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I-Liquid biopsy (LB) yingcamango efumana udumo ngokukhawuleza kwicandelo lezonyango. Le ngcamango isekelwe kakhulu ekufumaneni iziqwenga ze-DNA engaphandle kweseli (ccfDNA) ejikelezayo, ezikhutshwa kakhulu njengeziqwenga ezincinci emva kokufa kweeseli kwizicubu ezahlukeneyo. Inxalenye encinci yezi ziqwenga ivela kwizicubu zangaphandle (zangaphandle) okanye izinto eziphilayo. Kumsebenzi wangoku, sisebenzise le ngcamango kwi-mussels, uhlobo olulindi olwaziwa ngokuba namandla aphezulu okuhluza amanzi olwandle. Sisebenzisa amandla e-mussels ukuba asebenze njengezihluzo zendalo ukubamba iziqwenga ze-DNA yokusingqongileyo kwimithombo eyahlukeneyo ukubonelela ngolwazi malunga nokwahlukana kwezinto eziphilayo kwiindawo zolwandle. Iziphumo zethu zibonisa ukuba i-hemolymph ye-mussel iqulethe iziqwenga ze-DNA ezahluka kakhulu ngobukhulu, ukusuka kwi-1 ukuya kwi-5 kb. Ukulandelelana kwezibhamu kubonise ukuba inani elikhulu leziqwenga ze-DNA zivela kwiintsholongwane zangaphandle. Phakathi kwazo, sifumene iziqwenga ze-DNA ezivela kwiibhaktheriya, i-archaea, kunye neentsholongwane, kubandakanya iintsholongwane ezaziwa ngokusulela iintlobo ngeentlobo zezinto ezihlala zifumaneka kwiindawo zolwandle zonxweme. Ukuqukumbela, uphando lwethu lubonisa ukuba ingcamango ye-LB esetyenziswa kwiimbaza imele umthombo wolwazi otyebileyo kodwa ongekafunyanwa malunga nokwahlukahluka kweentsholongwane kwiindawo eziphilayo zaselunxwemeni lolwandle.
Impembelelo yotshintsho lwemozulu (CC) kwizinto eziphilayo zaselwandle yindawo ekhula ngokukhawuleza yophando. Ukufudumala kwehlabathi akupheleli nje ekubangeleni uxinzelelo olubalulekileyo lwempilo, kodwa kukwatyhala imida yokuzinza kobushushu bezinto eziphilayo zaselwandle, okuchaphazela indawo yokuhlala yeentlobo ngeentlobo, okubangela ukuba zifune iimeko ezilungileyo ngakumbi [1, 2]. Ukongeza ekuchaphazeleni izinto eziphilayo ze-metazoans, i-CC iphazamisa ulungelelwaniso olubuthathaka lokunxibelelana kwe-host-microbial. Le dysbacteriosis ye-microbial ibeka umngcipheko omkhulu kwiindawo zaselwandle njengoko yenza izinto zaselwandle zibe sesichengeni sokufumana izifo ezisulelayo [3, 4]. Kukholelwa ukuba i-SS idlala indima ebalulekileyo ekufeni kwabantu abaninzi, nto leyo eyingxaki enkulu kulawulo lweendawo zaselwandle zehlabathi [5, 6]. Le yingxaki ebalulekileyo xa kujongwa impembelelo yezoqoqosho, yendalo kunye neyondlo yeentlobo ezininzi zaselwandle. Oku kuyinyani ngakumbi kwi-bivalves ezihlala kwimimandla ye-polar, apho iziphumo ze-CK zikhawuleza kwaye zinzima [6, 7]. Enyanisweni, ii-bivalves ezifana ne-Mytilus spp. zisetyenziswa kakhulu ukujonga imiphumo ye-CC kwiindawo zaselwandle. Akumangalisi ukuba inani elikhulu lee-biomarker liye laphuhliswa ukuze lijonge impilo yazo, kusetyenziswa indlela enamanqanaba amabini ebandakanya ii-biomarker ezisebenzayo ezisekelwe kumsebenzi we-enzymatic okanye imisebenzi yeselula efana nokusebenza kweseli kunye nomsebenzi we-phagocytic [8]. Ezi ndlela ziquka nokulinganisa uxinzelelo lweempawu ezithile zoxinzelelo eziqokelelana kwizicubu ezithambileyo emva kokufunxwa kwamanzi amaninzi olwandle. Nangona kunjalo, amandla aphezulu okuhluza kunye nenkqubo yokujikeleza kwegazi evulekileyo kancinci inika ithuba lokuphuhlisa ii-biomarker ezintsha ze-hemolymph kusetyenziswa ingcamango ye-liquid biopsy (LB), indlela elula nengenamdla kangako yokulawula izigulane. [9, 10]. Nangona iintlobo ezininzi zeemolekyuli ezijikelezayo zinokufumaneka kwi-LB yomntu, le ngcamango isekelwe kakhulu kuhlalutyo lokulandelelana kwe-DNA yeziqwenga ze-DNA ezijikelezayo (ccfDNA) kwi-plasma. Enyanisweni, ubukho be-DNA ejikelezayo kwi-plasma yomntu buyaziwa ukususela phakathi kwenkulungwane yama-20 [11], kodwa kukwiminyaka yakutshanje kuphela apho ukufika kweendlela zokulandelelana eziphezulu kukhokelele ekuxilongweni kweklinikhi okusekelwe kwi-ccfDNA. Ubukho bezi ziqwenga zeDNA ezijikelezayo bubangelwa kukukhululwa kwe-genomic DNA (inyukliya kunye ne-mitochondrial) emva kokufa kweseli. Kubantu abaphilileyo, uxinaniso lwe-ccfDNA ludla ngokuba luphantsi (<10 ng/mL) kodwa lunokwandiswa ngamaxesha ama-5–10 kwizigulana ezineengxaki ezahlukeneyo okanye eziphantsi koxinzelelo, nto leyo ebangela umonakalo kwizicubu. Kubantu abaphilileyo, uxinaniso lwe-ccfDNA ludla ngokuba luphantsi (<10 ng/mL) kodwa lunokwandiswa ngamaxesha ama-5–10 kwizigulana ezineengxaki ezahlukeneyo okanye eziphantsi koxinzelelo, nto leyo ebangela umonakalo kwizicubu. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 подвергающихся стрессу, приводящему к повреждению тканей. Kubantu abaphilileyo, uxinaniso lwe-cccDNA ludla ngokuba phantsi (<10 ng/mL), kodwa lunokunyuka ngamaxesha ama-5–10 kwizigulane ezineengxaki ezahlukeneyo okanye eziphantsi koxinzelelo olukhokelela ekonakaleni 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。20 5-10 倍 , 从而 组织。。。Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут быть увеличены в 5-10 стрессом, что приводит к повреждению тканей. Amanqanaba e-ccfDNA adla ngokuba phantsi (<10 ng/ml) kubantu abaphilileyo, kodwa anokwandiswa ngokuphindwe ka-5-10 kwizigulane ezineengxaki ezahlukeneyo okanye uxinzelelo, nto leyo ebangela umonakalo kwizicubu.Ubungakanani beziqwenga ze-ccfDNA buyahluka kakhulu, kodwa ngokuqhelekileyo buqala kwi-150 ukuya kwi-200 bp. [12]. Uhlalutyo lwe-ccfDNA ezizivela ngokwazo, oko kukuthi, i-ccfDNA evela kwiiseli eziqhelekileyo okanye eziguquliweyo, lunokusetyenziselwa ukubona utshintsho lwe-genetic kunye ne-epigenetic olukhoyo kwi-genome yenyukliya kunye/okanye ye-mitochondrial, ngaloo ndlela lunceda oogqirha ukuba bakhethe unyango oluthile olujoliswe kwi-molecular [13]. Nangona kunjalo, i-ccfDNA inokufumaneka kwimithombo yangaphandle efana ne-ccfDNA evela kwiiseli ze-fetal ngexesha lokukhulelwa okanye kwizitho ezifakelwe kwenye indawo [14,15,16,17]. I-ccfDNA ikwangumthombo obalulekileyo wolwazi lokufumanisa ubukho be-nucleic acids ye-arhente esulelayo (yangaphandle), evumela ukufunyanwa okungangenisi kwizifo ezisasazekayo ezingachongwanga ziinkcubeko zegazi, kuthintelwe i-biopsy ehlaselayo yezicubu ezisulelekileyo [18]. Izifundo zamva nje zibonise ukuba igazi lomntu liqulathe umthombo ocebileyo wolwazi onokusetyenziselwa ukuchonga iintsholongwane kunye neentsholongwane, kwaye malunga ne-1% ye-ccfDNA efumaneka kwi-plasma yomntu ivela kwamanye amazwe [19]. Ezi zifundo zibonisa ukuba ubuninzi bezinto eziphilayo ezijikeleza i-microbiome yesidalwa bunokuvavanywa kusetyenziswa uhlalutyo lwe-ccfDNA. Nangona kunjalo, kude kube kutshanje, le ngcamango yayisetyenziswa kuphela ebantwini, kwaye, kancinci, nakwezinye izilwanyana ezinethambo lomqolo [20, 21].
Kwiphepha langoku, sisebenzisa amandla e-LB ukuhlalutya i-ccfDNA ye-Aulacomya atra, uhlobo oluhlala emazantsi olufumaneka kwiziqithi zaseKerguelen ezingaphantsi kwe-antarctic, iqela leziqithi eziphezu kwethafa elikhulu elakhiwa kwiminyaka eyi-35 yezigidi eyadlulayo. Ukuqhuma kwentaba-mlilo. Sisebenzisa inkqubo yovavanyo lwe-in vitro, sifumanise ukuba iziqwenga ze-DNA emanzini olwandle zithathwa ngokukhawuleza zii-mussels kwaye zingene kwindawo ye-hemolymph. Ukulandelelana kwezibhamu kubonise ukuba i-mussel hemolymph ccfDNA iqulethe iziqwenga ze-DNA ezizezazo nezingezizo ezazo, kubandakanya iibhaktheriya ezihambelanayo kunye neziqwenga ze-DNA ezivela kwii-biomes eziqhelekileyo kwiindawo ezibandayo zentaba-mlilo. I-Hemolymph ccfDNA ikwaqulathe ulandelelwano lweentsholongwane oluvela kwiintsholongwane ezineendawo ezahlukeneyo zokusingatha. Sikwafumene iziqwenga ze-DNA ezivela kwizilwanyana ezininzi ezifana neentlanzi ezinamathambo, ii-anemones zolwandle, i-algae kunye nezinambuzane. Ukuqukumbela, uphando lwethu lubonisa ukuba ingcamango ye-LB ingasetyenziswa ngempumelelo kwizilwanyana ezingenamqolo zaselwandle ukuvelisa i-repertoire etyebileyo ye-genomic kwiindawo ezihlala emanzini.
Iintaka ezindala (55-70 mm ubude) iMytilus platensis (M. platensis) kunye neAulacomya atra (A. atra) ziqokelelwe kunxweme olunamatye oluphakathi kolwandle lwasePort-au-France (049°21.235 S, 070°13.490 E .). Iziqithi zaseKerguelen ngoDisemba 2018. Ezinye iimbaza ezindala eziluhlaza okwesibhakabhaka (Mytilus spp.) zifunyenwe kumthengisi wezorhwebo (PEI Mussel King Inc., ePrince Edward Island, eKhanada) zaza zafakwa kwitanki enomoya olawulwayo (4°C) equlathe i-10–20 L ye-32‰ yolwelo lokwenziwa. (ityuwa yolwandle yokwenziwa iReef Crystal, e-Instant Ocean, eVirginia, e-USA). Kwilingo ngalinye, ubude kunye nobunzima beembaza nganye nganye kulinganiswa.
Iprotokholi yokufikelela evulekileyo yasimahla yale nkqubo ifumaneka kwi-intanethi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). Ngamafutshane, i-LB hemolymph yaqokelelwa kwimisipha ye-abductor njengoko kuchaziwe [22]. I-hemolymph yacaciswa nge-centrifugation kwi-1200×g imizuzu emi-3, i-supernatant yaqandiswa (-20°C) ide isetyenziswe. Ukuze kwahlulwe kwaye kucocwe i-cfDNA, iisampulu (1.5-2.0 ml) zanyibilikiswa zaza zacutshungulwa kusetyenziswa i-NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ngokwemiyalelo yomenzi. I-ccfDNA igcinwe kwi--80°C ide ihlalutywe ngakumbi. Kwezinye iimvavanyo, i-ccfDNA yahlulwa yaza yacocwa kusetyenziswa i-QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada). I-DNA ecociweyo yalinganiswa kusetyenziswa uvavanyo oluqhelekileyo lwePicoGreen. Ukusasazwa kweqhekeza le-ccfDNA ehlukanisiweyo kuhlalutywe nge-capillary electrophoresis kusetyenziswa i-Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) kusetyenziswa i-High Sensitivity DNA Kit. Uvavanyo lwenziwe kusetyenziswa i-1 µl yesampulu ye-ccfDNA ngokwemiyalelo yomenzi.
Ukulandelelanisa iziqwenga ze-hemolymph ccfDNA, iGénome Québec (eMontreal, eQuebec, eKhanada) ilungiselele iilayibrari ze-shotgun isebenzisa i-Illumina DNA Mix kit ye-Illumina MiSeq PE75 kit. Kusetyenziswe i-adapter eqhelekileyo (i-BioO). Iifayile zedatha eluhlaza ziyafumaneka kwi-NCBI Sequence Read Archive (SRR8924808 kunye ne-SRR8924809). Umgangatho wokufunda osisiseko uvavanywe kusetyenziswa i-FastQC [23]. I-Trimmomatic [24] isetyenziselwe ukunqumla ii-adapters kunye nokufundwa okungalunganga. Ukufundwa kwe-Shotgun okuneephelo ezidibeneyo kudityaniswe ne-FLASH kwii-reads ezinde ezine-overlap encinci ye-20 bp ukuthintela ukungafani [25]. Ukufundwa okudityanisiweyo kwabhalwa nge-BLASTN kusetyenziswa isiseko sedatha se-NCBI Taxonomy se-bivalve (ixabiso le-e < 1e−3 kunye ne-90% homology), kwaye ukufihla ulandelelwano oluphantsi kobunzima kwenziwa kusetyenziswa i-DUST [26]. Ukufundwa okudityanisiweyo kwabhalwa nge-BLASTN kusetyenziswa isiseko sedatha se-NCBI Taxonomy se-bivalve (ixabiso le-e < 1e−3 kunye ne-90% homology), kwaye ukufihla ulandelelwano oluphantsi kobunzima kwenziwa kusetyenziswa i-DUST [26]. Uluhlu lwenkcazelo BLASTN базы данных таксономии двустворчатых моллюсков% NCBI гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием Uthuli [26]. Ukufundwa okudibeneyo kwabhalwa nge-BLASTN kusetyenziswa i-database ye-NCBI bivalve taxonomy (ixabiso le-e < 1e-3 kunye ne-90% homology), kwaye kwenziwa i-sequence masking enobunzima obuphantsi kusetyenziswa 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 注释 合并读数 , 并 使用公物 [26]的。。。。Объединенные чтения были аннотированы с помощью BLASTN kunye ne-iOSP таксономической базы даных двустворчатых моллюс10-NCBI (9) гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием Uthuli [26]. Ukufundwa okudibeneyo kwabhalwa nge-BLASTN kusetyenziswa i-database ye-NCBI bivalve taxonomic (ixabiso le-e <1e-3 kunye ne-90% homology), kwaye kwenziwa i-sequence masking enobunzima obuphantsi kusetyenziswa i-DUST [26].IiReads zahlulwe zangamaqela amabini: ezinxulumene ne-bivalve sequences (apha zibizwa ngokuba zii-self-reads) kunye ne-non-self-reads (ezingezizo ii-self-reads). Amaqela amabini ahlanganiswe ngokwahlukeneyo kusetyenziswa i-MEGAHIT ukuvelisa ii-contigs [27]. Okwangoku, ukusasazwa kwe-taxonomic kwe-alien microbiome reads kwahlulwa kusetyenziswa iKraken2 [28] kwaye kwamelwa ngokwemizobo yitshathi yephayi yeKrona kwiGalaxy [29, 30]. Ii-kmers ezilungileyo zafunyaniswa ukuba zii-kmers-59 ezivela kuvavanyo lwethu lokuqala. Ii-self contigs zachongwa ngokulungelelaniswa ne-BLASTN (i-bivalve NCBI database, ixabiso le-e < 1e−10 kunye ne-60% homology) kwingcaciso yokugqibela. Ii-self contigs zachongwa ngokulungelelaniswa ne-BLASTN (i-bivalve NCBI database, ixabiso le-e < 1e−10 kunye ne-60% homology) kwingcaciso yokugqibela. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, значени10 e) для окончательной аннотации. Emva koko ii-self-contigs zachongwa ngokuthelekisa ne-BLASTN (i-NCBI bivalve database, ixabiso le-e <1e-10 kunye ne-60% homology) kwingcaciso yokugqibela.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60%同源性）对齐來识别自身重叠群以进行最终注释.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% Затем были идентифицированы собственные контиги для окончательной аннотации путем сопоставления с BLASTN (база данных NCBI ND значение e <1e-10 и гомология 60%). Emva koko kwachongwa izinto ezizimeleyo ukuze zichazwe ngokuhambelana neBLASTN (isiseko sedatha se-NCBI bivalve, ixabiso le-e <1e-10 kunye ne-60% homology). Ngaxeshanye, ii-nonself group contigs zichazwe nge-BLASTN (isiseko sedatha se-nt NCBI, ixabiso le-e < 1e−10 kunye ne-60% homology). Ngaxeshanye, ii-nonself group contigs zichazwe nge-BLASTN (isiseko sedatha se-nt NCBI, ixabiso le-e < 1e−10 kunye ne-60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 ne гомология 60%). Ngaxeshanye, amaqela angaphandle adityaniswe neBLASTN (isiseko sedatha se-NT NCBI, ixabiso le-e <1e-10 kunye ne-60% homology).平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。 Параллельно контиги, не относящиеся к собственной группе, были аннотированы с помощью BLASTN (база данных nt NCBI, знагогние 0 <6 e-%). Ngaxeshanye, amaqela angengawo azimeleyo achazwe nge-BLASTN (isiseko sedatha se-nt NCBI, ixabiso le-e <1e-10 kunye ne-60% homology). I-BLASTX ikwaqhutywe nakwii-nonself contigs kusetyenziswa ii-database ze-nr kunye ne-RefSeq protein NCBI (ixabiso le-e < 1e−10 kunye ne-60% homology). I-BLASTX ikwaqhutywe nakwii-nonself contigs kusetyenziswa ii-database ze-nr kunye ne-RefSeq protein NCBI (ixabiso le-e < 1e−10 kunye ne-60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 6%). I-BLASTX yenziwe nakwi-non-self contigs kusetyenziswa i-nr kunye ne-RefSeq NCBI protein databases (ixabiso le-e < 1e-10 kunye 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）。 BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 ne 60%). I-BLASTX yenziwe nakwi-non-self contigs kusetyenziswa i-nr kunye ne-RefSeq NCBI protein databases (ixabiso le-e <1e-10 kunye ne-60% homology).Ii-BLASTN kunye ne-BLASTX pools ze-non-self-contigs zimele ii-contigs zokugqibela (jonga ifayile eyongezelelweyo).
Iiprimers ezisetyenziselwa i-PCR zidweliswe kwiTheyibhile S1. I-Taq DNA polymerase (i-Bio Basic Canada, i-Markham, i-ON) yasetyenziswa ukukhulisa ii-genes ezijoliswe kwi-ccfDNA. Ezi meko zilandelayo zokusabela zisetyenzisiwe: i-denaturation kwi-95°C imizuzu emi-3, i-95°C umzuzu o-1, i-set annealing temperature umzuzu o-1, i-longation kwi-72°C umzuzu o-1, imijikelo engama-35, kwaye ekugqibeleni i-72°C ngaphakathi kwemizuzu eli-10. . Iimveliso ze-PCR zahlulwe yi-electrophoresis kwiijeli ze-agarose (1.5%) eziqulethe i-SYBRTM Safe DNA Gel Stain (i-Invitrogen, i-Burlington, i-ON, i-Canada) kwi-95 V.
IiMussels (Mytilus spp.) zaqhelana namanzi olwandle aneoksijini angama-500 ml (32 PSU) kangangeeyure ezingama-24 kwi-4°C. I-Plasmid DNA equlethe i-insert ebhala i-human galectin-7 cDNA sequence (inombolo yokungena kwe-NCBI L07769) yongezwa kwi-vial kuxinzelelo lokugqibela lwe-190 μg/μl. IiMussels ezifakwe phantsi kweemeko ezifanayo ngaphandle kokongezwa kwe-DNA zazilawula. Itanki yokulawula yesithathu yayine-DNA engenazo iiMussels. Ukujonga umgangatho we-DNA emanzini olwandle, iisampulu zamanzi olwandle (20 μl; ukuphindaphindwa kathathu) zathathwa kwi-tank nganye ngexesha elichaziweyo. Ukuze kulandelelwe i-plasmid DNA, iiMussels ze-LB zavunwa ngamaxesha achaziweyo kwaye zahlalutywa yi-qPCR kunye ne-ddPCR. Ngenxa yomxholo wetyuwa ephezulu emanzini olwandle, ii-aliquots zaxutywa emanzini asemgangathweni we-PCR (1:10) ngaphambi kokuba kwenziwe zonke iimvavanyo ze-PCR.
I-PCR yedroplet yedijithali (ddPCR) yenziwe kusetyenziswa iprotocol yeBioRad QX200 (eMississippi, eOntario, eKhanada). Sebenzisa iprofayili yobushushu ukuze ufumane ubushushu obufanelekileyo (Itheyibhile S1). Amathontsi aveliswa kusetyenziswa ijenereyitha yedrophu ye-QX200 (iBioRad). I-ddPCR yenziwe ngolu hlobo lulandelayo: 95°C imizuzu emi-5, imijikelo engama-50 yama-95°C imizuzwana engama-30 kunye nobushushu obunikiweyo bokutsalela ubushushu imizuzwana eli-1 kunye nama-72°C imizuzwana engama-30, i-4°C imizuzu emi-5 kunye nama-90°C kwimizuzu emi-5. Inani lamathontsi kunye neempendulo ezilungileyo (inani leekopi/µl) zilinganiswe kusetyenziswa i-QX200 drop reader (iBioRad). Iisampulu ezinamathontsi angaphantsi kwe-10,000 zaliwe. Ulawulo lwepateni aluzange lwenziwe ngalo lonke ixesha i-ddPCR isebenza.
I-qPCR yenziwe kusetyenziswa iRotor-Gene® 3000 (Corbett Research, eSydney, eOstreliya) kunye neeprimer ezithile ze-LGALS7. Zonke ii-PCR ezilinganiselweyo zenziwe kwi-20 µl kusetyenziswa iQuantiFast SYBR Green PCR Kit (QIAGEN). I-qPCR yaqalwa nge-15 min incubation kwi-95°C yalandelwa yi-40 cycles kwi-95°C imizuzwana eli-10 kunye ne-60°C imizuzwana engama-60 ngokuqokelelwa kwedatha enye. Ii-Melting curves zenziwe kusetyenziswa imilinganiselo elandelelanayo kwi-95°C imizuzwana emi-5, i-65°C imizuzwana engama-60, kunye ne-97°C ekupheleni kwe-qPCR. I-qPCR nganye yenziwe kwi-triplicate, ngaphandle kweesampulu zolawulo.
Ekubeni ii-mussels zaziwa ngesantya sazo esiphezulu sokuhluza, siqale saphanda ukuba zingazihluza na kwaye zigcine iinxalenye ze-DNA ezikhoyo emanzini olwandle. Sikwanomdla wokuba ezi ziqwenga ziqokelelana na kwinkqubo yazo ye-lymphatic evulelekileyo. Sisombulule le ngxaki ngokuvavanya ngokulandela isiphelo seziqwenga ze-DNA ezinyibilikayo ezongezwe kwiitanki zee-mussel eziluhlaza okwesibhakabhaka. Ukuze kube lula ukulandelela iinxalenye ze-DNA, sisebenzise i-DNA ye-plasmid yangaphandle (engeyiyo eyakho) equlethe i-human galectin-7 gene. I-ddPCR ilandelela iinxalenye ze-plasmid DNA emanzini olwandle nakwii-mussels. Iziphumo zethu zibonisa ukuba ukuba inani leziqwenga ze-DNA emanzini olwandle lihlala lingaguquki ngokuhamba kwexesha (ukuya kuthi ga kwiintsuku ezi-7) xa kungekho zi-mussels, ngoko xa kukho ii-mussels eli nqanaba liphantse lanyamalala ngokupheleleyo kwiiyure ezi-8 (Umzobo 1a,b). Iziqwenga ze-DNA yangaphandle zafunyanwa ngokulula kwimizuzu eli-15 kwi-intravalvular fluid kunye ne-hemolymph (Umzobo 1c). Ezi ziqwenga zisenokufunyanwa ukuya kuthi ga kwiiyure ezi-4 emva kokuvezwa. Lo msebenzi wokucoca malunga neziqwenga ze-DNA ufana nomsebenzi wokucoca iintsholongwane kunye ne-algae [31]. Ezi ziphumo zibonisa ukuba iimbaza zinokuhluza kwaye ziqokelele i-DNA yangaphandle kwiindawo zazo zolwelo.
Uxinzelelo oluhambelanayo lwe-plasmid DNA emanzini olwandle xa kukho (A) okanye kungekho (B) kweembaza, olulinganiswa yi-ddPCR. Kwi-A, iziphumo zibonakaliswa njengeepesenti, imida yeebhokisi imele i-75th kunye ne-25th percentile. I-logarithmic curve efakiweyo iboniswe ngombala obomvu, kwaye indawo enombala ongwevu imele i-95% confidence interval. Kwi-B, umgca obomvu umele i-mean kwaye umgca oluhlaza okwesibhakabhaka umele i-95% confidence interval yoxinzelelo. C Ukuqokelelwa kwe-plasmid DNA kwi-hemolymph kunye ne-valvular fluid yeembaza ngamaxesha ahlukeneyo emva kokongezwa kwe-plasmid DNA. Iziphumo ziboniswa njengeekopi ezipheleleyo ezifunyenweyo/mL (±SE).
Okulandelayo, siphande ngemvelaphi ye-ccfDNA kwiimbaza eziqokelelwe kwiibhedi zeembaza kwiZiqithi zaseKerguelen, iqela leziqithi ezikude ezinempembelelo encinci ebantwini. Ngenxa yesi sizathu, i-cccDNA evela kwiimbaza zeembaza yahlulwa yaza yacocwa ngeendlela ezisetyenziswa rhoqo ukucoca i-cccDNA yomntu [32, 33]. Sifumanise ukuba umyinge woxinzelelo lwe-ccfDNA ye-hemolymph kwiimbaza ukwi-micrograms ephantsi nge-ml ye-hemolymph (jonga iTheyibhile S2, Ulwazi olongezelelweyo). Olu luhlu loxinzelelo lukhulu kakhulu kunakwibantu abasempilweni (ii-nanograms eziphantsi nge-milliliter), kodwa kwiimeko ezingaqhelekanga, kwizigulane ezinomhlaza, inqanaba le-ccfDNA linokufikelela kwii-micrograms ezininzi nge-milliliter [34, 35]. Uhlalutyo lokusasazwa kobungakanani be-ccfDNA ye-hemolymph lubonise ukuba ezi ziqwenga zahluka kakhulu ngobukhulu, ukusuka kwi-1000 bp ukuya kwi-1000 bp. ukuya kwi-5000 bp (Umzobo 2). Iziphumo ezifanayo zifunyenwe kusetyenziswa i-silica-based QIAamp Investigator Kit, indlela esetyenziswa kakhulu kwisayensi ye-forensic ukwahlula nokucoca i-genomic DNA ngokukhawuleza kwiisampuli ze-DNA ezixineneyo eziphantsi, kuquka i-ccfDNA [36].
I-electrophoregram emele i-ccfDNA ye-hemolymph yeembaza. Ikhutshwe nge-NucleoSnap Plasma Kit (phezulu) kunye ne-QIAamp DNA Investigator Kit. I-B Violin plot ebonisa ukusasazwa kwe-hemolymph ccfDNA concentrations (±SE) kwiimbaza. Imigca emnyama nebomvu imele i-median kunye ne-first and third quartiles, ngokulandelelana.
Malunga ne-1% ye-ccfDNA ebantwini nakwii-primates inomthombo wangaphandle [21, 37]. Ngenxa yenkqubo yokujikeleza kwegazi evulekileyo kancinci yee-bivalves, amanzi olwandle atyebileyo kwiintsholongwane, kunye nokusasazwa kobungakanani be-ccfDNA yee-mussel, sicinge ukuba i-mussel hemolymph ccfDNA inokuba nedama elinobutyebi nolwahlukeneyo lwe-DNA yeentsholongwane. Ukuvavanya le ngcamango, silandelelanise i-ccfDNA ye-hemolymph hemolymph kwiisampulu ze-atra zase-Aulacomya eziqokelelwe kwiZiqithi zaseKerguelen, savelisa ngaphezulu kwe-10 yezigidi zeerandi zokufunda, i-97.6% yazo iphumelele ulawulo lomgangatho. Ukufundwa emva koko kwahlulwa ngokwemithombo yobuqu nengeyoyayo kusetyenziswa i-BLASTN kunye ne-NCBI bivalve databases (Umzobo S1, Ulwazi olongezelelweyo).
Kubantu, zombini i-DNA yenyukliya kunye ne-mitochondrial zinokukhutshwa zingene egazini [38]. Nangona kunjalo, kolu phononongo lwangoku, bekungenakwenzeka ukuchaza ngokweenkcukacha i-DNA ye-nuclear genomic yeembaza, kuba i-genome ye-A. atra ayilandelelwanga okanye ayichazwanga. Nangona kunjalo, sikwazile ukuchonga inani leziqwenga ze-ccfDNA zemvelaphi yethu sisebenzisa ilayibrari ye-bivalve (Umzobo S2, Ulwazi olongezelelweyo). Sikwaqinisekisile ukubakho kweziqwenga ze-DNA zemvelaphi yethu ngokukhulisa i-PCR ngqo kwezo jini ze-A. atra ezilandelelwanisiweyo (Umzobo 3). Ngokufanayo, ekubeni i-genome ye-mitochondrial ye-A. atra ifumaneka kwiidathabheyisi zikawonke-wonke, umntu unokufumana ubungqina bokubakho kweziqwenga ze-ccfDNA ze-mitochondrial kwi-hemolymph ye-A. atra. Ubukho beziqwenga ze-DNA ze-mitochondrial buqinisekiswe yi-PCR amplification (Umzobo 3).
Iijini ezahlukeneyo ze-mitochondrial zazikho kwi-hemolymph ye-A. atra (amachaphaza abomvu – inombolo yesitokhwe: SRX5705969) kunye ne-M. platensis (amachaphaza aluhlaza – inombolo yesitokhwe: SRX5705968) eyandisiweyo yi-PCR. Umfanekiso uhlengahlengiswe kwiBreton et al., 2011 B Ukwandiswa kwe-hemolymph supernatant evela kwi-A. atra Igcinwe kwiphepha le-FTA. Sebenzisa i-punch ye-3 mm ukongeza ngqo kwityhubhu ye-PCR equlethe umxube we-PCR.
Ngenxa yobuninzi bezinto ezincinci ezikumanzi olwandle, ekuqaleni sigxile ekuchazeni ulandelelwano lwe-DNA ye-microbial kwi-hemolymph. Ukuze senze oku, sisebenzisa amaqhinga amabini ahlukeneyo. Icebo lokuqala lisebenzise iKraken2, inkqubo yokwahlulahlula ulandelelwano olusekwe kwi-algorithm enokuthi ichonge ulandelelwano lwe-microbial ngokuchanekileyo okufana ne-BLAST kunye nezinye izixhobo [28]. Izifundo ezingaphezu kwama-6719 zafunyaniswa ukuba zivela kwi-bacterial, ngelixa ezili-124 kunye ne-64 zazivela kwi-archaea kunye nee-virus, ngokulandelelana (Umzobo 4). Iziqwenga ze-DNA ze-bacterial ezininzi zaziyi-Firmicutes (46%), i-Proteobacteria (27%), kunye ne-Bacteroidetes (17%) (Umzobo 4a). Olu sasazo luhambelana nezifundo zangaphambili ze-marine blue mussel microbiome [39, 40]. I-Gammaproteobacteria yayiludidi oluphambili lwe-Proteobacteria (44%), kuquka ii-Vibrionales ezininzi (Umzobo 4b). Indlela ye-ddPCR iqinisekisile ukubakho kweziqwenga ze-Vibrio DNA kwi-ccfDNA ye-A. atra hemolymph (Umzobo 4c) [41]. Ukuze kufunyanwe ulwazi oluthe kratya malunga nemvelaphi yebhaktiriya ye-ccfDNA, kuthathwe indlela eyongezelelweyo (Umzobo S2, Ulwazi olongezelelweyo). Kule meko, ii-reads ezifakwe ngaphezulu zihlanganiswe njengee-reads ze-paired-end kwaye zahlulwa njenge-self (bivalves) okanye i-nonself origin kusetyenziswa i-BLASTN kunye nexabiso le-e le-1e−3 kunye ne-cutoff ene-homology engaphezulu kwe-90%. Kule meko, ii-reads ezifakwe ngaphezulu zihlanganiswe njengee-reads ze-paired-end kwaye zahlulwa njenge-self (bivalves) okanye i-nonself origin kusetyenziswa i-BLASTN kunye nexabiso le-e le-1e−3 kunye ne-cutoff ene-homology engaphezulu kwe-90%. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственстворые (двузе) чужие по происхождению с использованием BLASTN kunye ne-значения e 1e-3 kwaye отсечения с гомологией> 90%. Kule meko, ukufundwa okugqithanayo kuqokelelwe njengokufundwa okunesiphelo esidibeneyo kwaye kwahlelwa njengokwasekuqaleni (i-bivalve) okanye okungeyonyani kusetyenziswa ixabiso le-BLASTN kunye ne-e le-1e-3 kwaye kwanqunyulwa nge-homology engaphezulu kwe-90%.在這种情况下，重叠的读数组装為配对末端話数，并使用BLASTN 和1e-3的值和>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 話数 , 使用 使用 1e-3 的值% ne> 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 (двустворье) несобственные по происхождению с использованием значений e BLASTN kunye ne-1e-3 kunye ne-90%. Kule meko, ukufundwa okugqithanayo kuqokelelwe njengee-reads ezipheleliswe ngababini kwaye kwahlulwa njengee-own (bivalves) okanye ezingezizo ezasekuqaleni kusetyenziswa amaxabiso e-e BLASTN kunye ne-1e-3 kunye nomda we-homology >90%.Ekubeni i-genome ye-A. atra ingekahlelwanga, sisebenzise icebo lokuhlanganisa i-de novo le-MEGAHIT Next Generation Sequencing (NGS) assembler. Ii-contigs ezili-147,188 zichongiwe njengee-bivalves ezixhomekeke kuzo. Ezi contigs zaqhuma nge-e-values ​​​​ze-1e-10 kusetyenziswa i-BLASTN kunye ne-BLASTX. Eli cebo lisivumele ukuba sichonge iziqwenga ezingama-482 ezingezizo ze-bivalve ezikhoyo kwi-A. atra ccfDNA. Ngaphezulu kwesiqingatha (57%) sezi ziqwenga ze-DNA zifunyenwe kwiibhaktheriya, ikakhulu kwii-gill symbionts, kubandakanya ii-sulfotrophic symbionts, nakwi-gill symbionts Solemya velum (Umzobo 5).
Ubuninzi obuthelekisekayo kwinqanaba lohlobo. B Ukwahluka kweentsholongwane ze-phyla ezimbini eziphambili (iiFirmicutes kunye neeProteobacteria). Ukwandiswa okubalulekileyo kwe-ddPCR C Vibrio spp. A. Iziqwenga ze-16S rRNA gene (eluhlaza okwesibhakabhaka) kwii-hemolymph ezintathu ze-atra.
Kuhlalutywe ii-contigs ezingama-482 zizonke. Iprofayili ngokubanzi yokusasazwa kwe-taxonomic kwee-metagenomic contig annotations (ii-prokaryotes kunye nee-eukaryotes). B Ukusasazwa okuneenkcukacha kweziqwenga ze-DNA yebhaktheriya ezichongiweyo yi-BLASTN kunye ne-BLASTX.
Uhlalutyo lweKraken2 lukwabonise ukuba i-ccfDNA ye-mussel yayineziqwenga ze-DNA zakudala, kuquka neziqwenga ze-DNA ze-Euryarchaeota (65%), iCrenarchaeota (24%), kunye neThaurmarcheota (11%) (Umzobo 6a). Ubukho beziqwenga ze-DNA ezithathwe kwi-Euryarchaeota kunye neCrenarchaeota, ezazifunyenwe ngaphambili kuluntu lwee-microbial lwee-mussels zaseCalifornia, akufuneki zimangalise [42]. Nangona i-Euryarchaeota idla ngokunxulunyaniswa neemeko eziqatha, ngoku kuyaqatshelwa ukuba zombini i-Euryarchaeota kunye neCrenarcheota ziphakathi kwee-prokaryotes eziqhelekileyo kwindawo ebangela i-cryogenic yolwandle [43, 44]. Ubukho bee-microorganisms ze-methanogenic kwii-mussels akumangalisi, ngenxa yeengxelo zakutshanje zokuvuza okukhulu kwe-methane ukusuka ekuvuza okusezantsi kwi-Kerguelen Plateau [45] kunye nokuveliswa kwe-methane ye-microbial okubonwe kunxweme lweZiqithi zaseKerguelen [46].
Ingqwalasela yethu yajika yaya kwiingxelo ezivela kwiintsholongwane zeDNA. Ngokolwazi lwethu, olu luphononongo lokuqala olungekho ngqo lomxholo wentsholongwane kwiimbaza. Njengoko bekulindelekile, sifumene iziqwenga zeDNA zebhakteriophages (Caudovirales) (Umzobo 6b). Nangona kunjalo, iDNA yentsholongwane eqhelekileyo ivela kwi-phylum ye-nucleocytoviruses, eyaziwa ngokuba yi-nuclear cytoplasmic large DNA virus (NCLDV), ene-genome enkulu kunazo zonke iintsholongwane. Ngaphakathi kwesi phylum, uninzi lwe-DNA sequences luvela kwiintsapho zeMimimidoviridae (58%) kunye nePoxviridae (21%), apho izidalwa zendalo ziquka izilwanyana ezinethambo lomqolo kunye ne-arthropods, ngelixa inxalenye encinci yale sequences yeDNA ikwi-algae eyaziwayo ye-virological. Ichaphazela i-algae yolwandle ye-eukaryotic. Ezi sequences zikwafunyenwe kwi-virus yePandora, intsholongwane enkulu enobukhulu be-genome obukhulu kunayo nayiphi na i-genera yentsholongwane eyaziwayo. Okunomdla kukuba, uluhlu lweendawo ezaziwa ngokuba zisulelekile yile ntsholongwane, njengoko kumiselwe yi-hemolymph ccfDNA sequencing, lwalukhulu kakhulu (Umfanekiso S3, Ulwazi olongezelelweyo). Ibandakanya iintsholongwane ezisulela izinambuzane ezifana neBaculoviridae kunye ne-Iridoviridae, kunye neentsholongwane ezisulela i-amoeba, i-algae kunye nezilwanyana ezinethambo lomqolo. Sikwafumene ulandelelwano oluhambelana ne-genome yePithovirus sibericum. Ii-Pitoviruses (ezaziwa ngokuba "zii-zombie viruses") zaqala ukuhlukaniswa kwi-permafrost eneminyaka engama-30,000 ubudala eSiberia [47]. Ke ngoko, iziphumo zethu zihambelana neengxelo zangaphambili ezibonisa ukuba ayizizo zonke iintlobo zanamhlanje zezi ntsholongwane eziphelileyo [48] kwaye ezi ntsholongwane zinokuba zikhona kwiindawo ezikude zolwandle.
Okokugqibela, sivavanye ukuze sibone ukuba singazifumana na iziqwenga zeDNA ezivela kwezinye izilwanyana ezineeseli ezininzi. I-BLASTN kunye ne-BLASTX zizonke ii-contigs zangaphandle ezingama-482 zichongiwe yi-BLASTN kunye ne-BLASTX kunye neelayibrari ze-nt, nr kunye ne-RefSeq (i-genomic kunye ne-protein). Iziphumo zethu zibonisa ukuba phakathi kweziqwenga zangaphandle ze-ccfDNA yezilwanyana ezineeseli ezininzi i-DNA yamathambo ethambo iphambili (Umzobo 5). Iziqwenga ze-DNA ezivela kwizinambuzane nakwezinye iintlobo nazo zifunyenwe. Inxalenye enkulu yeziqwenga ze-DNA ayikachongwa, mhlawumbi ngenxa yokungamelwa ngokwaneleyo kwenani elikhulu leentlobo zaselwandle kwiidathabheyisi ze-genomic xa kuthelekiswa neentlobo zasemhlabeni [49].
Kwiphepha langoku, sisebenzisa ingcamango ye-LB kwiimbaza, sithi ukulandelelana kwee-hemolymph ccfDNA shot kunokubonelela ngengqiqo malunga nokwakheka kweendawo eziphilayo zaselunxwemeni lolwandle. Ngokukodwa, sifumanise ukuba 1) i-hemolymph yeembaza iqulethe amazinga aphezulu (amanqanaba e-microgram) yeziqwenga ze-DNA ezinkulu (~1-5 kb) ezijikelezayo; 2) ezi ziqwenga ze-DNA zizimele kwaye azixhomekekanga 3) Phakathi kwemithombo yangaphandle yezi ziqwenga ze-DNA, sifumene i-DNA yebhaktiriya, i-archaeal kunye ne-viral, kunye ne-DNA yezinye izilwanyana ezininzi; 4) Ukuqokelelwa kwezi ziqwenga ze-ccfDNA zangaphandle kwi-hemolymph kwenzeka ngokukhawuleza kwaye kunegalelo kumsebenzi wokucoca wangaphakathi weembaza. Ukuqukumbela, uphando lwethu lubonisa ukuba ingcamango ye-LB, esele isetyenziswa kakhulu kwicandelo le-biomedicine, ifaka umthombo wolwazi ocebileyo kodwa ongakahlolwa onokusetyenziselwa ukuqonda ngcono ukusebenzisana phakathi kweentlobo ze-sentinel kunye nokusingqongileyo kwazo.
Ukongeza kwiiprimates, ukuhlukaniswa kwe-ccfDNA kuye kwaxelwa kwizilwanyana ezanyisayo, kuquka iimpuku, izinja, iikati, kunye namahashe [50, 51, 52]. Nangona kunjalo, ngokolwazi lwethu, uphando lwethu lolokuqala ukuxela ukufunyanwa kunye nokulandelelana kwe-ccfDNA kwiintlobo zaselwandle ezinenkqubo yokujikeleza kwegazi evulekileyo. Olu phawu lwe-anatomical kunye nokukwazi kokucoca ii-mussels, ubuncinane ngokuyinxenye, kunokuchaza iimpawu ezahlukeneyo zobukhulu beziqwenga ze-DNA ezijikelezayo xa kuthelekiswa nezinye iintlobo. Kubantu, uninzi lweziqwenga ze-DNA ezijikeleza egazini ziziqwenga ezincinci ezinobukhulu obuqala kwi-150 ukuya kwi-200 bp. kunye nencopho ephezulu ye-167 bp [34, 53]. Inxalenye encinci kodwa ebalulekileyo yeziqwenga ze-DNA iphakathi kwe-300 kunye ne-500 bp ngobukhulu, kwaye malunga ne-5% inde kune-900 bp. [54]. Isizathu solu sasazo lobungakanani kukuba umthombo ophambili we-ccfDNA kwi-plasma uvela ngenxa yokufa kweeseli, nokuba kungenxa yokufa kweeseli okanye ngenxa yokubola kweeseli ze-hematopoietic ezijikelezayo kubantu abaphilileyo okanye ngenxa yokuqhekeka kweeseli ze-tumor kwizigulane ezinomhlaza (ezaziwa ngokuba yi-circular tumor DNA). , ctDNA). Usasazo lobungakanani be-hemolymph ccfDNA esilufumene kwii-mussels lwaluqala kwi-1000 ukuya kwi-5000 bp, nto leyo ebonisa ukuba i-mussel ccfDNA inemvelaphi eyahlukileyo. Le yingcamango enengqondo, kuba ii-mussels zinenkqubo yemithambo yegazi evulekileyo kwaye zihlala kwiindawo zasemanzini zasemanzini eziqulathe ubuninzi be-microbial genomic DNA. Enyanisweni, uvavanyo lwethu lwelabhoratri olusebenzisa i-DNA yangaphandle lubonise ukuba ii-mussels ziqokelela iziqwenga ze-DNA emanzini olwandle, ubuncinci emva kweeyure ezimbalwa ziyonakaliswa emva kokuthathwa kweeseli kunye/okanye zikhutshwe kwaye/okanye zigcinwe kwimibutho eyahlukeneyo. Ngenxa yokunqaba kweeseli (zombini i-prokaryotic kunye ne-eukaryotic), ukusetyenziswa kweendawo ezingaphakathi kwevalvu kuya kunciphisa ubungakanani be-ccfDNA evela kwimithombo yazo kunye nemithombo yangaphandle. Sicinga ngokubaluleka kokuzikhusela kwemvelo kwe-bivalve kunye nenani elikhulu lee-phagocytes ezijikelezayo, sicinge ngakumbi ukuba ne-ccfDNA yangaphandle ityebile kwii-phagocytes ezijikelezayo eziqokelela i-DNA yangaphandle xa zingena kwiintsholongwane kunye/okanye ukungcola kweseli. Xa zizonke, iziphumo zethu zibonisa ukuba i-bivalve hemolymph ccfDNA yindawo yokugcina ulwazi lweemolekyuli kwaye iqinisa imeko yazo njengohlobo lwe-sentinel.
Idatha yethu ibonisa ukuba ukulandelelana kunye nohlalutyo lweengcezu ze-ccfDNA ze-hemolymph ezithathwe yibhaktiriya zinokubonelela ngolwazi oluphambili malunga nezityalo zebhaktiriya ezihlala kuyo kunye neebhaktiriya ezikhoyo kwindawo ejikelezileyo yolwandle. Iindlela zokulandelelana kweeshothi zityhile ulandelelwano lwebhaktiriya ye-commensal i-A. atra gill ebingayi kuphinda ifumaneke ukuba iindlela zokuchonga ze-16S rRNA eziqhelekileyo bezisetyenzisiwe, ngenxa yokuthambekela kwithala leencwadi elibhekisayo. Enyanisweni, ukusebenzisa kwethu idatha ye-LB eqokelelwe kwi-M. platensis kumaleko ofanayo weembaza eKerguelen kubonise ukuba ukwakheka kwee-symbionts zebhaktiriya ezinxulumene neembaza kwakufana kuzo zombini iintlobo zeembaza (Umzobo S4, Ulwazi olongezelelweyo). Oku kufana kweembaza ezimbini ezahlukeneyo ngokwemfuza kunokubonakalisa ukwakheka kweendawo zeebhaktiriya kwiindawo ezibandayo, ezinesulfure, kunye ne-volcanic zaseKerguelen [55, 56, 57, 58]. Amanqanaba aphezulu ee-microorganisms ezinciphisa i-sulfur achazwe kakuhle xa kuvunwa iimbaza kwiindawo ezinxwemeni ezine-bioturbated [59], njengonxweme lwasePort-au-France. Enye into enokwenzeka kukuba izityalo ze-commensal mussel zinokuchaphazeleka kukudluliselwa okuthe tyaba [60, 61]. Kufuneka uphando oluthe kratya ukuze kuchongwe unxibelelwano phakathi kwendalo yolwandle, umphezulu wolwandle, kunye nokwakheka kwebhaktheriya ehambelanayo kwiimbaza. Ezi zifundo ziyaqhubeka okwangoku.
Ubude kunye noxinzelelo lwe-hemolymph ccfDNA, ukulula kwayo ukucocwa, kunye nomgangatho ophezulu ukuvumela ukulandelelana ngokukhawuleza kwe-shotgun zezinye zeenzuzo ezininzi zokusebenzisa i-mussel ccfDNA ukuvavanya ubume bezinto eziphilayo kwiindawo eziphilayo zaselwandle. Le ndlela iyasebenza ngakumbi ekuchazeni uluntu olunentsholongwane (ii-viromes) kwindawo ethile yendalo [62, 63]. Ngokungafaniyo neebhaktheriya, ii-archaea, kunye nee-eukaryotes, ii-genomes zentsholongwane azinazo ii-genes ezigcinwe yi-phylogenetically ezifana ne-16S sequences. Iziphumo zethu zibonisa ukuba ii-biopsies zolwelo ezivela kwiintlobo zeempawu ezifana nee-mussels zinokusetyenziselwa ukuchonga inani elikhulu leenxalenye ze-ccfDNA virus ezaziwa ngokuba ziyosulela i-hosts ezihlala kwiindawo eziphilayo zaselwandle zaselunxwemeni. Oku kuquka iintsholongwane ezaziwa ngokuba ziyosulela i-protozoa, ii-arthropods, izinambuzane, izityalo, kunye neentsholongwane zebhaktheriya (umz., ii-bacteriophages). Ukusasazwa okufanayo kufunyenwe xa sihlola i-hemolymph ccfDNA virome yee-blue mussels (M. platensis) eziqokelelwe kumaleko ofanayo wee-mussel eKerguelen (Itheyibhile S2, Ulwazi olongezelelweyo). Ukulandelelana kwezibhamu ze-ccfDNA ngokwenene yindlela entsha efumana amandla okufunda i-virome yabantu okanye ezinye iintlobo [21, 37, 64]. Le ndlela iluncedo kakhulu ekufundeni iintsholongwane ze-DNA ezinemicu emibini, kuba akukho jini inye egciniweyo phakathi kwazo zonke iintsholongwane ze-DNA ezinemicu emibini, ezimele udidi lweentsholongwane ezahlukeneyo nezibanzi eBaltimore [65]. Nangona uninzi lwezi ntsholongwane lungahlelwanga kwaye lunokubandakanya iintsholongwane ezivela kwindawo engaziwayo ngokupheleleyo kwihlabathi leentsholongwane [66], sifumanise ukuba ii-viromes kunye noluhlu lwe-host ye-mussels i-A. atra kunye ne-M. platensis ziwela phakathi kwezi ntlobo zimbini. ngokufanayo (jonga umfanekiso S3, ulwazi olongezelelweyo). Oku kufana akumangalisi, njengoko kunokubonisa ukungabikho kokukhetha ekuthathweni kwe-DNA ekhoyo kwindalo esingqongileyo. Izifundo zexesha elizayo ezisebenzisa i-RNA ecocekileyo okwangoku ziyafuneka ukuze kuchazwe i-RNA virome.
Kwisifundo sethu, sisebenzise umbhobho onzima kakhulu oguqulwe kumsebenzi kaKowarski kunye noogxa bakhe [37], abasebenzise ukususwa kwamanyathelo amabini kwezinto ezidityanisiweyo kunye nee-contigs ngaphambi nasemva kokuhlanganiswa kwe-ccfDNA yendalo, nto leyo eyabangela ukuba kubekho inani elikhulu lezinto ezingafakwanga kwimephu. Ke ngoko, asinakuyikhanyela into yokuba ezinye zezi zinto zingafakwanga kwimephu zisenokuba zisenemvelaphi yazo, ikakhulu kuba asinayo i-genome yesalathiso sale ntlobo yeembaza. Sikwasebenzise lo mbhobho kuba sasixhalabile malunga nee-chimeras phakathi kwezinto ezizifunayo nezingezizo ezendalo kunye nobude bokufunda obuveliswa yi-Illumina MiSeq PE75. Esinye isizathu sokuba uninzi lwezinto ezingafakwanga kwimephu kukuba uninzi lweentsholongwane zaselwandle, ngakumbi kwiindawo ezikude ezifana neKerguelen, azikachazwa. Sisebenzise i-Illumina MiSeq PE75, sicinga ukuba ubude beengceba ze-ccfDNA bufana ne-ccfDNA yomntu. Kwizifundo ezizayo, ngenxa yeziphumo zethu ezibonisa ukuba i-hemolymph ccfDNA ifundeka ixesha elide kunabantu kunye/okanye izilwanyana ezincancisayo, sicebisa ukusebenzisa iqonga lokulandelelanisa elifanelekileyo ngakumbi kwiziqwenga ze-ccfDNA ezinde. Olu qheliselo luya kwenza kube lula kakhulu ukuchonga izibonakaliso ezingaphezulu zohlalutyo olunzulu. Ukufumana ulandelelwano olupheleleyo lwe-A. atra nuclear genome olufumanekayo okwangoku kuya kunceda kakhulu ukwahlula i-ccfDNA kwimithombo yobuqu kunye neyodwa. Ngenxa yokuba uphando lwethu lugxile ekusetyenzisweni kwengcamango ye-liquid biopsy kwi-mussels, sinethemba lokuba njengoko le ngcamango isetyenziswa kuphando oluzayo, izixhobo ezintsha kunye nemibhobho ziya kuphuhliswa ukwandisa amandla ale ndlela yokufunda ulwahluko lweentsholongwane ze-mussels.
Njenge-biomarker yeklinikhi engangenisi ntsholongwane, amanqanaba aphezulu e-ccfDNA kwiplasma yomntu anxulunyaniswa nezifo ezahlukeneyo, umonakalo wezicubu, kunye neemeko zoxinzelelo [67,68,69]. Olu nyuko lunxulunyaniswa nokukhululwa kweziqwenga zeDNA ezivela kuzo emva komonakalo wezicubu. Sithethe ngalo mbandela sisebenzisa uxinzelelo lobushushu olukhawulezileyo, apho ii-mussels zavezwa okwethutyana kubushushu obungama-30 °C. Senze olu hlalutyo kwiintlobo ezintathu ezahlukeneyo zee-mussels kwiimvavanyo ezintathu ezizimeleyo. Nangona kunjalo, asifumananga naluphi na utshintsho kumanqanaba e-ccfDNA emva koxinzelelo lobushushu olukhawulezileyo (jonga uMfanekiso S5, ulwazi olongezelelweyo). Olu fumaniso lunokuchaza, ubuncinane ngokuyinxenye, inyani yokuba ii-mussels zinenkqubo yokujikeleza kwegazi evulekileyo kwaye ziqokelela inani elikhulu le-DNA yangaphandle ngenxa yomsebenzi wazo wokucoca ophezulu. Kwelinye icala, ii-mussels, njengezinye izilwanyana ezingenamqolo ezininzi, zinokumelana ngakumbi nomonakalo wezicubu obangelwa luxinzelelo, ngaloo ndlela zithintela ukukhululwa kwe-ccfDNA kwi-hemolymph yazo [70, 71].
Ukuza kuthi ga ngoku, uhlalutyo lwe-DNA lwezinto eziphilayo kwiindawo ezihlala emanzini lugxile kakhulu kwi-DNA yokusingqongileyo (eDNA) metabarcoding. Nangona kunjalo, le ndlela idla ngokunqongophala kuhlalutyo lwezinto eziphilayo xa kusetyenziswa ii-primers. Ukusetyenziswa kwe-shotgun sequencing kuthintela imida ye-PCR kunye nokukhethwa okukhethiweyo kwee-primer sets. Ngoko ke, ngandlela ithile, indlela yethu isondele kwindlela ye-high-throughput eDNA Shotgun sequencing esetyenzisiweyo kutshanje, ekwaziyo ukulandelelanisa ngokuthe ngqo i-DNA eqhekekileyo kwaye ihlalutye phantse zonke izinto eziphilayo [72, 73]. Nangona kunjalo, kukho imiba emininzi esisiseko eyahlula i-LB kwiindlela eziqhelekileyo ze-eDNA. Kakade ke, umahluko ophambili phakathi kwe-eDNA kunye ne-LB kukusetyenziswa kwezixhobo zendalo zokuhluza. Ukusetyenziswa kweentlobo zezilwanyana zaselwandle ezifana neesponge kunye nee-bivalves (Dresseina spp.) njengesihluzo sendalo sokufunda i-eDNA kuye kwaxelwa [74, 75]. Nangona kunjalo, uphando lukaDreissena lusebenzise ii-tissue biopsies apho i-DNA yakhutshwa khona. Uhlalutyo lwe-ccfDNA oluvela kwi-LB aludingi i-tissue biopsy, izixhobo ezikhethekileyo kwaye ngamanye amaxesha ezibizayo kunye ne-logistics edibene ne-eDNA okanye i-tissue biopsy. Enyanisweni, kutshanje sibike ukuba i-ccfDNA evela kwi-LB inokugcinwa kwaye ihlalutywe ngenkxaso ye-FTA ngaphandle kokugcina uchungechunge olubandayo, oluyingxaki enkulu kuphando kwiindawo ezikude [76]. Ukukhupha i-ccfDNA kwi-liquid biopsies nako kulula kwaye kubonelela nge-DNA esemgangathweni ophezulu yokulandelelana kwe-shotgun kunye nohlalutyo lwe-PCR. Le yinzuzo enkulu xa kujongwa ezinye zeengxaki zobugcisa ezinxulumene nohlalutyo lwe-eDNA [77]. Ukulula kunye neendleko eziphantsi zendlela yokuthatha iisampulu zikwafanelekile ngokukodwa kwiinkqubo zokubeka iliso ixesha elide. Ukongeza kwisakhono sazo sokucoca esiphezulu, olunye uphawu olwaziwayo lwee-bivalves kukwakheka kwe-chemical mucopolysaccharide kwi-mucus yazo, ekhuthaza ukufunxwa kweentsholongwane [78, 79]. Oku kwenza ii-bivalves zibe sisihluzo sendalo esifanelekileyo sokuchonga ubume bezinto eziphilayo kunye nempembelelo yotshintsho lwemozulu kwindawo ethile ye-ecosystem yasemanzini. Nangona ubukho beziqwenga zeDNA ezithathwe ngumninimzi bunokubonwa njengomda wendlela xa kuthelekiswa ne-eDNA, ixabiso elinxulumene nokuba ne-ccfDNA yendalo xa kuthelekiswa ne-eDNA liyaqondakala ngaxeshanye kulwazi oluninzi olufumanekayo kwizifundo zempilo. Oku kubandakanya ubukho be-viral sequences ezidityaniswe kwi-genome yomninimzi. Oku kubaluleke kakhulu kwii-mussels, ngenxa yokuba kukho ii-retroviruses ze-leukemic ezidluliselwa ngokuthe tye kwi-bivalves [80, 81]. Enye inzuzo ye-LB ngaphezu kwe-eDNA kukuba isebenzisa umsebenzi we-phagocytic weeseli zegazi ezijikelezayo kwi-hemolymph, egubungela ii-microorganisms (kunye nee-genomes zazo). I-Phagocytosis ngumsebenzi ophambili weeseli zegazi kwi-bivalves [82]. Okokugqibela, le ndlela isebenzisa amandla aphezulu okucoca ii-mussels (umyinge we-1.5 l/h wamanzi olwandle) kunye nokujikeleza kweentsuku ezimbini, okwandisa ukuxubana kweengqimba ezahlukeneyo zamanzi olwandle, okuvumela ukubanjwa kwe-heterologous eDNA. [83, 84]. Ngoko ke, uhlalutyo lwe-ccfDNA ye-mussel yindlela enomdla xa kujongwa impembelelo yesondlo, yezoqoqosho, neyendalo yeembaza. Ngokufana nohlalutyo lwe-LB oluqokelelwe ebantwini, le ndlela ikwavula ithuba lokulinganisa utshintsho lwe-genetic kunye ne-epigenetic kwi-host DNA ukuphendula kwizinto zangaphandle. Umzekelo, ubuchwepheshe bokulandelelana kwesizukulwana sesithathu bunokucingelwa ukwenza uhlalutyo lwe-methylation ye-genome kwi-ccfDNA yendalo kusetyenziswa ulandelelwano lwe-nanopore. Le nkqubo kufuneka yenziwe lula kukuba ubude beengcezu ze-ccfDNA ze-mussel buhambelana kakuhle namaqonga okulandelelana afundwa ixesha elide avumela uhlalutyo lwe-methylation ye-DNA ye-genome-wide ukusuka kwindlela enye yokulandelelana ngaphandle kwesidingo sokuguqulwa kweekhemikhali.85,86] Oku kunokwenzeka okunomdla, njengoko kubonisiwe ukuba iipateni ze-methylation ye-DNA zibonisa impendulo kuxinzelelo lwendalo kwaye zihlala kwizizukulwana ezininzi. Ke ngoko, inokubonelela ngengqiqo ebalulekileyo kwiindlela ezilawula impendulo emva kokuchaphazeleka kutshintsho lwemozulu okanye ungcoliseko [87]. Nangona kunjalo, ukusetyenziswa kwe-LB akunamingcele. Akufuneki ukuba kuthethwe, oku kufuna ukubakho kweentlobo zesalathisi kwi-ecosystem. Njengoko kukhankanyiwe apha ngasentla, ukusebenzisa i-LB ukuvavanya ubume bezinto eziphilayo kwindawo ethile yendalo kufuna umbhobho oqinileyo we-bioinformatics oqwalasela ubukho beziqwenga ze-DNA ezivela kumthombo. Enye ingxaki enkulu kukufumaneka kwee-genomes zesalathiso seentlobo zezilwanyana zaselwandle. Kuthenjwa ukuba amanyathelo afana neMarine Mammal Genomes Project kunye neprojekthi yeFish10k esandula ukusekwa [88] aya kwenza kube lula uhlalutyo olunjalo kwixesha elizayo. Ukusetyenziswa kwengcamango ye-LB kwizinto eziphilayo ezihluza ukutya zaselwandle kuyahambelana nenkqubela phambili yamva nje kwitekhnoloji yokulandelelana, okwenza ukuba ifaneleke kakuhle kuphuhliso lwee-biomarkers ezininzi ze-ohm ukubonelela ngolwazi olubalulekileyo malunga nempilo yeendawo zokuhlala zaselwandle ukuphendula uxinzelelo lokusingqongileyo.
Idatha yokulandelelana kweejini ifakiwe kwi-NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 phantsi kwe-Bioprojects SRR8924808.
IBrierley AS, Kingsford MJ Impembelelo yotshintsho lwemozulu kwizinto eziphilayo zaselwandle kunye ne-ecosystem. Cole Biology. 2009; 19: P602–P614.
UGissi E, uManea E, uMazaris AD, uFraschetti S, uAlmpanidou V, uBevilacqua S, nabanye. Cinga ngempembelelo edibeneyo yotshintsho lwemozulu kunye nezinye izinto ezibangela uxinzelelo lwengingqi kwindalo yaselwandle. okusingqongileyo kwesayensi ngokubanzi. 2021;755:142564.
Carella F, Antuofermo E, Farina S, Salati F, Mandas D, Prado P, et al. ). Inzululwazi yokuqala kaMatshi. 2020;7:48.
USeront L, uNicastro CR, uZardi GI, uGoberville E. Ukunciphisa ukunyamezela ubushushu phantsi kweemeko zoxinzelelo lobushushu oluphindaphindayo kuchaza ukufa okuphezulu kwe-blue mussels ehlotyeni. Ingxelo yesayensi ka-2019; 9:17498.
UFey SB, uSiepielski AM, uNussle S, uCervantes-Yoshida K, uHwan JL, uHuber ER, nabanye. Utshintsho lwamva nje kwinani, unobangela kunye nobungakanani bokufa kwezilwanyana. Proc Natl Acad Sci USA. 2015;112:1083-8.
Scarpa F, Sanna D, Azzena I, Mughetti D, Cerruti F, Hosseini S, et al. Iintsholongwane ezininzi ezingezizo ezohlobo oluthile zisenokuba zibangele ukufa kwabantu abaninzi be-Pinna nobilis. Ubomi. 2020;10:238.
UBradley M, uCoutts SJ, uJenkins E, uO'Hara TM. Impembelelo enokubakho yotshintsho lwemozulu kwizifo zezilwanyana zaseArctic. Int J Circumpolar health. 2005; 64:468–77.
UBeyer J., uGreene NW, uBrooks S., uAllan IJ, uRuus A., uGomez T. et al. Iimbaza eziluhlaza (iMytilus edulis spp.) njengezinto eziphilayo ezibonisa uphawu ekujongeni ungcoliseko lolwandle: uphononongo. Mar Environ Res 2017; 130:338-65.
USiravegna G, uMarsoni S, uSiena S, uBardelli A. Ukuhlanganiswa kwe-biopsy yolwelo kunyango lomhlaza. Nat Rev Clean Oncol. 2017; 14:531–48.
UWan JCM, uMassie C, uGarcia-Corbacho J, uMouliere F, uBrenton JD, uCaldas C, nabanye. Ukuvuthwa kwe-biopsy yolwelo: Ivumela i-DNA yethumba ukuba ijikeleze. Umhlaza weNat Rev. 2017;17:223–38.
UMandel P., uMetais P. Ii-Nucleic acids kwi-plasma yomntu. Imizuzu yentlanganiso yeenkampani eziphantsi kweSoc Biol. 1948; 142:241-3.
IBronkhorst AJ, Ungerer W, Holdenrieder S. Indima entsha ye-DNA engenaseli njengophawu lweemolekyuli zonyango lomhlaza. Ukulinganiswa kohlalutyo lwe-biomolar. 2019;17:100087.
U-Ignatiadis M., uSledge GW, uJeffrey SS I-Liquid biopsy ingena ekliniki - imiba yokuphunyezwa kunye nemingeni yexesha elizayo. Nat Rev Clin Oncol. 2021; 18:297–312.
ULo YM, uCorbetta N., uChamberlain PF, uRai W., uSargent IL, uRedman CW nabanye. I-DNA yosana olungekazalwa ikhona kwiplasma nakwi-serum yomama. I-Lancet. 1997; 350:485-7.
UMufarray MN, uWong RJ, uShaw GM, uStevenson DK, uQuake SR Uphononongo lwenkqubo yokukhulelwa kunye neengxaki zayo kusetyenziswa i-RNA engaphandle kwegazi ejikeleza egazini labasetyhini ngexesha lokukhulelwa. I-Dopediatrics. 2020;8:605219.
U-Ollerich M, uSherwood K, uKeown P, uSchütz E, uBeck J, uStegbauer J, nabanye. I-Liquid biopsy: i-DNA engenazo iiseli zomnikeli isetyenziselwa ukubona izilonda ze-allogeneic kwi-graft yezintso. UNat Rev Nephrol. 2021; 17:591–603.
UJuan FC, uLo YM Innovations kwi-prenatal diagnostics: ukulandelelana kwe-genome ye-plasma yomama. UAnna MD. 2016;67:419-32.
UGu W, uDeng X, uLee M, uSucu YD, uArevalo S, uStryke D, nabanye. Ukufunyanwa kwezifo ngokukhawuleza kunye nokulandelelana kwe-metagenomic yesizukulwana esilandelayo solwelo lomzimba olusulelekileyo. I-Nat Medicine. 2021;27:115-24.