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I-Liquid biopsy (LB) ingumqondo othola ukuthandwa ngokushesha emkhakheni wezokwelapha. Lo mqondo usekelwe kakhulu ekutholakaleni kwezingcezu ze-DNA engaphandle kweseli ejikelezayo (ccfDNA), ezikhishwa kakhulu njengezingcezu ezincane ngemva kokufa kweseli ezicutshini ezahlukahlukene. Ingxenye encane yalezi zingcezu ivela ezicutshini zakwamanye amazwe (zakwamanye amazwe) noma izinto eziphilayo. Emsebenzini wamanje, sisebenzise lo mqondo ezicutshini, uhlobo oluqaphile olwaziwa ngomthamo walo wokuhlunga amanzi olwandle ophezulu. Sisebenzisa ikhono lezingcezu ze-mussel ukusebenza njengezihlungi zemvelo ukuze sibambe izingcezu ze-DNA yemvelo ezivela emithonjeni eyahlukahlukene ukuze sinikeze ulwazi mayelana nokuhlukahluka kwezinto eziphilayo zasogwini lolwandle. Imiphumela yethu ikhombisa ukuthi i-hemolymph yezingcezu ze-mussel iqukethe izingcezu ze-DNA ezihluka kakhulu ngobukhulu, kusukela ku-1 kuya ku-5 kb. Ukulandelana kwezibhamu kubonise ukuthi inani elikhulu lezingcezu ze-DNA zivela kumagciwane angaphandle. Phakathi kwazo, sithole izingcezu ze-DNA ezivela kumabhaktheriya, i-archaea, kanye namagciwane, kufaka phakathi amagciwane aziwa ngokuthelela izinhlobo ezahlukene zezinto ezivame ukutholakala ezindaweni zemvelo zasogwini lolwandle. Ekuphetheni, ucwaningo lwethu lubonisa ukuthi umqondo we-LB osetshenziswa ezimbazeni umele umthombo wolwazi ocebile kodwa ongakahlolwa mayelana nokuhlukahluka kwamagciwane ezindaweni zemvelo zasogwini lolwandle.
Umthelela wokushintsha kwesimo sezulu (CC) ezinhlobonhlobo zezinto eziphilayo zasolwandle uyindawo ekhula ngokushesha yocwaningo. Ukufudumala komhlaba akugcini nje ngokubangela ukucindezeleka okubalulekile kwezemvelo, kodwa futhi kusunduza imikhawulo yokuzinza kokushisa kwezinto eziphilayo zasolwandle, kuthinta indawo yokuhlala yezinhlobo eziningana, okuzibangela ukuba zifune izimo ezinhle kakhulu [1, 2]. Ngaphezu kokuthinta ukuhlukahluka kwezinto eziphilayo ze-metazoan, i-CC iphazamisa ibhalansi ebucayi yokusebenzisana kwe-host-microbial. Le dysbacteriosis yamagciwane ibeka usongo olukhulu ezintweni eziphilayo zasolwandle njengoba yenza izinto eziphilayo zasolwandle zibe sengozini enkulu yezifo ezithathelwanayo [3, 4]. Kukholakala ukuthi i-SS idlala indima ebalulekile ekufeni kwabantu abaningi, okuyinkinga enkulu ekuphathweni kwezinto eziphilayo zasolwandle zomhlaba [5, 6]. Lena inkinga ebalulekile uma kubhekwa imiphumela yezomnotho, yezemvelo kanye neyokudla kwezinhlobo eziningi zasolwandle. Lokhu kuyiqiniso ikakhulukazi kuma-bivalve ahlala ezindaweni ezibandayo, lapho imiphumela ye-CK ishesha futhi inzima kakhulu [6, 7]. Eqinisweni, ama-bivalve afana ne-Mytilus spp. asetshenziswa kabanzi ukuqapha imiphumela ye-CC ezintweni eziphilayo zasolwandle. Akumangazi ukuthi inani elikhulu kakhulu lama-biomarker lakhiwe ukuze liqaphe impilo yawo, ngokuvamile kusetshenziswa indlela enezigaba ezimbili ehilela ama-biomarker asebenzayo asekelwe emsebenzini we-enzymatic noma imisebenzi yamaseli njengokuphila kweseli kanye nomsebenzi we-phagocytic [8]. Lezi zindlela zifaka phakathi nokulinganisa ukuhlushwa kwezinkomba ezithile zokucindezela eziqongelela ezicutshini ezithambile ngemva kokumuncwa kwamanzi amaningi olwandle. Kodwa-ke, amandla aphezulu okuhlunga kanye nohlelo lokujikeleza kwegazi oluvulekile lwe-bivalves kunikeza ithuba lokuthuthukisa ama-biomarker amasha e-hemolymph kusetshenziswa umqondo we-liquid biopsy (LB), indlela elula futhi engangenisi kakhulu yokuphathwa kweziguli. amasampula egazi [9, 10]. Nakuba izinhlobo eziningana zama-molecule ajikelezayo zingatholakala ku-LB yomuntu, lo mqondo usekelwe kakhulu ekuhlaziyweni kokulandelana kwe-DNA kwezingcezu ze-DNA yangaphandle (ccfDNA) ezijikelezayo ku-plasma. Eqinisweni, ukuba khona kwe-DNA ejikelezayo ku-plasma yomuntu kuye kwaziwa kusukela maphakathi nekhulu lama-20 [11], kodwa eminyakeni yamuva nje kuphela lapho ukufika kwezindlela zokulandelana okuphezulu kuholele ekuxilongweni kwezokwelapha okusekelwe ku-ccfDNA. Ukuba khona kwalezi zingcezu ze-DNA ezijikelezayo kungenxa yokukhululwa kwe-DNA ye-genomic (i-nuclear kanye ne-mitochondrial) ngemva kokufa kweseli. Kubantu abaphilile, ukuhlushwa kwe-ccfDNA ngokuvamile kuphansi (<10 ng/mL) kodwa kungandiswa ngokuphindwe ka-5-10 ezigulini ezihlushwa izifo ezahlukahlukene noma ezibhekene nokucindezeleka, okuholela ekulimaleni kwezicubu. Kubantu abaphilile, ukuhlushwa kwe-ccfDNA ngokuvamile kuphansi (<10 ng/mL) kodwa kungandiswa ngokuphindwe ka-5-10 ezigulini ezihlushwa izifo ezahlukahlukene noma ezibhekene nokucindezeleka, okuholela ekulimaleni kwezicubu. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться ku 5–10 раз у больныхй пилика и разлика подвергающихся стрессу, приводящему к повреждению тканей. Kubantu abaphilile, ukuhlushwa kwe-cccDNA kuvame ukuba phansi (<10 ng/mL), kodwa kunganda ngokuphindwe ka-5-10 ezigulini ezinezifo ezahlukahlukene noma ezingaphansi kokucindezeleka okuholela ekulimaleni kwezicubu.在健康个体中，ccfDNA的浓度通常较低（<10 ng/mL），但在患有各种病理或承受压力的患者力的患者倍，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。。. 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤Концентрации ccfDNA обычно низкие (<10 нг/мл) у здоровых людей, но могут быть увеличены в 5-10 раз у пациентонтовы пациентоны стрессом, что приводит к повреждению тканей. Amazinga e-ccfDNA ngokuvamile aphansi (<10 ng/ml) kubantu abaphilile, kodwa angandiswa ngokuphindwe ka-5-10 ezigulini ezinezifo ezahlukahlukene noma ukucindezeleka, okuholela ekulimaleni kwezicubu.Usayizi wezingcezu ze-ccfDNA uyahlukahluka kakhulu, kodwa ngokuvamile usukela ku-150 kuya ku-200 bp. [12]. Ukuhlaziywa kwe-ccfDNA ezitholakele yona, okungukuthi, i-ccfDNA evela kumaseli ajwayelekile noma aguquliwe, kungasetshenziswa ukuthola izinguquko zofuzo kanye ne-epigenetic ezikhona ku-genome yenuzi kanye/noma i-mitochondrial, ngaleyo ndlela kusize odokotela ukuthi bakhethe izindlela zokwelapha ezithile eziqondiswe kuma-molecule [13]. Kodwa-ke, i-ccfDNA ingatholakala emithonjeni yangaphandle njenge-ccfDNA evela kumaseli e-fetus ngesikhathi sokukhulelwa noma ezithweni ezitshaliwe [14,15,16,17]. I-ccfDNA nayo ingumthombo obalulekile wolwazi wokuthola ukuba khona kwama-nucleic acid e-ejenti ethelelanayo (yangaphandle), okuvumela ukutholakala okungangenisi ukutheleleka okusabalele okungabonwa ngamasiko egazi, okugwema i-biopsy ehlaselayo yezicubu ezithelelekile [18]. Izifundo zakamuva zikhombisile ukuthi igazi lomuntu liqukethe umthombo ocebile wolwazi ongasetshenziswa ukuhlonza amagciwane amagciwane namagciwane, nokuthi cishe u-1% we-ccfDNA etholakala ku-plasma yomuntu ivela kwamanye amazwe [19]. Lezi zifundo zibonisa ukuthi ukuhlukahluka kwezinhlobo zezinto eziphilayo ezijikelezayo kungahlolwa kusetshenziswa ukuhlaziywa kwe-ccfDNA. Kodwa-ke, kuze kube muva nje, lo mqondo wawusetshenziswa kuphela kubantu, futhi, ngezinga elincane, kwezinye izilwane ezinomgogodla [20, 21].
Kuleli phepha lamanje, sisebenzisa amandla e-LB ukuhlaziya i-ccfDNA ye-Aulacomya atra, uhlobo oluseningizimu oluvame ukutholakala eziQhingini zaseKerguelen ezingaphansi kwe-antarctic, iqembu 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 engxenyeni ye-hemolymph. Ukulandelana kwezibhamu kukhombisile ukuthi i-mussel hemolymph ccfDNA iqukethe izingcezu ze-DNA zayo kanye nezingezona ezazo, kufaka phakathi amabhaktheriya e-symbiotic kanye nezingcezu ze-DNA ezivela kuma-biomes ajwayelekile ezindaweni zemvelo zasogwini lwentaba-mlilo ezibandayo. I-Hemolymph ccfDNA iphinde iqukethe ukulandelana kwamagciwane okususelwa kumagciwane anezinhlobo ezahlukene zomsingathi. Siphinde sathola izingcezu ze-DNA ezivela ezilwaneni ezinamaseli amaningi njengezinhlanzi ezinamathambo, ama-anemones olwandle, ulwelwe kanye nezinambuzane. Ekuphetheni, ucwaningo lwethu lubonisa ukuthi umqondo we-LB ungasetshenziswa ngempumelelo ezilwaneni ezingenamgogodla zasolwandle ukuze kukhiqizwe i-repertoire ecebile ye-genomic ezindaweni zemvelo zasolwandle.
I-Mytilus platensis (M. platensis) ezindala (55-70 mm ubude) kanye ne-Aulacomya atra (A. atra) zaqoqwa ogwini olunamadwala oluphakathi kwamaza asePort-au-France (049°21.235 S, 070°13.490 E .). Iziqhingi zaseKerguelen ngoDisemba 2018. Ezinye izimbaza eziluhlaza okwesibhakabhaka ezindala (Mytilus spp.) zatholakala kumhlinzeki wezentengiselwano (PEI Mussel King Inc., ePrince Edward Island, eCanada) zafakwa ethangini elinomoya olawulwa yizinga lokushisa (4°C) eliqukethe amalitha angu-10–20 kasawoti wokwenziwa ongu-32‰. (usawoti wolwandle wokwenziwa i-Reef Crystal, i-Instant Ocean, eVirginia, e-USA). Kulolo nalolo cwaningo, ubude nesisindo samagobolondo ngamanye kwalinganiswa.
Iphrothokholi yokufinyelela evulekile yamahhala yalolu hlelo iyatholakala ku-inthanethi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). Kafushane, i-LB hemolymph yaqoqwa emisipheni ye-abductor njengoba kuchaziwe [22]. I-hemolymph yacaciswa nge-centrifugation ku-1200×g imizuzu emi-3, i-supernatant yaqandiswa (-20°C) kuze kube yilapho isetshenziswa. Ukuze kuhlukaniswe futhi kuhlanzwe i-cfDNA, amasampula (1.5-2.0 ml) ancibilikiswa futhi acutshungulwa kusetshenziswa i-NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ngokwemiyalelo yomenzi. I-ccfDNA yagcinwa ku--80°C kuze kube yilapho kuhlaziywa okwengeziwe. Kwezinye izivivinyo, i-ccfDNA yahlukaniswa futhi yahlanzwa kusetshenziswa i-QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada). I-DNA ehlanziwe yalinganiswa kusetshenziswa i-PicoGreen assay ejwayelekile. Ukusatshalaliswa kwengxenye ye-ccfDNA ehlukanisiwe kwahlaziywa yi-capillary electrophoresis kusetshenziswa i-Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) kusetshenziswa i-High Sensitivity DNA Kit. Ukuhlolwa kwenziwa kusetshenziswa i-1 µl yesampula ye-ccfDNA ngokwemiyalelo yomenzi.
Ukuze kulandelwe izingcezu ze-hemolymph ccfDNA, iGénome Québec (eMontreal, eQuebec, eCanada) yalungisa imitapo yolwazi ye-shotgun isebenzisa ikhithi ye-Illumina DNA Mix yekhithi ye-Illumina MiSeq PE75. Kusetshenziswe i-adaptha ejwayelekile (i-BioO). Amafayela edatha aluhlaza atholakala ku-NCBI Sequence Read Archive (SRR8924808 kanye ne-SRR8924809). Ikhwalithi yokufunda eyisisekelo ihlolwe kusetshenziswa i-FastQC [23]. I-Trimmomatic [24] isetshenziswe ekunqumeni ama-adaptha kanye nokufundwa kwekhwalithi embi. Ukufundwa kwe-Shotgun okuneziphetho ezibhangqiwe kwahlanganiswa i-FLASH kwaba ukufundwa okukodwa okude okunokudlulana okungenani okungu-20 bp ukuze kugwenywe ukungalingani [25]. Ukufundwa okuhlanganisiwe kwabhalwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI Taxonomy se-bivalve (inani le-e < 1e−3 kanye ne-90% homology), futhi ukufihla ukulandelana okuyinkimbinkimbi okuphansi kwenziwa kusetshenziswa i-DUST [26]. Ukufundwa okuhlanganisiwe kwabhalwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI Taxonomy se-bivalve (inani le-e < 1e−3 kanye ne-90% homology), futhi ukufihla ukulandelana okuyinkimbinkimbi okuphansi kwenziwa kusetshenziswa i-DUST [26]. I-BLASTN iphinde ikhiphe i-BLASTN базы данных таксономии двустворчатых моллюсков NCBI (i-NCBI) гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ukufundwa okuhlanganisiwe kwabhalwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI bivalve taxonomy (inani le-e < 1e-3 kanye ne-90% homology), futhi kwenziwa i-sequence masking eyinkimbinkimbi ephansi 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 用 注释 合并 读数 ， 并 使用如 列祖 [26] . .I-BLASTN iphinde ikhiphe i-BLASTN ngohlelo lokusebenza olusha lwe-BLASTN lwe-NCBI <9% гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ukufundwa okuhlanganisiwe kwabhalwa nge-BLASTN kusetshenziswa isizindalwazi se-NCBI bivalve taxonomic (inani le-e <1e-3 kanye ne-90% homology), futhi kwenziwa i-sequence masking eyinkimbinkimbi ephansi kusetshenziswa i-DUST [26].Ama-Read ahlukaniswe ngamaqembu amabili: ahlobene nokulandelana kwama-bivalve (lapha okubizwa ngokuthi ama-self-reading) kanye nama-non-self-reading (okungafundwanga). Amaqembu amabili ahlanganiswa ngokwehlukana kusetshenziswa i-MEGAHIT ukukhiqiza ama-contigs [27]. Phakathi naleso sikhathi, ukusatshalaliswa kwe-taxonomic kwama-alien microbiome reads kwahlukaniswa kusetshenziswa i-Kraken2 [28] futhi kwamelwa ngemidwebo yishadi lephayi le-Krona ku-Galaxy [29, 30]. Ama-kmers afanele kakhulu atholwe ukuthi angama-kmers-59 kusukela ekuhlolweni kwethu kokuqala. Ama-self contig abe esehlonzwa ngokuhambisana ne-BLASTN (i-bivalve NCBI database, inani le-e < 1e−10 kanye ne-60% homology) ukuze kuchazwe isichasiselo sokugcina. Ama-self contig abe esehlonzwa ngokuhambisana ne-BLASTN (i-bivalve NCBI database, inani le-e < 1e−10 kanye ne-60% homology) ukuze kuchazwe isichasiselo sokugcina. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, значени10 e) для окончательной аннотации. Ama-self-contig abe esehlonzwa ngokuqhathanisa ne-BLASTN (isizindalwazi se-NCBI bivalve, inani le-e <1e-10 kanye ne-homology engu-60%) ukuze kuchazwe 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 NCBI для , значение e <1e-10 futhi гомология 60%). Ama-self-contig abe esehlonzwa ukuze achazwe ngokugcina ngokuqhathanisa ne-BLASTN (isizindalwazi se-NCBI bivalve, inani le-e <1e-10 kanye ne-homology engu-60%). Ngesikhathi esifanayo, ama-nonself group contigs abhalwe nge-BLASTN (isizindalwazi se-nt NCBI, inani le-e < 1e−10 kanye ne-homology engu-60%). Ngesikhathi esifanayo, ama-nonself group contigs abhalwe nge-BLASTN (isizindalwazi se-nt NCBI, inani le-e < 1e−10 kanye ne-homology engu-60%). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 and гомология 60%). Ngesikhathi esifanayo, izixhumanisi zamaqembu angaphandle zachazwa nge-BLASTN (isizindalwazi se-NT NCBI, inani le-e <1e-10 kanye ne-homology engu-60%).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%). Ngesikhathi esifanayo, izixhumanisi zeqembu ezingezona ezakho zachazwa nge-BLASTN (isizindalwazi se-nt NCBI, inani le-e <1e-10 kanye ne-homology engu-60%). I-BLASTX iphinde yenziwa kuma-nonself contigs kusetshenziswa izizindalwazi ze-nr kanye ne-RefSeq protein NCBI (inani le-e < 1e−10 kanye ne-60% homology). I-BLASTX iphinde yenziwa kuma-nonself contigs kusetshenziswa izizindalwazi ze-nr kanye ne-RefSeq protein NCBI (inani le-e < 1e−10 kanye ne-60% 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 nayo yenziwe kuma-non-self contigs kusetshenziswa i-nr kanye ne-RefSeq NCBI protein database (inani le-e < 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 nayo yenziwe kuma-non-self contigs kusetshenziswa i-nr kanye ne-RefSeq NCBI protein databases (inani le-e <1e-10 kanye ne-60% homology).Amachibi e-BLASTN kanye ne-BLASTX ezinto ezingezona ezizimele amelela izixhumanisi zokugcina (bheka ifayela elingeziwe).
Ama-primer asetshenziswa ku-PCR abhalwe kuThebula S1. I-Taq DNA polymerase (i-Bio Basic Canada, i-Markham, i-ON) yasetshenziswa ukukhulisa izakhi zofuzo ze-ccfDNA eziqondiwe. Kusetshenziswe izimo zokusabela ezilandelayo: ukuguquka kwesimo sezulu ku-95°C imizuzu emi-3, u-95°C umzuzu o-1, ukusetha izinga lokushisa lomswakama umzuzu o-1, ukunwebeka ku-72°C umzuzu o-1, imijikelezo engu-35, futhi ekugcineni ku-72°C kungakapheli imizuzu eyi-10. . Imikhiqizo ye-PCR yahlukaniswa nge-electrophoresis kuma-gel e-agarose (1.5%) aqukethe i-SYBRTM Safe DNA Gel Stain (i-Invitrogen, i-Burlington, i-ON, i-Canada) ku-95 V.
Izimbaza (i-Mytilus spp.) zajwayelana namanzi olwandle angu-500 ml afakwe umoya-mpilo (32 PSU) amahora angama-24 ku-4°C. I-Plasmid DNA equkethe i-insert ebhala i-human galectin-7 cDNA sequence (inombolo yokungena ye-NCBI L07769) yanezelwa ebhodleleni ngokuhlushwa kokugcina okungu-190 μg/μl. Izimbaza ezifakwe ngaphansi kwezimo ezifanayo ngaphandle kokwengezwa kwe-DNA kwakuyizo ezilawulayo. Ithangi lokulawula lesithathu laliqukethe i-DNA engenazo izimbaza. Ukuqapha ikhwalithi ye-DNA emanzini olwandle, amasampula amanzi olwandle (20 μl; ukuphindaphinda kathathu) athathwa ethangini ngalinye ngesikhathi esibonisiwe. Ukuze kulandeleke i-plasmid DNA, izimbaza ze-LB zavunwa ngezikhathi ezibonisiwe futhi zahlaziywa yi-qPCR kanye ne-ddPCR. Ngenxa yokuqukethwe kosawoti ophezulu emanzini olwandle, ama-aliquot ancishisiwe emanzini ekhwalithi ye-PCR (1:10) ngaphambi kwazo zonke izivivinyo ze-PCR.
I-PCR ye-droplet yedijithali (ddPCR) yenziwe kusetshenziswa iphrothokholi ye-BioRad QX200 (e-Mississippiuga, e-Ontario, eCanada). Sebenzisa iphrofayili yokushisa ukuthola izinga lokushisa elifanele (Ithebula S1). Amaconsi akhiqizwe kusetshenziswa i-QX200 drop generator (i-BioRad). I-ddPCR yenziwe kanje: 95°C imizuzu emi-5, imijikelezo engama-50 engama-95°C imizuzwana engama-30 kanye nezinga lokushisa elinikeziwe lokufaka umswakama umzuzu o-1 kanye no-72°C imizuzwana engama-30, u-4°C imizuzu emi-5 kanye no-90°C zingakapheli imizuzu emi-5. Inani lamaconsi kanye nokusabela okuhle (inani lamakhophi/µl) kwalinganiswa kusetshenziswa i-QX200 drop reader (i-BioRad). Amasampula anamaconsi angaphansi kuka-10,000 enqatshiwe. Ukulawulwa kwephethini akwenziwanga njalo lapho i-ddPCR isebenza.
I-qPCR yenziwe kusetshenziswa i-Rotor-Gene® 3000 (Corbett Research, eSydney, e-Australia) kanye nama-primer athile e-LGALS7. Wonke ama-PCR e-quantitative enziwe ngama-20 µl kusetshenziswa i-QuantiFast SYBR Green PCR Kit (QIAGEN). I-qPCR yaqalwa nge-incubation yemizuzu eyi-15 ku-95°C kwalandela imijikelezo engama-40 ku-95°C imizuzwana eyi-10 kanye naku-60°C imizuzwana engama-60 ngokuqoqwa kwedatha okukodwa. Ama-Melting curves akhiqizwa kusetshenziswa izilinganiso ezilandelanayo ku-95°C imizuzwana emi-5, 65°C imizuzwana engama-60, kanye no-97°C ekupheleni kwe-qPCR. I-qPCR ngayinye yenziwe ngama-triplicate, ngaphandle kwamasampula okulawula.
Njengoba ama-mussels aziwa ngesilinganiso sawo sokuhlunga esiphezulu, siqale sahlola ukuthi angakwazi yini ukuhlunga nokugcina izingcezu ze-DNA ezikhona emanzini olwandle. Sasinesithakazelo futhi sokuthi lezi zingcezu ziqoqana yini ohlelweni lwazo lwe-lymphatic oluvulekile. Sixazulule le nkinga ngokuhlola ngokulandela isiphetho sezingcezu ze-DNA ezincibilikayo ezifakwe ematangini e-mussel aluhlaza okwesibhakabhaka. Ukuze kube lula ukulandelela izingcezu ze-DNA, sisebenzise i-DNA ye-plasmid yangaphandle (hhayi eyakho) equkethe i-human galectin-7 gene. I-ddPCR ilandelela izingcezu ze-DNA ye-plasmid emanzini olwandle kanye nama-mussels. Imiphumela yethu ikhombisa ukuthi uma inani lezingcezu ze-DNA emanzini olwandle lihlala lingaguquguquki ngokuhamba kwesikhathi (kuze kube yizinsuku eziyi-7) uma kungekho zingcezu, khona-ke lapho kukhona izingcezu, leli zinga licishe lanyamalala ngokuphelele zingakapheli amahora ayi-8 (Isithombe 1a,b). Izingcezu ze-DNA yangaphandle zatholakala kalula zingakapheli imizuzu eyi-15 ku-intravalvular fluid kanye ne-hemolymph (Isithombe 1c). Lezi zingcezu zisengatholakala zingakapheli amahora ama-4 ngemva kokuvezwa. Lo msebenzi wokuhlunga maqondana nezingcezu ze-DNA ufana nomsebenzi wokuhlunga wamabhaktheriya kanye ne-algae [31]. Le miphumela iphakamisa ukuthi izimbaza zingahlunga futhi ziqongelele i-DNA yangaphandle ezingxenyeni zazo zoketshezi.
Ukuhlushwa okuhlobene kwe-plasmid DNA emanzini olwandle lapho kukhona (A) noma kungekho (B) kwe-mussels, kulinganiswa yi-ddPCR. Ku-A, imiphumela ivezwa njengamaphesenti, lapho imingcele yamabhokisi imele ama-percentile angu-75 no-25. Ijika le-logarithmic elifakiwe liboniswe ngombala obomvu, kanti indawo enombala ompunga imele isikhawu sokuzethemba esingu-95%. Ku-B, umugqa obomvu umele isilinganiso kanti umugqa oluhlaza okwesibhakabhaka umele isikhawu sokuzethemba esingu-95% sokuhlushwa. C Ukuqongelela kwe-plasmid DNA ku-hemolymph kanye noketshezi lwe-valvular lwe-mussels ngezikhathi ezahlukene ngemva kokwengezwa kwe-plasmid DNA. Imiphumela ivezwa njengamakhophi aphelele atholakele/mL (±SE).
Okulandelayo, sihlole imvelaphi ye-ccfDNA ezimbazeni eziqoqwe emibhedeni yezimbaza eziQhingini zaseKerguelen, iqembu leziqhingi ezikude ezinethonya elincane lokuvela kwabantu. Ngale njongo, i-cccDNA evela kuma-hemolymph e-mussel yahlukaniswa futhi yahlanzwa ngezindlela ezivame ukusetshenziswa ukuhlanza i-cccDNA yabantu [32, 33]. Sithole ukuthi amazinga ajwayelekile e-hemolymph ccfDNA ezimbazeni asezingeni eliphansi lama-microgram nge-ml ye-hemolymph (bheka Ithebula S2, Ulwazi Olungeziwe). Lolu hlu lwamazinga likhulu kakhulu kunakubantu abanempilo (ama-nanogram aphansi nge-milliliter), kodwa ezimweni ezingavamile, ezigulini ezinomdlavuza, izinga le-ccfDNA lingafinyelela kuma-microgram amaningana nge-milliliter [34, 35]. Ukuhlaziywa kokusatshalaliswa kosayizi we-hemolymph ccfDNA kubonise ukuthi lezi zingcezu ziyahlukahluka kakhulu ngobukhulu, kusukela ku-1000 bp kuya ku-1000 bp. kuze kufike ku-5000 bp (Isithombe 2). Imiphumela efanayo yatholakala kusetshenziswa i-silica-based QIAamp Investigator Kit, indlela evame ukusetshenziswa kwesayensi ye-forensic ukuhlukanisa nokuhlanza ngokushesha i-genomic DNA kumasampula e-DNA aphansi, kufaka phakathi i-ccfDNA [36].
I-electrophoregram emele i-ccfDNA ye-hemolymph ye-mussel. Ikhishwe nge-NucleoSnap Plasma Kit (phezulu) kanye ne-QIAamp DNA Investigator Kit. I-B Violin plot ekhombisa ukusatshalaliswa kwe-hemolymph ccfDNA concentrations (±SE) kuma-mussels. Imigqa emnyama nebomvu imelela ama-quartiles aphakathi nendawo kanye ne-first and third, ngokulandelana.
Cishe u-1% we-ccfDNA kubantu nasezinhlotsheni zezilwane unomthombo wangaphandle [21, 37]. Njengoba sibheka uhlelo lokujikeleza kwegazi oluvulekile lwe-bivalves, amanzi olwandle acebile ngamagciwane, kanye nokusatshalaliswa kosayizi we-ccfDNA ye-mussel, sicabange ukuthi i-mussel hemolymph ccfDNA ingase ibe nedamu elicebile nelihlukahlukene le-DNA yamagciwane. Ukuze sihlole lo mbono, silandele i-hemolymph ccfDNA evela kumasampula e-Aulacomya atra aqoqwe eziQhingini zaseKerguelen, sathola ukufundwa okungaphezu kwezigidi eziyi-10, okungu-97.6% kwawo kudlule ekulawulweni kwekhwalithi. Ukufundwa kwabe sekuhlelwa ngokwemithombo yomuntu siqu nengeyona eyakhe kusetshenziswa i-database ye-BLASTN ne-NCBI bivalve (Isithombe S1, Ulwazi Olungeziwe).
Kubantu, i-DNA yenuzi kanye ne-mitochondrial ingakhishwa egazini [38]. Kodwa-ke, kulesi sifundo samanje, bekungenakwenzeka ukuchaza ngokuningiliziwe i-DNA yenuzi ye-genomic yama-mussels, njengoba i-genome ye-A. atra ingakahlelwa noma ichazwe. Kodwa-ke, sikwazile ukuhlonza izingcezu eziningana ze-ccfDNA ezivela kithi sisebenzisa umtapo wolwazi we-bivalve (Isithombe S2, Ulwazi Olwengeziwe). Siphinde saqinisekisa ukuba khona kwezingcezu ze-DNA ezivela kithi ngokukhuliswa kwe-PCR okuqondisiwe kwalezo zakhi zofuzo ze-A. atra ezahlelwa (Isithombe 3). Ngokufanayo, njengoba i-genome ye-mitochondrial ye-A. atra itholakala kudathabheyisi yomphakathi, umuntu angathola ubufakazi bokuba khona kwezingcezu ze-ccfDNA ze-mitochondrial ku-hemolymph ye-A. atra. Ukuba khona kwezingcezu ze-DNA ye-mitochondrial kwaqinisekiswa ngokukhuliswa kwe-PCR (Isithombe 3).
Izakhi zofuzo ezahlukene ze-mitochondrial zazikhona ku-hemolymph ye-A. atra (amachashazi abomvu - inombolo yesitoko: SRX5705969) kanye ne-M. platensis (amachashazi aluhlaza okwesibhakabhaka - inombolo yesitoko: SRX5705968) ekhuliswe yi-PCR. Isithombe sithathwe ku-Breton et al., 2011 B Ukwandiswa kwe-hemolymph supernatant evela ku-A. atra Kugcinwe ephepheni le-FTA. Sebenzisa i-punch engu-3 mm ukuze ungeze ngqo ku-PCR tube equkethe ingxube ye-PCR.
Njengoba sibheka okuqukethwe okuningi kwamagciwane emanzini olwandle, ekuqaleni sigxile ekuchazeni ukulandelana kwe-DNA yamagciwane ku-hemolymph. Ukuze senze lokhu, sisebenzisa amasu amabili ahlukene. Isu lokuqala lisebenzise i-Kraken2, uhlelo lokuhlukanisa ukulandelana olusekelwe ku-algorithm olungakwazi ukubona ukulandelana kwamagciwane ngokunemba okufana ne-BLAST namanye amathuluzi [28]. Kutholakale ukuthi ukufunda okungaphezu kuka-6719 kuvela kumagciwane, kanti okungu-124 no-64 kwakuvela ku-archaea namagciwane, ngokulandelana (Isithombe 4). Izingcezu ze-DNA yamagciwane eziningi kakhulu kwakuyi-Firmicutes (46%), i-Proteobacteria (27%), kanye ne-Bacteroidetes (17%) (Isithombe 4a). Lokhu kusatshalaliswa kuhambisana nezifundo zangaphambilini ze-marine blue mussel microbiome [39, 40]. I-Gammaproteobacteria yayiyisigaba esiyinhloko se-Proteobacteria (44%), kufaka phakathi ama-Vibrionales amaningi (Isithombe 4b). Indlela ye-ddPCR iqinisekisile ukuba khona kwezingcezu ze-Vibrio DNA ku-ccfDNA ye-A. atra hemolymph (Isithombe 4c) [41]. Ukuze kutholakale ulwazi olwengeziwe mayelana nomsuka webhaktheriya we-ccfDNA, kwathathwa indlela eyengeziwe (Isithombe S2, Ulwazi Olwengeziwe). Kulesi simo, ama-reads ahlanganisiwe ahlanganiswe njenge-paired-end reads futhi ahlukaniswa njenge-self (bivalves) noma i-nonself origin kusetshenziswa i-BLASTN kanye nenani le-e elingu-1e−3 kanye ne-cutoff ene-homology engu->90%. Kulesi simo, ama-reads ahlanganisiwe ahlanganiswe njenge-paired-end reads futhi ahlukaniswa njenge-self (bivalves) noma i-nonself origin kusetshenziswa i-BLASTN kanye nenani le-e elingu-1e−3 kanye ne-cutoff ene-homology engu->90%. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственные обственные (двуста) чужие по происхождению с использованием BLASTN futhi значения e 1e-3 futhi отсечения с гомологией> 90%. Kulesi simo, ukufundwa okugqagqene kuqoqwe njengokufundwa okunezinhlangothi ezimbili futhi kwahlukaniswa njengokwasendaweni (i-bivalve) noma okungewona okwangempela kusetshenziswa inani le-BLASTN kanye ne-e elingu-1e-3 kanye nokunqunywa okune-homology engaphezulu kuka-90%.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的值 kanye>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 使用 blastn kanye 1e-3 的 值% > 90%自身 （双 壳类） 非 自身。。。。。. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицированы как собственные (двуствор в парными концами) несобственные по происхождению с использованием значений e BLASTN kanye ne-1e-3 kanye ne-гомологии> 90%. Kulesi simo, ukufundwa okugqagqene kuqoqwe njengokufundwa okunezinhlangothi ezimbili futhi kwahlukaniswa njengokwakho (ama-bivalve) noma okungewona okwangempela kusetshenziswa amanani e-e BLASTN kanye ne-1e-3 kanye nomkhawulo we-homology >90%.Njengoba i-genome ye-A. atra ingakahlelwanga, sisebenzise isu lokuhlanganisa i-de novo le-MEGAHIT Next Generation Sequencing (NGS) assembler. Sekukonke ama-contig angu-147,188 akhonjwe njengemvelaphi encike kuwo (ama-bivalves). La ma-contig abe eseqhunyiswa ngama-e-values ​​​​angu-1e-10 kusetshenziswa i-BLASTN ne-BLASTX. Leli su lisivumele ukuthi sithole izingcezu ezingama-non-bivalve ezingu-482 ezikhona ku-A. atra ccfDNA. Ngaphezu kwengxenye (57%) yalezi zingcezu ze-DNA zitholakale kumabhaktheriya, ikakhulukazi kuma-gill symbionts, kufaka phakathi ama-sulfotrophic symbionts, kanye naku-gill symbionts Solemya velum (Isithombe 5).
Ubuningi obuhlobene ezingeni lohlobo. B Ukwehlukahlukana kwamagciwane e-phyla amabili amakhulu (ama-Firmicutes nama-Proteobacteria). Ukwandiswa okumelelayo kwe-ddPCR C Vibrio spp. A. Izingcezu ze-gene ye-16S rRNA (eluhlaza okwesibhakabhaka) kuma-hemolymph amathathu e-atra.
Kuhlaziywe ama-contig angu-482 aqoqwe. Iphrofayili ejwayelekile yokusatshalaliswa kwe-taxonomic kwama-metagenomic contig annotations (ama-prokaryotes nama-eukaryotes). B Ukusatshalaliswa okuningiliziwe kwezingcezu ze-DNA yebhaktheriya ezitholwe yi-BLASTN kanye ne-BLASTX.
Ukuhlaziywa kwe-Kraken2 kuphinde kwabonisa ukuthi i-ccfDNA ye-mussel yayiqukethe izingcezu ze-DNA zakudala, okuhlanganisa izingcezu ze-DNA ze-Euryarchaeota (65%), i-Crenarchaeota (24%), kanye ne-Thaurmarcheota (11%) (Isithombe 6a). Ukuba khona kwezingcezu ze-DNA ezithathwe ku-Euryarchaeota kanye ne-Crenarchaeota, ezazitholakala emphakathini we-microbial we-mussels yaseCalifornia, akufanele kumangaze [42]. Nakuba i-Euryarchaeota ivame ukuhlotshaniswa nezimo ezimbi kakhulu, manje sekuyaziwa ukuthi zombili i-Euryarchaeota kanye ne-Crenarcheota ziphakathi kwama-prokaryotes avame kakhulu endaweni ebangela ukuvuza kolwandle [43, 44]. Ukuba khona kwezingcezu ezincane ze-methanogenic ezingcezu ze-mussels akumangazi, uma kubhekwa imibiko yakamuva yokuvuza okukhulu kwe-methane okuvela ekuvuza okuphansi e-Kerguelen Plateau [45] kanye nokukhiqizwa okungenzeka kwe-methane yamagciwane okubonwe ogwini lweziQhingi zaseKerguelen [46].
Ukunaka kwethu kwabe sekuguqukela ekufundweni kwamagciwane e-DNA. Ngokwazi kwethu okuhle, lolu ucwaningo lokuqala olungeyona inhloso lokuqukethwe kwegciwane kuma-mussels. Njengoba bekulindelekile, sithole izingcezu ze-DNA zama-bacteriophages (Caudovirales) (Isithombe 6b). Kodwa-ke, i-DNA yegciwane evame kakhulu ivela ku-phylum yama-nucleocytovirus, eyaziwa nangokuthi i-nuclear cytoplasmic large DNA virus (NCLDV), ene-genome enkulu kunanoma yiliphi igciwane. Ngaphakathi kwale phylum, iningi lama-DNA sequences lingemindeni i-Mimimidoviridae (58%) kanye ne-Poxviridae (21%), lapho izidalwa zemvelo zifaka khona izilwane ezinomgogodla kanye nama-arthropod, kanti ingxenye encane yalezi sequences ze-DNA ingeye-algae eyaziwayo ye-virological. Ithelela i-algae yasolwandle ye-eukaryotic. Ama-sequences nawo atholakale ku-Pandora virus, igciwane elikhulu elinobukhulu be-genome obukhulu kunanoma yiluphi uhlobo lwegciwane olwaziwayo. Ngokuthakazelisayo, uhla lwezindawo ezaziwa ukuthi zithelelekile ngaleli gciwane, njengoba kunqunywe yi-hemolymph ccfDNA sequencing, lwalulukhulu kakhulu (Isithombe S3, Ulwazi Olungeziwe). Luhlanganisa amagciwane atheleleka ngezinambuzane ezifana neBaculoviridae kanye ne-Iridoviridae, kanye namagciwane atheleleka nge-amoeba, i-algae kanye nezilwane ezinethambo lomgogodla. Sithole futhi ukulandelana okufana ne-genome ye-Pithovirus sibericum. Ama-Pitovirus (aziwa nangokuthi “ama-zombie virus”) aqala ukuhlukaniswa ne-permafrost eneminyaka engu-30,000 ubudala eSiberia [47]. Ngakho-ke, imiphumela yethu ihambisana nemibiko yangaphambilini ekhombisa ukuthi akuzona zonke izinhlobo zanamuhla zala magciwane eziphelile [48] nokuthi la magciwane angase abe khona ezindaweni zemvelo zasolwandle ezikude.
Ekugcineni, sihlole ukuze sibone ukuthi singathola yini izingcezu ze-DNA ezivela kwezinye izilwane ezinamaseli amaningi. I-BLASTN ne-BLASTX zizonke izingxenye zakwamanye amazwe ezingu-482 zatholakala ngemitapo yolwazi ye-nt, nr kanye ne-RefSeq (i-genomic kanye ne-protein). Imiphumela yethu ikhombisa ukuthi phakathi kwezingcezu zakwamanye amazwe ze-ccfDNA zezilwane ezinamaseli amaningi i-DNA yamathambo amathambo ibusa kakhulu (Isithombe 5). Izingcezu ze-DNA ezivela ezinambuzaneni nakwezinye izinhlobo nazo zitholakale. Ingxenye enkulu kakhulu yezingcezu ze-DNA ayikatholakali, mhlawumbe ngenxa yokungamelwa kahle kwenani elikhulu lezinhlobo zasolwandle kuma-database e-genomic uma kuqhathaniswa nezinhlobo zasemhlabeni [49].
Kuleli phepha lamanje, sisebenzisa umqondo we-LB kuma-mussel, sithi ukulandelana kwe-hemolymph ccfDNA shot kunganikeza ukuqonda ngokwakhiwa kwezindawo zemvelo zasogwini lolwandle. Ikakhulukazi, sithole ukuthi 1) i-hemolymph yama-mussel iqukethe amazinga aphezulu (amazinga e-microgram) ezingcezu ze-DNA ezinkulu kakhulu (~1-5 kb) ezijikelezayo; 2) lezi zingcezu ze-DNA zizimele futhi azizimele 3) Phakathi kwemithombo yangaphandle yalezi zingcezu ze-DNA, sithole i-DNA yamagciwane, yama-archaeal kanye neyamagciwane, kanye ne-DNA yezinye izilwane ezinamaseli amaningi; 4) Ukuqongelela kwalezi zingcezu ze-ccfDNA zangaphandle ku-hemolymph kwenzeka ngokushesha futhi kunegalelo emsebenzini wokuhlunga wangaphakathi wama-mussel. Ekuphetheni, ucwaningo lwethu lubonisa ukuthi umqondo we-LB, osetshenziswe kakhulu emkhakheni we-biomedicine kuze kube manje, uhlanganisa umthombo wolwazi ocebile kodwa ongakahlolwa ongasetshenziswa ukuqonda kangcono ukusebenzisana phakathi kwezinhlobo ze-sentinel nendawo ezikuyo.
Ngaphezu kwezilwane ezincelisayo, ukuhlukaniswa kwe-ccfDNA kubikwe ezincelisayo, okuhlanganisa amagundane, izinja, amakati, kanye namahhashi [50, 51, 52]. Kodwa-ke, ngokwazi kwethu, ucwaningo lwethu lulokuqala ukubika ukutholakala nokulandelana kwe-ccfDNA ezinhlotsheni zasolwandle ezinesistimu yokujikeleza kwegazi evulekile. Lesi sici sokwakheka komzimba kanye nekhono lokuhlunga lama-mussels, okungenani ngokwengxenye, kungachaza izici zobukhulu ezahlukene zezingcezu ze-DNA ezijikelezayo uma kuqhathaniswa nezinye izinhlobo. Kubantu, izingcezu eziningi ze-DNA ezijikeleza egazini ziyizingcezu ezincane ezinobukhulu obusukela ku-150 kuya ku-200 bp. kanye nenani eliphakeme kakhulu le-167 bp [34, 53]. Ingxenye encane kodwa ebalulekile yezingcezu ze-DNA iphakathi kuka-300 no-500 bp ngobukhulu, futhi cishe u-5% ubude kuno-900 bp. [54]. Isizathu salokhu kusabalala kosayizi ukuthi umthombo oyinhloko we-ccfDNA ku-plasma uvela ngenxa yokufa kweseli, kungaba ngenxa yokufa kweseli noma ngenxa yokufa kwamaseli e-hematopoietic ajikelezayo kubantu abaphilile noma ngenxa yokuqhekeka kwamaseli esimila ezigulini zomdlavuza (okwaziwa ngokuthi i-circulating tumor DNA). , ctDNA). Ukusatshalaliswa kosayizi we-hemolymph ccfDNA esikuthole kuma-mussels kwakusukela ku-1000 kuya ku-5000 bp, okuphakamisa ukuthi i-mussel ccfDNA inemvelaphi ehlukile. Lokhu kuyinkolelo-mbono enengqondo, njengoba ama-mussels enesistimu yemithambo yegazi evulekile kancane futhi ahlala ezindaweni zasemanzini zasolwandle eziqukethe amazinga aphezulu e-DNA ye-microbial genomic. Eqinisweni, izivivinyo zethu zelabhorethri ezisebenzisa i-DNA yangaphandle zikhombisile ukuthi ama-mussels aqoqa izingcezu ze-DNA emanzini olwandle, okungenani ngemva kwamahora ambalwa ayawohloka ngemva kokuthathwa kwamaseli kanye/noma ukukhishwa kanye/noma ukugcinwa ezinhlanganweni ezahlukene. Njengoba kungavamile ukuba khona kwamangqamuzana (kokubili i-prokaryotic ne-eukaryotic), ukusetshenziswa kwezingxenye zangaphakathi kwe-valvu kuzonciphisa inani le-ccfDNA evela emithonjeni ezimele kanye nemithombo yangaphandle. Uma sibheka ukubaluleka kokuzivikela komzimba okungokwemvelo kwe-bivalve kanye nenani elikhulu lama-phagocyte ajikelezayo, siphinde sacabanga ukuthi ngisho ne-ccfDNA yangaphandle icebile kuma-phagocyte ajikelezayo aqoqa i-DNA yangaphandle lapho idla ama-microorganisms kanye/noma udoti weseli. Uma sibheka ndawonye, ​​​​imiphumela yethu ikhombisa ukuthi i-bivalve hemolymph ccfDNA iyindawo yokugcina ulwazi lwama-molecule eyingqayizivele futhi iqinisa isimo sayo njengohlobo lwe-sentinel.
Idatha yethu ikhombisa ukuthi ukulandelana nokuhlaziywa kwezingcezu ze-ccfDNA ze-hemolymph ezithathwe yibhaktheriya kunganikeza ulwazi olubalulekile mayelana nezitshalo zebhaktheriya ezihlala kuyo kanye namabhaktheriya akhona endaweni ezungezile yezilwane zasolwandle. Amasu okulandelana kwezibhamu aveze ukulandelana kwebhaktheriya i-commensal i-A. atra gill obekungeke kutholakale ukube izindlela zokuhlonza ze-16S rRNA ezivamile bezisetshenzisiwe, ngenxa yokubandlululwa komtapo wolwazi. Eqinisweni, ukusebenzisa kwethu idatha ye-LB eqoqwe ku-M. platensis ongqimbeni olufanayo lwezimbaza eKerguelen kubonise ukuthi ukwakheka kwe-symbiont yebhaktheriya ehlobene ne-gill kwakufana kuzo zombili izinhlobo zezimbaza (Isithombe S4, Ulwazi Olwengeziwe). Lokhu kufana kwezimbaza ezimbili ezihlukene ngokwezakhi zofuzo kungabonisa ukwakheka kwemiphakathi yamabhaktheriya ezindaweni ezibandayo, ezine-sulfurous, kanye nezintaba-mlilo zaseKerguelen [55, 56, 57, 58]. Amazinga aphezulu ezilokazane ezincane ezinciphisa isibabule achazwe kahle lapho kuvunwa izimbaza ezindaweni ezisogwini ezigcwele i-bioturbated [59], njengogwini lwasePort-au-France. Okunye okungenzeka ukuthi izitshalo ze-commensal mussel zingase zithinteke ngokudluliswa okuvundlile [60, 61]. Kudingeka ucwaningo olwengeziwe ukuze kutholakale ukuhlobana phakathi kwendawo yasolwandle, ubuso bolwandle, kanye nokwakheka kwamagciwane ahambisanayo ezimbazeni. Lezi zifundo ziyaqhubeka okwamanje.
Ubude kanye nokuhlushwa kwe-hemolymph ccfDNA, ukulula kwayo ukuhlanzwa, kanye nekhwalithi ephezulu yokuvumela ukulandelana okusheshayo kwesibhamu kungenye yezinzuzo eziningi zokusebenzisa i-ccfDNA ye-mussel ukuhlola ukuhlukahluka kwezinto eziphilayo ezindaweni zemvelo zasogwini lolwandle. Le ndlela isebenza kahle kakhulu ekuchazeni imiphakathi yamagciwane (ama-virome) endaweni ethile yemvelo [62, 63]. Ngokungafani namabhaktheriya, ama-archaea, nama-eukaryotes, ama-genome egciwane awaqukethe izakhi zofuzo ezigcinwe yi-phylogenetically njenge-16S sequences. Imiphumela yethu ikhombisa ukuthi ama-biopsies e-liquid avela ezinhlotsheni zezinkomba ezifana nama-mussels angasetshenziswa ukuhlonza inani elikhulu kakhulu lezingcezu zegciwane le-ccfDNA ezaziwa ukuthi zithelela izidalwa ezivame ukuhlala ezindaweni zemvelo zasogwini lolwandle. Lokhu kufaka phakathi amagciwane aziwa ukuthi athelela ama-protozoa, ama-arthropod, izinambuzane, izitshalo, kanye namagciwane ebhaktheriya (isb., ama-bacteriophages). Ukusatshalaliswa okufanayo kutholakale lapho sihlola i-hemolymph ccfDNA virome yama-blue mussels (M. platensis) aqoqwe kungqimba olufanayo lwe-mussel eKerguelen (Ithebula S2, Ulwazi Olwengeziwe). Ukulandelana kwezibhamu ze-ccfDNA empeleni kuyindlela entsha ekhula ngamandla okutadisha i-virome yabantu noma ezinye izinhlobo [21, 37, 64]. Le ndlela iwusizo kakhulu ekutadisheni amagciwane e-DNA anezintambo ezimbili, njengoba kungekho gene eyodwa elondolozwe phakathi kwawo wonke amagciwane e-DNA anezintambo ezimbili, emele uhlobo lwamagciwane oluhlukahlukene kakhulu nolubanzi eBaltimore [65]. Nakuba iningi lala magciwane lihlala lingahlukaniswanga futhi lingafaka amagciwane avela engxenyeni engaziwa ngokuphelele yezwe lamagciwane [66], sithole ukuthi ama-virome kanye nohlu lwababambi be-mussels i-A. atra kanye ne-M. platensis ziwela phakathi kwalezi zinhlobo ezimbili. ngokufanayo (bheka isithombe S3, ulwazi olwengeziwe). Lokhu kufana akumangazi, njengoba kungabonisa ukuntuleka kokukhetha ekuthathweni kwe-DNA ekhona emvelweni. Izifundo zesikhathi esizayo ezisebenzisa i-RNA ehlanziwe okwamanje ziyadingeka ukuze kuchazwe i-RNA virome.
Esifundweni sethu, sisebenzise ipayipi eliqinile kakhulu elithathwe emsebenzini kaKowarski nozakwethu [37], abasebenzise ukususa okunezinyathelo ezimbili kokufundwa okuhlanganisiwe kanye nama-contig ngaphambi nangemva kokuhlanganiswa kwe-ccfDNA yomdabu, okwaholela engxenyeni ephezulu yokufundwa okungakahlelwanga. Ngakho-ke, asinakuphika ukuthi ezinye zalezi zincwadi ezingakahlelwanga zingase zibe nomsuka wazo, ikakhulukazi ngoba asinayo i-genome yokubhekisela yalolu hlobo lwezimbaza. Siphinde sasebenzisa le payipi ngoba sasikhathazekile ngama-chimera phakathi kokufundwa komuntu siqu nokungahleliwe kanye nobude bokufundwa obukhiqizwa yi-Illumina MiSeq PE75. Esinye isizathu sobuningi bokufundwa okungahleliwe ukuthi iningi lamagciwane asolwandle, ikakhulukazi ezindaweni ezikude njengeKerguelen, alukachazwanga. Sisebenzise i-Illumina MiSeq PE75, sicabanga ukuthi ubude bezingcezu ze-ccfDNA bufana ne-ccfDNA yomuntu. Ezifundweni zesikhathi esizayo, njengoba imiphumela yethu ikhombisa ukuthi i-ccfDNA ye-hemolymph inokufundwa isikhathi eside kunabantu kanye/noma izilwane ezincelisayo, sincoma ukusebenzisa ipulatifomu yokulandelela efaneleka kakhulu ngezingcezu ze-ccfDNA ezinde. Lo mkhuba uzokwenza kube lula kakhulu ukuhlonza izinkomba ezengeziwe zokuhlaziywa okujulile. Ukuthola ukulandelana kwe-genome ye-nuclear ephelele ye-A. atra okwamanje kuzosiza kakhulu ukuhlukaniswa kwe-ccfDNA emithonjeni yomuntu siqu kanye nengeyona eyakhe. Njengoba ucwaningo lwethu lugxile ekwelashweni kokusebenzisa umqondo we-biopsy yoketshezi kuma-mussels, sithemba ukuthi njengoba lo mqondo usetshenziswa ocwaningweni lwesikhathi esizayo, kuzothuthukiswa amathuluzi amasha kanye namapayipi ukuze kwandiswe amandla ale ndlela okutadisha ukuhlukahluka kwamagciwane kwama-mussels.
Njenge-biomarker yezokwelapha engangenisi uhlaselo, amazinga aphezulu e-ccfDNA egazini lomuntu ahlotshaniswa nezifo ezahlukahlukene, ukulimala kwezicubu, kanye nezimo zokucindezeleka [67,68,69]. Lokhu kwanda kuhlotshaniswa nokukhululwa kwezingcezu ze-DNA ezivela kuzo ngemuva kokulimala kwezicubu. Sibhekane nale nkinga sisebenzisa ukucindezeleka kokushisa okukhulu, lapho izimbaza zavezwa khona okwesikhashana ekushiseni okungu-30 °C. Senze lokhu kuhlaziya ezinhlotsheni ezintathu ezahlukene zezimbaza ekuhlolweni okuzimele okuthathu. Kodwa-ke, asitholanga ushintsho emazingeni e-ccfDNA ngemuva kokucindezeleka kokushisa okukhulu (bheka Umfanekiso S5, ulwazi olwengeziwe). Lokhu kutholakala kungachaza, okungenani ngokwengxenye, iqiniso lokuthi izimbaza zinesistimu yokujikeleza kwegazi evulekile kancane futhi ziqoqa inani elikhulu le-DNA yangaphandle ngenxa yomsebenzi wazo wokuhlunga ophezulu. Ngakolunye uhlangothi, izimbaza, njengezilwane eziningi ezingenamgogodla, zingase zimelane kakhulu nomonakalo wezicubu obangelwa ukucindezeleka, ngaleyo ndlela zinciphise ukukhishwa kwe-ccfDNA ku-hemolymph yazo [70, 71].
Kuze kube manje, ukuhlaziywa kwe-DNA kwezinto eziphilayo ezindaweni zemvelo zasemanzini kugxile kakhulu ku-metabarcoding ye-DNA yemvelo (i-eDNA). Kodwa-ke, le ndlela ivame ukukhawulelwa ekuhlaziyweni kwezinto eziphilayo lapho kusetshenziswa ama-primer. Ukusetshenziswa kwe-shotgun sequencing kugwema ukulinganiselwa kwe-PCR kanye nokukhethwa okubandlululayo kwamasethi e-primer. Ngakho-ke, ngandlela thile, indlela yethu iseduze nendlela ye-high-throughput esetshenziswa kakhulu ye-eDNA Shotgun sequencing, ekwazi ukulandelanisa ngqo i-DNA ehlukanisiwe futhi ihlaziye cishe zonke izinto eziphilayo [72, 73]. Kodwa-ke, kunezinkinga eziningi eziyisisekelo ezihlukanisa i-LB nezindlela ezijwayelekile ze-eDNA. Yiqiniso, umehluko omkhulu phakathi kwe-eDNA ne-LB ukusetshenziswa kwezindawo zokuhlunga zemvelo. Ukusetshenziswa kwezinhlobo zasolwandle ezifana neziponji nama-bivalves (i-Dresseina spp.) njengesihlungi semvelo sokutadisha i-eDNA kubikiwe [74, 75]. Kodwa-ke, ucwaningo lukaDreissena lusebenzise ama-biopsies ezicubu lapho i-DNA ikhishwe khona. Ukuhlaziywa kwe-ccfDNA kusuka ku-LB akudingi i-tissue biopsy, imishini ekhethekile futhi ngezinye izikhathi ebizayo kanye ne-logistics ehlobene ne-eDNA noma i-tissue biopsy. Eqinisweni, muva nje sibike ukuthi i-ccfDNA kusuka ku-LB ingagcinwa futhi ihlaziywe ngokusekelwa kwe-FTA ngaphandle kokugcina uchungechunge olubandayo, okuyinselele enkulu ocwaningweni ezindaweni ezikude [76]. Ukukhishwa kwe-ccfDNA kusuka ku-liquid biopsies nakho kulula futhi kunikeza i-DNA esezingeni eliphezulu yokulandelana kwe-shotgun kanye nokuhlaziywa kwe-PCR. Lokhu kuyinzuzo enkulu uma kubhekwa eminye yemikhawulo yobuchwepheshe ehlobene nokuhlaziywa kwe-eDNA [77]. Ubulula kanye nezindleko eziphansi zendlela yokwenza isampula nakho kufanelekile kakhulu ezinhlelweni zokuqapha zesikhathi eside. Ngaphezu kwekhono labo lokuhlunga eliphezulu, esinye isici esaziwayo sama-bivalves ukwakheka kwamakhemikhali e-mucopolysaccharide emucwini wawo, okukhuthaza ukumuncwa kwamagciwane [78, 79]. Lokhu kwenza ama-bivalves abe yisihlungi semvelo esifanele sokuchaza ukuhlukahluka kwezinto eziphilayo kanye nomthelela wokushintsha kwesimo sezulu endaweni ethile yasemanzini. Nakuba ukuba khona kwezingcezu ze-DNA ezithathwe ngumnikazi kungabonakala njengomkhawulo wendlela uma kuqhathaniswa ne-eDNA, izindleko ezihambisana nokuba ne-ccfDNA enjalo yemvelo uma kuqhathaniswa ne-eDNA ziyaqondakala ngesikhathi esifanayo ngenani elikhulu lolwazi olutholakalayo lwezifundo zezempilo. Lokhu kufaka phakathi ukuba khona kwezinhlobo zegciwane ezihlanganiswe ku-genome yomnikazi womnikazi. Lokhu kubaluleke kakhulu kuma-mussels, uma kubhekwa ukuba khona kwama-retrovirus e-leukemic adluliselwa ngokuvundlile kuma-bivalves [80, 81]. Enye inzuzo ye-LB kune-eDNA ukuthi isebenzisa umsebenzi we-phagocytic wamaseli egazi ajikelezayo ku-hemolymph, egubha ama-microorganisms (kanye nama-genome awo). I-Phagocytosis ngumsebenzi oyinhloko wamaseli egazi kuma-bivalves [82]. Okokugcina, le ndlela isebenzisa amandla aphezulu okuhlunga ama-mussels (isilinganiso esingu-1.5 l/h samanzi olwandle) kanye nokujikeleza kwezinsuku ezimbili, okwandisa ukuxubana kwezingqimba ezahlukene zamanzi olwandle, okuvumela ukubanjwa kwe-heterologous eDNA. [83, 84]. Ngakho-ke, ukuhlaziywa kwe-ccfDNA ye-mussel kuyindlela ethakazelisayo uma kubhekwa imiphumela yokudla okunempilo, ezomnotho, kanye nemvelo ye-mussels. Ngokufana nokuhlaziywa kwe-LB eqoqwe kubantu, le ndlela iphinde ivule ithuba lokulinganisa izinguquko zezakhi zofuzo kanye ne-epigenetic ku-DNA yomnikazi ngokuphendula izinto zangaphandle. Isibonelo, ubuchwepheshe bokulandelana kwesizukulwane sesithathu bungacatshangelwa ukwenza ukuhlaziywa kwe-methylation ye-genome ku-ccfDNA yomdabu kusetshenziswa ukulandelelana kwe-nanopore. Le nqubo kufanele yenziwe lula iqiniso lokuthi ubude bezingcezu ze-ccfDNA ye-mussel buhambisana kahle namapulatifomu okulandelana afundwa isikhathi eside avumela ukuhlaziywa kwe-methylation ye-DNA ye-genome-wide kusuka ekulandeleni okukodwa ngaphandle kwesidingo sokuguqulwa kwamakhemikhali.85,86] Lokhu kungenzeka okuthakazelisayo, njengoba kuboniswe ukuthi amaphethini e-methylation ye-DNA abonisa impendulo ekucindezelekeni kwemvelo futhi aqhubeka ezizukulwaneni eziningi. Ngakho-ke, inganikeza ukuqonda okubalulekile ngezindlela eziyisisekelo ezilawula impendulo ngemva kokuchayeka ekushintsheni kwesimo sezulu noma ukungcola [87]. Kodwa-ke, ukusetshenziswa kwe-LB akunamingcele. Akudingeki ukusho, lokhu kudinga ukuba khona kwezinhlobo zezinkomba endaweni ezungezile. Njengoba kushiwo ngenhla, ukusebenzisa i-LB ukuhlola ukuhlukahluka kwezinto eziphilayo endaweni ethile kudinga futhi ipayipi eliqinile le-bioinformatics elicabangela ukuba khona kwezingcezu ze-DNA ezivela emthonjeni. Enye inkinga enkulu ukutholakala kwama-genome okubhekisela ezilwaneni zasolwandle. Kuthenjwa ukuthi izinhlelo ezifana ne-Marine Mammal Genomes Project kanye nephrojekthi ye-Fish10k esanda kusungulwa [88] kuzokwenza kube lula ukuhlaziywa okunjalo esikhathini esizayo. Ukusetshenziswa komqondo we-LB ezintweni eziphilayo ezihlunga izihlungi zasolwandle nakho kuhambisana nentuthuko yakamuva kwezobuchwepheshe bokulandelana, okwenza kube kuhle kakhulu ekuthuthukisweni kwama-biomarker amaningi ukuze kuhlinzekwe ngolwazi olubalulekile mayelana nempilo yezindawo zokuhlala zasolwandle ekuphenduleni ukucindezeleka kwemvelo.
Idatha yokulandelana kwezakhi zofuzo ifakwe ku-NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 ngaphansi kwe-Bioprojects SRR8924808.
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