwazi se-bivalve NCBI Taxonomy (inani elingu-< 1e−3 kanye ne-90% ye-homology), kanye nokufihlwa kokulandelana okuyinkimbinkimbi kwenziwa kusetshenziswa i-DUST [26]. Ukufundwa okuhlanganisiwe kwachasiselwa nge-BLASTN kusetshenziswa isizindalwazi se-bivalve NCBI Taxonomy (inani elingu-< 1e−3 kanye ne-90% ye-homology), kanye nokufihlwa kokulandelana okuyinkimbinkimbi kwenziwa kusetshenziswa i-DUST [26]. I-BLASTN isekela i-BLASTN ngokuphathelene ne-данных таксономии двустворчатых моллюсков NCBI-09 маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ukufundwa okuhlanganisiwe kwachasiselwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI bivalve taxonomy (inani le-< 1e-3 kanye ne-90% homology), kanye nokufihla okuphansi okuyinkimbinkimbi kokulandelana kwenziwa kusetshenziswa i-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，并使平DUST的并恶 [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 使用公物 [26] .掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩Объединенные чтения были аннотированы с помощью BLASTN с использованием таксономической базы даных двустворчатых молюс10 NCBI а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ukufundwa okuhlanganisiwe kwachasiselwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI bivalve taxonomic (inani elingu-<1e-3 kanye ne-90% homology), kanye nokufihla okuphansi okuyinkimbinkimbi kokulandelana kwenziwa kusetshenziswa i-DUST [26].Ukufundwa kwehlukaniswe ngamaqembu amabili: okuhlobene nokulandelana kwe-bivalve (lapha okubizwa ngokuthi ukuzifundela) nokungahlobene (okungazifundanga ngokwakho).Amaqembu amabili aqoqwe ngokwehlukana kusetshenziswa i-MEGAHIT ukukhiqiza ama-contigs [27].Ngaleso sikhathi, ukusatshalaliswa kwe-taxonomic kokufundwa kwe-microbiome yangaphandle kwahlukaniswa kusetshenziswa i-Kraken2 [28] futhi imelelwa ngokucacile ishadi likaphayi we-Krona ku-Galaxy [29, 30].Ama-kmers alungile anqunywe ukuthi abe ama-kmers-59 kusukela ekuhloleni kwethu kokuqala. Ama-self contigs abe esehlonzwa ngokuhambisana ne-BLASTN (isizindalwazi se-bivalve NCBI, inani elingu-< 1e−10 kanye ne-60% homology) ukuze kube nesichasiselo sokugcina. Ama-self contigs abe esehlonzwa ngokuhambisana ne-BLASTN (isizindalwazi se-bivalve NCBI, inani elingu-< 1e−10 kanye ne-60% homology) ukuze kube nesichasiselo sokugcina. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, зная-010 e) нчательной аннотации. Ama-self-contigs abe esehlonzwa ngokuqhathanisa ne-BLASTN (i-NCBI bivalve database, inani elingu-<1e-10 kanye ne-60% homology) ukuze uthole isichasiselo sokugcina.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% 同源性）对齐來识别自身里行。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 NCBI , е e <1e-10 futhi гомология 60%). Ama-self-contigs abe esehlonzwa ukuze athole isichasiselo sokugcina ngokufanisa ne-BLASTN (i-NCBI bivalve database, inani elingu-<1e-10 kanye ne-60% homology). Ngokuhambisanayo, ama-contig weqembu okungelona uqobo achazwe nge-BLASTN (isizindalwazi se-nt NCBI, inani elingu-< 1e−10 kanye ne-60% ye-homology). Ngokuhambisanayo, ama-contig weqembu okungelona uqobo achazwe nge-BLASTN (isizindalwazi se-nt NCBI, inani elingu-< 1e−10 kanye ne-60% ye-homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 and гомология 60%). Ngokuhambisanayo, ama-contig weqembu langaphandle achazwe nge-BLASTN (isizindalwazi se-NT NCBI, inani elingu-<1e-10 kanye no-60% we-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, знагогние e и 1e-1%). Ngokuhambisanayo, ama-contig weqembu okungezona ngokwakho achazwe nge-BLASTN (isizindalwazi se-nt NCBI, inani elingu-<1e-10 kanye ne-60% ye-homology). I-BLASTX iphinde yenziwa kuma-nonself contigs kusetshenziswa izizindalwazi ze-nr kanye ne-RefSeq protein NCBI (inani elingu-< 1e−10 kanye ne-60% ye-homology). I-BLASTX iphinde yenziwa kuma-nonself contigs kusetshenziswa izizindalwazi ze-nr kanye ne-RefSeq protein NCBI (inani elingu-< 1e−10 kanye ne-60% ye-homology). I-BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 6%). I-BLASTX iphinde yenziwa kuma-non-self contigs kusetshenziswa i-nr kanye ne-RefSeq NCBI yolwazi lwamaphrotheni (i-e value <1e-10 kanye ne-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）。 I-BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 60%). I-BLASTX iphinde yenziwa kuma-contig okungezona wena kusetshenziswa i-nr kanye ne-RefSeq NCBI yolwazi lwamaprotheni (inani elingu-<1e-10 kanye ne-60% ye-homology).I-BLASTN kanye ne-BLASTX yamachibi okungezona i-self-contigs amele ama-contigs okugcina (bona ifayela lokungezelela).
Iziqalo ezisetshenziselwa i-PCR zikleliswe kuThebula S1.I-Taq DNA polymerase (Bio Basic Canada, Markham, ON) yasetshenziswa ukukhulisa izakhi zofuzo eziqondiwe ze-ccfDNA.Kusetshenziswe izimo zokusabela ezilandelayo: i-denaturation ku-95°C imizuzu engu-3, ​​95°C iminithi elingu-1, setha izinga lokushisa le-annealing iminithi elingu-1, ubude libe ngu-72°C iminithi elingu-1, imijikelezo engu-35, futhi ekugcineni libe ngu-72°C phakathi nemizuzu eyi-10..Imikhiqizo ye-PCR yahlukaniswa nge-electrophoresis kumajeli e-agarose (1.5%) aqukethe i-SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Canada) ku-95 V.
Izimbaza (Mytilus spp.) zajwayelaniswa no-500 ml wamanzi olwandle afakwe umoya-mpilo (32 PSU) amahora angama-24 ku-4°C.I-Plasmid DNA equkethe into efaka ikhodi yokulandelana kwe-galectin-7 cDNA yomuntu (inombolo yokungena ye-NCBI L07769) yengezwe ku-vial ekuhlanganiseni kokugcina okungu-190 μg/μl.Izimbaza ezifukanyelwe ngaphansi kwezimo ezifanayo ngaphandle kokwengezwa kwe-DNA zaziwukulawula.Ithangi lesithathu lokulawula laliqukethe i-DNA engenazo izimbaza.Ukuqapha ikhwalithi ye-DNA emanzini olwandle, amasampula amanzi olwandle (20 μl; ukuphindaphinda okuthathu) athathwe ethangini ngalinye ngesikhathi esibonisiwe.Ukuze uthole ukulandeleka kwe-plasmid DNA, izimbaza ze-LB zavunwa ngezikhathi ezibonisiwe futhi zahlaziywa yi-qPCR ne-ddPCR.Ngenxa yokuqukethwe okuphezulu kukasawoti emanzini olwandle, ama-aliquots ahlanjululwe emanzini ekhwalithi ye-PCR (1:10) ngaphambi kwazo zonke izivivinyo ze-PCR.
I-Digital droplet PCR (ddPCR) yenziwe kusetshenziswa iphrothokholi ye-BioRad QX200 (Mississauga, Ontario, Canada).Sebenzisa iphrofayela yezinga lokushisa ukuze unqume izinga lokushisa eliphezulu (Ithebula S1).Amaconsi akhiqizwa kusetshenziswa i-QX200 drop generator (BioRad).I-ddPCR yenziwa kanje: 95°C imizuzu emi-5, imijikelezo engu-50 engu-95°C amasekhondi angu-30 kanye nezinga lokushisa elinikeziwe le-annealing iminithi elingu-1 no-72°C ama-30 s, 4°C imizuzu engu-5 no-90°C phakathi kwemizuzu emi-5.Inombolo yokwehla nokusabela okuhle (inombolo yamakhophi/µl) kukalwe kusetshenziswa i-QX200 drop reader (BioRad).Amasampula anamaconsi angaphansi kuka-10,000 anqatshiwe.Ukulawulwa kwephethini akwenziwanga ngaso sonke isikhathi lapho i-ddPCR isetshenziswa.
I-qPCR yenziwe kusetshenziswa i-Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) kanye neziqalo ezithile ze-LGALS7.Wonke ama-PCR omthamo enziwe ngo-20 µl kusetshenziswa i-QuantiFast SYBR Green PCR Kit (QIAGEN).I-qPCR yaqalwa nge-incubation yemizuzu eyi-15 ku-95°C kulandele imijikelezo engama-40 ku-95°C imizuzwana eyi-10 kanye naku-60°C imizuzwana engama-60 ngokuqoqwa kwedatha eyodwa.Amajika okuncibilika akhiqizwa kusetshenziswa izilinganiso ezilandelanayo ku-95°C kuma-5 s, u-65°C ama-60 s, kanye no-97°C ekupheleni kwe-qPCR.I-qPCR ngayinye yenziwe ngokuphindwe kathathu, ngaphandle kwamasampuli okulawula.
Njengoba izimbaza zaziwa ngezinga lazo eliphezulu lokuhluza, siqale saphenya ukuthi zingakwazi yini ukuhlunga futhi zigcine izingcezu ze-DNA ezikhona emanzini olwandle.Siphinde saba nentshisekelo yokuthi lezi zingcezu ziyanqwabelana yini ohlelweni lwazo lwe-semi-open lymphatic.Sixazulule le nkinga ngokuhlola ngokulandela isiphetho sezingcezu ze-DNA ezincibilikayo ezengezwe emathangeni embaza eluhlaza okwesibhakabhaka.Ukuze kube lula ukulandelela izingcezu ze-DNA, sisebenzise i-plasmid DNA yangaphandle (hhayi yona uqobo) equkethe isakhi sofuzo se-galectin-7.I-ddPCR ilandelela izingcezu ze-plasmid DNA emanzini olwandle nasezimbazeni.Imiphumela yethu ibonisa ukuthi uma inani lezingcezu ze-DNA emanzini olwandle lihlala lihlala lingashintshi ngokuhamba kwesikhathi (kuze kube yizinsuku ezingu-7) lapho kungekho izimbaza, khona-ke phambi kwezimbaza leli zinga cishe lanyamalala ngokuphelele phakathi namahora angu-8 (Fig. 1a,b).Izingcezu ze-DNA exogenous zatholwa kalula phakathi kwemizuzu engu-15 ku-intravalvular fluid kanye ne-hemolymph (Fig. 1c).Lezi zingcezu zisengatholwa kufika emahoreni angu-4 ngemva kokuchayeka.Lo msebenzi wokuhlunga maqondana nezingcezu ze-DNA ufaniswa nomsebenzi wokuhlunga wamabhaktheriya nolwelwe [31].Le miphumela iphakamisa ukuthi izimbaza zingakwazi ukuhlunga futhi ziqongelele i-DNA yangaphandle ezindaweni zazo zoketshezi.
Ukugxila okuhlobene kwe-plasmid DNA emanzini olwandle lapho kukhona (A) noma ukungabikho (B) kwezimbaza, kukalwa nge-ddPCR.Ku-A, imiphumela ichazwa njengamaphesenti, nemingcele yamabhokisi amele amaphesenti angama-75 kanye nama-25.Ijika le-logarithmic elifakiwe liboniswa ngokubomvu, futhi indawo enomthunzi ngokumpunga imelela isikhawu sokuzithemba esingu-95%.Ku-B, umugqa obomvu umele incazelo kanti ulayini oluhlaza okwesibhakabhaka umele isikhawu sokuzethemba esingu-95% sokugxilisa ingqondo.C Ukuqoqwa kwe-plasmid DNA ku-hemolymph kanye noketshezi lwe-valvular lwezimbaza ngezikhathi ezihlukene ngemva kokwengezwa kwe-plasmid DNA.Imiphumela yethulwa njengamakhophi aphelele atholiwe/mL (±SE).
Okulandelayo, siphenye umsuka we-ccfDNA ezimbazeni eziqoqwe emibhedeni yezimbaza e-Kerguelen Islands, iqembu elikude leziqhingi ezinethonya elilinganiselwe le-anthropogenic.Ngale njongo, i-cccDNA evela kuma-mussel hemolymphs yahlukaniswa futhi yahlanzwa ngezindlela ezivame ukusetshenziselwa ukuhlanza i-cccDNA yomuntu [32, 33].Sithole ukuthi ukugxila kwe-hemolymph ccfDNA ezimbaza kungama-micrograms aphansi ngobubanzi be-hemolymph (bheka Ithebula S2, Ulwazi Olwengeziwe).Lolu hlu lokugxila lukhulu kakhulu kunabantu abanempilo (ama-nanogram aphansi ngemililitha), kodwa ezimweni ezingavamile, ezigulini ezinomdlavuza, izinga le-ccfDNA lingafinyelela ama-micrograms amaningana ngemililitha ngayinye [34, 35].Ukuhlaziywa kosayizi wokusatshalaliswa kwe-hemolymph ccfDNA kubonise ukuthi lezi zingcezu zihluka kakhulu ngosayizi, kusukela ku-1000 bp kuya ku-1000 bp.kufika ku-5000 bp (Fig. 2).Imiphumela efanayo yatholwa kusetshenziswa i-silica-based QIAamp Investigator Kit, indlela evame ukusetshenziswa kwisayensi yezobunhloli ukuhlukanisa ngokushesha nokuhlanza i-genomic DNA kumasampula e-DNA okuhlushwa okuphansi, okuhlanganisa i-ccfDNA [36].
Omele i-ccfDNA electrophoregram ye-mussel hemolymph.Kukhishwe nge-NucleoSnap Plasma Kit (phezulu) kanye ne-QIAamp DNA Investigator Kit.Isakhiwo seviyolin esibonisa ukusatshalaliswa kwe-hemolymph ccfDNA concentrations (±SE) ezimbazeni.Imigqa emnyama nebomvu imele i-median kanye ne-quartiles yokuqala neyesithathu, ngokulandelanayo.
Cishe i-1% ye-ccfDNA kubantu nasezinyameni inomthombo wangaphandle [21, 37].Uma kubhekwa uhlelo lokujikeleza kwegazi oluvulekile lwe-bivalves, amanzi olwandle anothe nge-microbial, kanye nosayizi wokusatshalaliswa kwezimbaza ze-ccfDNA, sacabanga ukuthi i-mussel hemolymph ccfDNA ingase iqukathe iphuli ecebile nehlukahlukene ye-DNA encane.Ukuhlola le nkoleloze, silandelenisa i-hemolymph ccfDNA kumasampula e-Aulacomya atra aqoqwe e-Kerguelen Islands, enikeza ukufundwa okungaphezu kwezigidi ezingu-10, u-97.6% wakho ophumelele ukulawula ikhwalithi.Ukufundwa kwabe sekuhlukaniswa ngokuya ngemithombo yakho siqu kanye nengezona uqobo kusetshenziswa i-BLASTN kanye ne-NCBI bivalve yolwazi (Fig. S1, Ulwazi Olwengeziwe).
Kubantu, i-DNA yenuzi ne-mitochondrial ingadedelwa egazini [38].Kodwa-ke, ocwaningweni lwamanje, akukwazanga ukuchaza ngokuningiliziwe i-nuclear genomic DNA yezimbaza, uma kubhekwa ukuthi i-A. atra genome ayizange ilandelwe noma ichazwe.Nokho, sikwazile ukuhlonza inombolo yezingcezu ze-ccfDNA zomsuka wethu sisebenzisa umtapo wezincwadi we-bivalve (Fig. S2, Ulwazi Olwengeziwe).Siphinde saqinisekisa ukuba khona kwezingcezu ze-DNA zomsuka wethu ngokukhulisa i-PCR eqondisiwe yalezo zakhi zofuzo ze-A. atra ezazilandelana (Umfanekiso 3).Ngokufanayo, uma kubhekwa ukuthi igenome ye-mitochondrial ye-A. atra iyatholakala kuzigcinilwazi zomphakathi, umuntu angathola ubufakazi bokuba khona kwezingcezu ze-mitochondrial ccfDNA ku-hemolymph ye-A. atra.Ukuba khona kwezingcezu ze-DNA ye-mitochondrial kwaqinisekiswa yi-PCR amplification (Fig. 3).
Izakhi zofuzo ezihlukahlukene ze-mitochondrial zazikhona ku-hemolymph ye-A. atra (amachashazi abomvu - inombolo yesitoko: SRX5705969) kanye ne-M. platensis (amachashazi aluhlaza - inombolo yesitoko: SRX5705968) ekhuliswe yi-PCR.Umfanekiso uthathwe ku-Breton et al., 2011 B Ukukhuliswa kwe-hemolymph supernatant kusuka ku-A. atra Kugcinwe ephepheni le-FTA.Sebenzisa i-punch engu-3 mm ukwengeza ngqo kushubhu le-PCR eliqukethe imiksi ye-PCR.
Njengoba kunikezwe okuqukethwe okuningi kwama-microbial emanzini olwandle, ekuqaleni sagxila ekuhlukaniseni ama-microbial DNA ukulandelana ku-hemolymph.Ukwenza lokhu, sisebenzisa amasu amabili ahlukene.Isu lokuqala lasebenzisa i-Kraken2, uhlelo lokuhlukanisa ngokulandelana okusekelwe ku-algorithm olungakhomba ukulandelana kwamagciwane ngokunemba okuqhathaniswa ne-BLAST namanye amathuluzi [28].Ukufundwa okungaphezu kuka-6719 kunqunywe ukuthi kunemvelaphi yebhaktheriya, kuyilapho i-124 ne-64 yayivela ku-archaea kanye namagciwane, ngokulandelana (Fig. 4).Izingcezu ze-DNA zebhaktheriya eziningi kakhulu kwakuyi-Firmicutes (46%), i-Proteobacteria (27%), ne-Bacteroidetes (17%) (Fig. 4a).Lokhu kusatshalaliswa kuhambisana nezifundo zangaphambilini ze-marine blue mussel microbiome [39, 40].I-Gammaproteobacteria yayiyikilasi eliyinhloko le-Proteobacteria (44%), kuhlanganise nama-Vibrionales amaningi (Fig. 4b).Indlela ye-ddPCR iqinisekise ukuba khona kwezingcezu ze-Vibrio DNA ku-ccfDNA ye-A. atra hemolymph (Fig. 4c) [41].Ukuze uthole ulwazi olwengeziwe mayelana nemvelaphi yebhaktheriya ye-ccfDNA, kuthathwe indlela eyengeziwe (Fig. S2, Ulwazi Olwengeziwe). Kulesi simo, ukufundwa okugqagqene kwahlanganiswa njengokufundiwe ekugcineni futhi kwahlukaniswa njengokungezakho (i-bivalves) noma imvelaphi engeyona yona kusetshenziswa i-BLASTN kanye nenani elingu-e elingu-1e−3 kanye nokunqamuka nge->90% ye-homology. Kulesi simo, ukufundwa okugqagqene kwahlanganiswa njengokufundiwe ekugcineni futhi kwahlukaniswa njengokungezakho (i-bivalves) noma imvelaphi engeyona yona kusetshenziswa i-BLASTN kanye nenani elingu-e elingu-1e−3 kanye nokunqamuka nge->90% ye-homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственные обственные (высокое) происхождению с использованием BLASTN futhi значения e 1e-3 futhi отсечения с гомологией> 90%. Kulesi simo, ukufunda okugqagqene kwaqoqwa njengokufundiwe okumataniswe futhi kwahlukaniswa njengokomdabu (i-bivalve) noma okungekona okoqobo kusetshenziswa i-BLASTN kanye nenani elingu-e elingu-1e-3 kanye nokunqanyulwa kokuthi >90% i-homology.在這种情况下，重叠的读数组装为配对末端读数，并使用BLASTN 和1e-3 idolobha kanye>90%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 使的情况 和 1e-3 的 值% > 90 （双 壳类） 非 自身。。。。。. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицированы как собственные (двуствонство) происхождению с использованием значений e BLASTN kanye ne-1e-3 kanye ne-прога гомологии> 90%. Kulokhu, ukufundwa okugqagqene kwaqoqwa njengokufunda okuhamba ngamabili futhi kwahlukaniswa njengokwakho (ama-bivalves) noma okungekona okoqobo kusetshenziswa i-e BLASTN kanye namanani we-1e-3 kanye nomkhawulo we-homology>90%.Njengoba i-A. atra genome ingakalandeli, sisebenzise isu lokuhlanganisa le-de novo lomhlanganisi we-MEGAHIT Next Generation Sequencing (NGS).Isamba sama-contig ayi-147,188 akhonjwe njengancike (bivalves) yemvelaphi.Lawa ma-contigs abe eseqhunyiswa ngamanani we-e we-1e-10 kusetshenziswa i-BLASTN ne-BLASTX.Lelisu lisivumele ukuthi sihlonze izingcezu ezingama-482 ezingezona i-bivalve ezikhona ku-A. atra ccfDNA.Ngaphezu kwengxenye (57%) yalezi zingcezu ze-DNA zitholwe kumagciwane, ikakhulukazi kuma-gill symbionts, kuhlanganise nama-sulfotrophic symbionts, kanye nama-gill symbionts i-Solemya velum (Fig. 5).
Ubuningi obuhlobene ezingeni lohlobo.B Ukuhlukahluka kwe-Microbial ye-phyla emibili eyinhloko (i-Firmicutes ne-Proteobacteria).Ukukhulisa omele i-ddPCR C Vibrio spp.A. Izingcezu zofuzo lwe-16S rRNA (oluluhlaza okwesibhakabhaka) kuma-atra hemolymphs amathathu.
Ingqikithi yama-contigs eqoqiwe angama-482 yahlaziywa.Iphrofayili evamile yokusatshalaliswa kwe-taxonomic yezichasiselo ze-metagenomic contig (ama-prokaryotes nama-eukaryote).B Ukusatshalaliswa okuningiliziwe kwezingcezu zebhaktheriya ze-DNA ezikhonjwe yi-BLASTN ne-BLASTX.
Ukuhlaziywa kwe-Kraken2 kuphinde kwabonisa ukuthi i-ccfDNA yezimbaza iqukethe izingcezu ze-archaeal DNA, okuhlanganisa izingcezu ze-DNA ze-Euryarchaeota (65%), i-Crenarchaeota (24%), ne-Thaurmarcheota (11%) (Fig. 6a).Ukuba khona kwezingcezu ze-DNA ezithathwe ku-Euryarchaeota kanye ne-Crenarchaeota, ngaphambilini ezatholakala emphakathini wezilwane ezincane zezimbaza zaseCalifornian, akufanele kumangaze [42].Nakuba i-Euryarchaeota ivame ukuhlotshaniswa nezimo ezimbi kakhulu, manje sekuyaziwa ukuthi kokubili i-Euryarchaeota ne-Crenarcheota ziphakathi kwama-prokaryote avame kakhulu endaweni ye-marine cryogenic [43, 44].Ukuba khona kwama-methane microorganisms ezimbaza akumangazi, uma kubhekwa imibiko yakamuva yokuvuza okukhulu kwe-methane ekuvuzeni okuphansi kwe-Kerguelen Plateau [45] kanye nokukhiqizwa okungenzeka kwe-methane emincane emincane ebonwe ogwini lweziQhingi zaseKerguelen [46].
Ukunaka kwethu kwabe sekushintshela ekufundeni amagciwane e-DNA.Ngokwazi kwethu, lolu wucwaningo lokuqala olungahlosiwe lokuqukethwe kwegciwane ezimbaza.Njengoba bekulindelekile, sithole izingcezu ze-DNA ze-bacteriophages (Caudovirales) (Fig. 6b).Kodwa-ke, i-DNA yegciwane ejwayeleke kakhulu ivela ku-phylum ye-nucleocytoviruses, eyaziwa nangokuthi i-nuclear cytoplasmic big DNA virus (NCLDV), ene-genome enkulu kunanoma yiliphi igciwane.Ngaphakathi kwale phylum, ukulandelana kwe-DNA okuningi kungokwemindeni i-Mimimidoviridae (58%) kanye ne-Poxviridae (21%), okusingathwa kwayo kwemvelo okuhlanganisa ama-vertebrates nama-arthropods, kuyilapho ingxenye encane yalokhu kulandelana kwe-DNA ingeye-algae eyaziwayo.Ithelela ulwelwe lwasolwandle lwe-eukaryotic.Ukulandelana kwaphinde kwatholakala egciwaneni le-Pandora, igciwane elikhulu elinosayizi wofuzo omkhulu kunawo wonke wanoma iyiphi i-viral genera eyaziwayo.Kuyathakazelisa ukuthi uhla lwababungazi abaziwa ukuthi banegciwane, njengoba kunqunywa ukulandelana kwe-hemolymph ccfDNA, belulukhulu uma kuqhathaniswa (Umfanekiso S3, Ulwazi Olwengeziwe).Kuhlanganisa amagciwane ahlasela izinambuzane ezifana ne-Baculoviridae ne-Iridoviridae, kanye namagciwane ahlasela i-amoeba, ulwelwe kanye nezilwane ezinomgogodla.Siphinde sathola ukulandelana okuhambisana ne-Pithovirus sibericum genome.Ama-pitoviruses (aziwa nangokuthi "ama-zombie viruses") aqala ukuhlukaniswa ne-permafrost eneminyaka engu-30,000 ubudala eSiberia [47].Ngakho-ke, imiphumela yethu ihambisana nemibiko yangaphambilini ebonisa ukuthi akuzona zonke izinhlobo zesimanje zalawa magciwane ezingasekho [48] nokuthi la magciwane angase abe khona ezindaweni ezikude zasolwandle ezingaphansi kwe-subarctic.
Ekugcineni, sahlola ukuze sibone ukuthi singazithola yini izingcezu ze-DNA kwezinye izilwane ezinamangqamuzana amaningi.Isamba sama-contigs angaphandle angama-482 akhonjwe yi-BLASTN kanye ne-BLASTX enemitapo yolwazi ye-nt, nr kanye ne-RefSeq (i-genomic namaprotheni).Imiphumela yethu ibonisa ukuthi phakathi kwezingcezu zakwamanye amazwe ze-ccfDNA zezilwane ezinama-multicellular DNA yamathambo amathambo idla kakhulu (Fig. 5).Kutholakale izingcezu ze-DNA zezinambuzane nezinye izinhlobo zezilwane.Ingxenye enkulu kakhulu yezingcezu ze-DNA ayikahlonzwa, mhlawumbe ngenxa yokungamelwa kahle kwenani elikhulu lezinhlobo zasolwandle kulwazi lwe-genomic uma kuqhathaniswa nezinhlobo zasemhlabeni [49].
Ephepheni lamanje, sisebenzisa umqondo we-LB ezimbazeni, siphikisa ngokuthi ukulandelana kwesibhamu se-hemolymph ccfDNA kunganikeza ukuqonda ekubunjweni kwezinto eziphilayo zasogwini.Ikakhulukazi, sithole ukuthi i-1) i-hemolymph yembaza iqukethe ukugxila okuphezulu kakhulu (amazinga e-microgram) enkulu kakhulu (~1-5 kb) izingcezu ze-DNA ezijikelezayo;2) lezi zingcezu ze-DNA zizimele futhi azizimele 3) Phakathi kwemithombo yangaphandle yalezi zingcezu ze-DNA, sithole i-bacterial, archaeal kanye ne-viral DNA, kanye ne-DNA yezinye izilwane eziningi;4) Ukuqoqwa kwalezi zingcezu ze-ccfDNA zangaphandle ku-hemolymph kwenzeka ngokushesha futhi kunomthelela emsebenzini wokuhlunga wangaphakathi wezimbaza.Ekuphetheni, ucwaningo lwethu lubonisa ukuthi umqondo we-LB, osetshenziswe kuze kube manje ikakhulukazi emkhakheni we-biomedicine, uhlanganisa umthombo wolwazi ocebile kodwa ongahloliwe ongasetshenziselwa ukuqonda kangcono ukusebenzisana phakathi kwezinhlobo ze-sentinel kanye nendawo yazo.
Ngokungeziwe kuma-primates, ukuhlukaniswa kwe-ccfDNA kuye kwabikwa ezilwaneni ezincelisayo, okuhlanganisa amagundane, izinja, amakati, namahhashi [50, 51, 52].Kodwa-ke, ngokwazi kwethu, ucwaningo lwethu lungowokuqala ukubika ukutholwa nokulandelana kwe-ccfDNA ezinhlotsheni zasolwandle ngohlelo lokujikeleza oluvulekile.Lesi sici se-anatomical nekhono lokuhlunga lezimbaza, okungenani ngokwengxenye, lingase lichaze izici ezihlukene zosayizi wezingcezu ze-DNA ezijikelezayo uma kuqhathaniswa nezinye izinhlobo.Kubantu, izingcezu ze-DNA eziningi ezizungeza egazini ziyizingxenyana ezincane ezinobukhulu obusuka ku-150 kuya ku-200 bp.nenani eliphakeme kakhulu lika-167 bp [34, 53].Ingxenye encane kodwa ebalulekile yezingcezu ze-DNA iphakathi kuka-300 no-500 bp ngosayizi, futhi cishe u-5% yinde kuno-900 bp.[54].Isizathu salokhu kusatshalaliswa kosayizi ukuthi umthombo oyinhloko we-ccfDNA ku-plasma uvela ngenxa yokufa kwamangqamuzana noma ngenxa ye-necrosis yamangqamuzana e-hematopoietic ajikelezayo kubantu abanempilo noma ngenxa ye-apoptosis yamaseli wesimila ezigulini ezinomdlavuza (okwaziwa ngokuthi i-circulating tumor DNA)., ctDNA).Ukusatshalaliswa kosayizi we-hemolymph ccfDNA esikuthole ezimbazeni kusuka ku-1000 kuya ku-5000 bp, okuphakamisa ukuthi i-ccfDNA yezimbaza inomsuka ohlukile.Lena inkolelo-mbono enengqondo, njengoba izimbaza zinesimiso semithambo evulekile kancane futhi zihlala ezindaweni zasemanzini zasolwandle eziqukethe ukugxila okuphezulu kwe-microbial genomic DNA.Eqinisweni, ukuhlola kwethu kwaselabhorethri kusetshenziswa i-DNA yangaphandle kubonise ukuthi izimbaza ziqongelela izingcezu ze-DNA emanzini olwandle, okungenani ngemva kwamahora ambalwa ziwohloka ngemva kokuthathwa kwamaselula kanye/noma ukukhululwa kanye/noma ukugcinwa ezinhlanganweni ezihlukahlukene.Uma kubhekwa ukutholakala kwamaseli (kokubili i-prokaryotic ne-eukaryotic), ukusetshenziswa kwamakhompathimenti e-intravalvular kuzonciphisa inani le-ccfDNA evela emithonjeni ngokwayo kanye nakweminye imithombo.Uma sicabangela ukubaluleka kwe-bivalve innate immune immune kanye nenani elikhulu lama-phagocyte ajikelezayo, saphinde sacabanga ukuthi ngisho ne-ccfDNA yangaphandle inothiswe ekujikelezeni kwama-phagocyte aqoqa i-DNA yangaphandle lapho kudliwa amagciwane kanye/noma imfucumfucu yamaselula.Uma sihlanganiswa, imiphumela yethu ibonisa ukuthi i-bivalve hemolymph ccfDNA iyinqolobane eyingqayizivele yolwazi lwamangqamuzana futhi iqinisa isimo sayo njengohlobo lwezilwane ezilindayo.
Idatha yethu ikhombisa ukuthi ukulandelana nokuhlaziywa kwezingcezwana ze-hemolymph ccfDNA ezitholakala kubhaktheriya zinganikeza ulwazi olubalulekile mayelana nesitshalo esibambile sebhaktheriya kanye namagciwane akhona ku-ecosystem yasolwandle ezungezile.Amasu okulandelanisa amashothi embule ukulandelana kwebhaktheriya ye-commensal A. atra gill ebiyogeja ukube izindlela ezijwayelekile zokuhlonza i-16S rRNA bezisetshenzisiwe, ngenxa yengxenye yokuchema kwelabhulali yereferensi.Eqinisweni, ukusebenzisa kwethu idatha ye-LB eqoqwe ku-M. platensis kungqimba lwezimbaza ezifanayo e-Kerguelen kubonise ukuthi ukwakheka kwama-symbionts ebhaktheriya ahlobene ne-gill kwakufana kuzo zombili izinhlobo zezimbaza (Fig. S4, Ulwazi Olwengeziwe).Lokhu kufana kwezimbaza ezimbili ezihlukene ngokwezakhi zofuzo kungase kubonise ukwakheka kwemiphakathi yamagciwane ezindaweni ezibandayo, ezisabalele, nezintaba-mlilo zaseKerguelen [55, 56, 57, 58].Amazinga aphezulu ezinto ezincane ezinciphisa isibabule achazwe kahle lapho evuna izimbaza ezindaweni ezisogwini ezine-bioturbated [59], njengogu lwasePort-au-France.Okunye okungenzeka ukuthi i-commensal mussel flora ingase ithinteke ngokudluliswa okuvundlile [60, 61].Ucwaningo olwengeziwe luyadingeka ukuze kutholwe ukuhlobana phakathi kwemvelo yasolwandle, indawo engaphansi kolwandle, kanye nokwakheka kwamagciwane e-symbiotic ezimbazeni.Lezi zifundo ziyaqhubeka njengamanje.
Ubude nokugxila kwe-hemolymph ccfDNA, ukuhlanzeka kwayo kalula, kanye nekhwalithi ephezulu ukuvumela ukulandelana kwesibhamu esisheshayo ezinye zezinzuzo eziningi zokusebenzisa i-ccfDNA yezimbaza ukuhlola ukuhlukahluka kwemvelo endaweni yasogwini lolwandle.Le ndlela iphumelela kakhulu ekuboniseni imiphakathi enegciwane (viromes) ku-ecosystem enikeziwe [62, 63].Ngokungafani namabhaktheriya, i-archaea, ne-eukaryote, ama-genome egciwane awaqukethe izakhi zofuzo ezilondolozwe nge-phylogenetically ezifana nokulandelana kwe-16S.Imiphumela yethu ikhombisa ukuthi ama-liquid biopsies avela ezinhlotsheni zezinkomba ezifana nezimbaza angasetshenziswa ukuhlonza inani elikhulu kakhulu lezicucu zegciwane le-ccfDNA ezaziwa ukuthelela ababungazi abahlala ezindaweni eziphila ogwini.Lokhu kuhlanganisa amagciwane aziwa ngokuthelela i-protozoa, ama-arthropods, izinambuzane, izitshalo, namagciwane ebhaktheriya (isb, ama-bacteriophages).Ukusatshalaliswa okufanayo kutholwe lapho sihlola i-hemolymph ccfDNA virome yezimbaza eziluhlaza okwesibhakabhaka (M. platensis) eqoqwe kungqimba olufanayo lwezimbaza e-Kerguelen (Ithebula S2, Ulwazi Olwengeziwe).Ukulandelana kwesibhamu se-ccfDNA kuyindlela entsha yokuthola umfutho ocwaningweni lwe-virome yabantu noma ezinye izinhlobo zezilwane [21, 37, 64].Le ndlela iwusizo ikakhulukazi ekutadisheni amagciwane e-DNA anemicu ephindwe kabili, ngoba alukho isakhi sofuzo esisodwa esilondolozwe kuwo wonke amagciwane e-DNA anemicu ephindwe kabili, emele isigaba samagciwane ahluke kakhulu futhi abanzi e-Baltimore [65].Nakuba iningi lala magciwane lihlala lingahlukanisiwe futhi lingahlanganisa amagciwane avela endaweni engaziwa ngokuphelele yomhlaba wegciwane [66], sithole ukuthi ama-virome kanye nebanga lezimbaza i-A. atra ne-M. platensis ziwela phakathi kwalezi zinhlobo ezimbili.ngokufanayo (bheka umfanekiso S3, ulwazi olwengeziwe).Lokhu kufana akumangalisi, njengoba kungase kubonise ukuntuleka kokukhetha ekuthathweni kwe-DNA ekhona endaweni ezungezile.Izifundo zesikhathi esizayo ezisebenzisa i-RNA ehlanzekile ziyadingeka okwamanje ukuze zibonise i-RNA virome.
Ocwaningweni lwethu, sisebenzise ipayipi eliqinile elithathelwe emsebenzini ka-Kowarski nozakwabo [37], abasebenzisa ukususwa okuyizinyathelo ezimbili kokufundwa okuhlanganisiwe kanye nama-contigs ngaphambi nangemuva kokuhlanganiswa kwe-ccfDNA yomdabu, okuholele ekutheni kube nenani eliphezulu elingafundwanga.Ngakho-ke, ngeke sikukhiphele ngaphandle ukuthi okunye kwalokhu okufundwayo okungahleliwe kusengaba nemvelaphi yakho, ngokuyinhloko ngenxa yokuthi asinalo i-genome eyinkomba yalolu hlobo lwezimbaza.Siphinde sasebenzisa leli payipi ngoba besikhathazeke ngama-chimeras phakathi kokuzifundela nokungafundi kanye nobude obufundiwe obukhiqizwe i-Illumina MiSeq PE75.Esinye isizathu sokufunda okuningi okungahleliwe ukuthi amagciwane amaningi olwandle, ikakhulukazi ezindaweni ezikude njenge-Kerguelen, awakachazelwa.Sisebenzise i-Illumina MiSeq PE75, sithatha ubude bengcezu ye-ccfDNA efana ne-ccfDNA yomuntu.Ocwaningweni oluzayo, uma kubhekwa imiphumela yethu ekhombisa ukuthi i-hemolymph ccfDNA ifundeka isikhathi eside kunabantu kanye/noma izilwane ezincelisayo, sincoma ukuthi kusetshenziswe inkundla yokulandelana efaneleke kakhulu izingcezu ze-ccfDNA ezinde.Lo mkhuba uzokwenza kube lula kakhulu ukukhomba izinkomba ezengeziwe zokuhlaziya okujulile.Ukuthola ukulandelana kwegenome ye-nuclear ye-A. atra okungatholakali njengamanje kungasiza kakhulu ukucwaswa kwe-ccfDNA emithonjeni yakhona kanye nengeyona yona.Njengoba ucwaningo lwethu lugxile ekutheni kungenzeka kusetshenziswe umqondo we-liquid biopsy ezimbazeni, sithemba ukuthi njengoba lo mqondo usetshenziswa ocwaningweni lwangomuso, kuzothuthukiswa amathuluzi amasha namapayipi ukuze kwandiswe amandla ale ndlela okucwaninga ukuhlukahluka kwamagciwane ezimbaza.i-ecosystem yasolwandle.
Njenge-biomarker yomtholampilo engahlaseli, amazinga aphezulu e-plasma yomuntu we-ccfDNA ahlotshaniswa nezifo ezihlukahlukene, ukulimala kwezicubu, nezimo zokucindezeleka [67,68,69].Lokhu kwanda kuhlotshaniswa nokukhululwa kwezingcezu ze-DNA zemvelaphi yayo ngemva kokulimala kwezicubu.Sibhekane nalolu daba sisebenzisa ingcindezi yokushisa ebabazekayo, lapho izimbaza zavezwa khona isikhashana ezingeni lokushisa elingu-30 °C.Senze lokhu kuhlaziya ezinhlotsheni ezintathu ezihlukene zezimbaza ekuhloleni okuthathu okuzimele.Kodwa-ke, asizange sithole ushintsho kumazinga e-ccfDNA ngemva kokucindezeleka kokushisa okukhulu (bona Umfanekiso S5, ulwazi olwengeziwe).Lokhu kutholakala kungase kuchaze, okungenani ngokwengxenye, iqiniso lokuthi izimbaza zinesimiso sokujikeleza kwegazi esivulekile kancane futhi ziqongelela inani elikhulu le-DNA yangaphandle ngenxa yomsebenzi wazo ophezulu wokuhlunga.Ngakolunye uhlangothi, izimbaza, njengezilwane eziningi ezingenamgogodla, zingase zimelane kakhulu nokulimala kwezicubu ezibangelwa ukucindezeleka, ngaleyo ndlela zinciphise ukukhululwa kwe-ccfDNA ku-hemolymph yazo [70, 71].
Kuze kube manje, ukuhlaziya i-DNA yezinto eziphilayo ezinhlobonhlobo zemvelo yasemanzini kugxile kakhulu ku-metabarcoding ye-DNA yemvelo (eDNA).Kodwa-ke, le ndlela ivamise ukukhawulelwa ekuhlaziyeni izinhlobonhlobo zezinto eziphilayo lapho kusetshenziswa iziqalo.Ukusetshenziswa kokulandelanisa isibhamu kweqa imikhawulo ye-PCR kanye nokukhethwa okuchemile kwamasethi okuqala.Ngakho-ke, ngomqondo othile, indlela yethu iseduze nendlela yokulandelana kwe-eDNA Shotgun esanda kusetshenziswa, ekwazi ukulandelana ngokuqondile i-DNA ehlukene futhi ihlaziye cishe zonke izinto eziphilayo [72, 73].Kodwa-ke, kunezinkinga ezimbalwa eziyisisekelo ezihlukanisa i-LB ezindleleni ezijwayelekile ze-eDNA.Yiqiniso, umehluko omkhulu phakathi kwe-eDNA ne-LB ukusetshenziswa kwamahostela okuhlunga emvelo.Ukusetshenziswa kwezinhlobo zasolwandle njengezipontshi nama-bivalves (Dresseina spp.) njengesihlungi semvelo sokutadisha i-eDNA kuye kwabikwa [74, 75].Nokho, ucwaningo lukaDreissena lwasebenzisa ama-tissue biopsies okwakhishwa kuwo i-DNA.Ukuhlaziywa kwe-ccfDNA evela ku-LB akudingi i-tissue biopsy, amathuluzi akhethekile futhi kwesinye isikhathi abizayo kanye nezinto ezihambisana ne-eDNA noma i-tissue biopsy.Eqinisweni, sisanda kubika ukuthi i-ccfDNA evela ku-LB ingagcinwa futhi ihlaziywe ngokusekelwa kwe-FTA ngaphandle kokugcina uchungechunge olubandayo, okuyinselele enkulu yocwaningo ezindaweni ezikude [76].Ukukhishwa kwe-ccfDNA ku-liquid biopsies nakho kulula futhi kunikeza i-DNA yekhwalithi ephezulu yokulandelana kwesibhamu nokuhlaziywa kwe-PCR.Lokhu kuyinzuzo enkulu uma kunikezwe eminye imikhawulo yezobuchwepheshe ehambisana nokuhlaziywa kwe-eDNA [77].Ubulula kanye nezindleko eziphansi zendlela yokusampula nakho kufanele ikakhulukazi ezinhlelweni zokuqapha zesikhathi eside.Ngaphezu kwekhono labo eliphezulu lokuhlunga, esinye isici esaziwayo sama-bivalves ukwakheka kwekhemikhali ye-mucopolysaccharide yamafinyila awo, okukhuthaza ukumuncwa kwamagciwane [78, 79].Lokhu kwenza i-bivalves ibe isihlungi semvelo esikahle sokubonisa izinhlobonhlobo zezinto eziphilayo kanye nomthelela wokuguquka kwesimo sezulu endaweni ethile yasemanzini.Nakuba ukuba khona kwezingcezu ze-DNA ezitholakala kumsingathi kungabonakala njengomkhawulo wendlela uma kuqhathaniswa ne-eDNA, izindleko ezihlobene nokuba ne-ccfDNA yomdabu enjalo uma kuqhathaniswa ne-eDNA ziqondakala ngesikhathi esisodwa ngenani elikhulu lolwazi olutholakalayo lwezifundo zezempilo.i-offset host.Lokhu kufaka phakathi ukuba khona kokulandelana kwegciwane okuhlanganiswe ku-genome yomsingathi.Lokhu kubaluleke kakhulu ezimbazeni, uma kubhekwa ukuba khona kwe-leukemic retroviruses evundlile kuma-bivalves [80, 81].Enye inzuzo ye-LB ngaphezu kwe-eDNA ukuthi isebenzisa umsebenzi we-phagocytic wokujikeleza kwamaseli egazi ku-hemolymph, ehlanganisa ama-microorganisms (kanye nama-genomes).I-phagocytosis iwumsebenzi oyinhloko wamaseli egazi kuma-bivalves [82].Ekugcineni, le ndlela isebenzisa amandla aphezulu okusefa kwezimbaza (isilinganiso esingu-1.5 l/h wamanzi olwandle) kanye nokujikeleza kwezinsuku ezimbili, okwandisa ukuxutshwa kwezingqimba ezihlukene zamanzi olwandle, okuvumela ukubanjwa kwe-eDNA ene-heterologous.[83, 84].Ngakho-ke, ukuhlaziya i-ccfDNA yezimbaza kuyindlela ethokozisayo uma kubhekwa imithelela yokudla okunempilo, umnotho, kanye nemvelo yezimbaza.Ngokufanayo nokuhlaziywa kwe-LB eqoqwe kubantu, le ndlela iphinde ivule amathuba okulinganisa izinguquko zofuzo kanye ne-epigenetic ku-DNA ephethe ngokuphendula izinto ezingaphandle.Isibonelo, ubuchwepheshe bokulandelana kwesizukulwane sesithathu bungacatshangwa ukwenza ukuhlaziywa kwe-genome-wide methylation ku-ccfDNA yomdabu kusetshenziswa ukulandelana kwe-nanopore.Le nqubo kufanele ihanjiswe yiqiniso lokuthi ubude bezingcezu ze-mussel ccfDNA buhambisana kahle nezinkundla zokulandelana ezifundwe isikhathi eside ezivumela ukuhlaziywa kwe-DNA methylation ye-genome-wide kusuka ekugijimeni okukodwa kokulandelana ngaphandle kwesidingo sokuguqulwa kwamakhemikhali.85,86] Lokhu kuyinto engenzeka ezithakazelisayo, njengoba kuboniswe ukuthi impendulo ye-DNA phezu kwamaphethini wokucindezeleka kwemvelo ibonisa amaphethini amaningi we-DNA methylistation.Ngakho-ke, inganikeza ukuqonda okubalulekile ezindleleni ezingaphansi ezilawula impendulo ngemva kokuchayeka ekushintsheni kwesimo sezulu noma ukungcola [87].Kodwa-ke, ukusetshenziswa kwe-LB akukhona ukulinganiselwa.Akudingekile ukusho, lokhu kudinga ukuba khona kwezinhlobo zezinkomba ku-ecosystem.Njengoba kushiwo ngenhla, ukusebenzisa i-LB ukuhlola ukuhlukahluka kwemvelo kwesimiso sezinto eziphilayo esinikezwe kudinga futhi ipayipi eliqinile le-bioinformatics elicabangela ukuba khona kwezingcezu ze-DNA ezivela emthonjeni.Enye inkinga enkulu ukutholakala kwe-reference genomes yezinhlobo zasolwandle.Kwethenjwa ukuthi imizamo efana ne-Marine Mammal Genomes Project kanye nephrojekthi esanda kusungulwa ye-Fish10k [88] izokwenza lula ukuhlaziya okunjalo esikhathini esizayo.Ukusetshenziswa komqondo we-LB ezintweni eziphilayo ezidla izihlungi zasolwandle nakho kuyahambisana nentuthuko yakamuva ekulandeleni ubuchwepheshe, okwenza ifaneleke kahle ukuthuthukiswa kwama-multi-ohm biomarkers ukunikeza ulwazi olubalulekile mayelana nempilo yezindawo zasolwandle ekuphenduleni ukucindezeleka kwendawo ezungezile.
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