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Biopsy yemvura (LB) ipfungwa iri kukurumidza kufarirwa mumunda wezvekurapa. Pfungwa iyi inonyanya kubva pakuona zvidimbu zveDNA iri kunze kwemasero (ccfDNA), izvo zvinonyanya kuburitswa sezvidimbu zvidiki mushure mekufa kwemasero mumatishu akasiyana-siyana. Chikamu chidiki chezvidimbu izvi chinobva mumatishu ekunze (ekunze) kana zvipenyu. Mubasa razvino, tashandisa pfungwa iyi kune mussels, rudzi rwemurindi runozivikanwa nekugona kwarwo kupinza mvura yegungwa zvakanyanya. Tinoshandisa kugona kwemussels kushanda semafirita echisikigo ekubata zvidimbu zveDNA yezvakatipoteredza kubva kunzvimbo dzakasiyana-siyana kuti tipe ruzivo nezve kusiyana-siyana kwezvipenyu zvegungwa. Mhedzisiro yedu inoratidza kuti mussel hemolymph ine zvidimbu zveDNA zvakasiyana-siyana muhukuru, kubva pa1 kusvika ku5 kb. Kutevedzana kwepfuti kwakaratidza kuti huwandu hwakawanda hwezvidimbu zveDNA zvinobva kuhutachiona hwekune dzimwe nyika. Pakati pazvo, takawana zvidimbu zveDNA kubva kubhakitiriya, archaea, uye mavhairasi, kusanganisira mavhairasi anozivikanwa nekutapukira marudzi akasiyana-siyana evanogara anowanikwa muzvipenyu zvegungwa zvegungwa. Mukupedzisa, chidzidzo chedu chinoratidza kuti pfungwa yeLB inoshandiswa kune hozhwa inomiririra ruzivo rwakapfuma asi rwusati rwaongororwa nezve kusiyana kwehutachiona muzvisikwa zvegungwa.
Kukanganiswa kwekushanduka kwemamiriro ekunze (CC) pahupenyu hwezvisikwa zvemugungwa inzvimbo iri kukura nekukurumidza yekutsvagisa. Kudziya kwepasi rose hakungokonzere chete kushushikana kwakakosha kwemuviri, asiwo kunosundidzira miganhu yekushanduka-shanduka kwekugadzikana kwekupisa kwezvisikwa zvemugungwa, kuchikanganisa nzvimbo yekugara kwemarudzi akawanda, zvichivaita kuti vatsvage mamiriro akanaka [1, 2]. Pamusoro pekukanganisa hupenyu hwemarudzi akasiyana-siyana emetazoans, CC inokanganisa kuenzana kwakasimba kwekudyidzana kwemakemikari nemakemikari. Dysbacteriosis iyi inotyisidzira zvakanyanya zvisikwa zvemugungwa sezvo ichiita kuti zvisikwa zvemugungwa zvive nyore kubatwa nezvirwere zvinotapukira [3, 4]. Zvinotendwa kuti SS inoita basa rakakosha mukufa kwevanhu vazhinji, iro riri dambudziko guru rekutarisira zvisikwa zvemugungwa zvepasi rose [5, 6]. Iyi inyaya yakakosha tichifunga nezvemhedzisiro yehupfumi, zvakatipoteredza uye yekudya kwemarudzi mazhinji emugungwa. Izvi zvinonyanya kuitika kune ma bivalve anogara munzvimbo dzepolar, uko mhedzisiro yeCK iri nekukurumidza uye yakakomba [6, 7]. Kutaura zvazviri, ma bivalve akadai seMytilus spp. anoshandiswa zvakanyanya kutarisa mhedzisiro yeCC pazvisikwa zvemugungwa. Hazvishamisi kuti huwandu hwakawanda hwema biomarker hwakagadzirwa kuti hutarise hutano hwavo, kazhinji vachishandisa nzira ine matanho maviri inosanganisira ma biomarker anoshanda zvichibva pakuita kwe enzymatic kana mabasa emasero akadai sekuchengetedza masero uye phagocytic activity [8]. Nzira idzi dzinosanganisirawo kuyerwa kwekuwanda kwezviratidzo zvekumanikidzwa zvinoungana mumatishu akapfava mushure mekunwa mvura yakawanda yegungwa. Zvisinei, kugona kwakanyanya kupfapfaidza uye semi-open circulating system yema bivalve zvinopa mukana wekugadzira ma biomarker matsva e hemolymph uchishandisa pfungwa ye liquid biopsy (LB), nzira iri nyore uye isinganyanyi kukuvadza murwere. [9, 10]. Kunyangwe mhando dzakasiyana dzema circulating molecules dzichigona kuwanikwa mu human LB, pfungwa iyi inonyanya kubva pakuongorora DNA sequencing ye circulating extracellular DNA (ccfDNA) zvidimbu mu plasma. Kutaura zvazviri, kuvapo kwe circulating DNA mu human plasma kwave kuchizivikanwa kubvira pakati pezana remakore rechi20 [11], asi mumakore achangopfuura chete ndipo pakatanga kuwanikwa nzira dze high-throughput sequencing dzakakonzera kuongororwa kwekiriniki zvichibva pa ccfDNA. Kuvapo kwezvidimbu zveDNA izvi zvinotenderera kunokonzerwa nekuburitswa kweDNA ye genomic (yenyukireya neye mitochondrial) mushure mekufa kwesero. Muvanhu vane hutano hwakanaka, huwandu hweccfDNA hunowanzova hwakaderera (<10 ng/mL) asi hunogona kuwedzerwa ne5-10 nguva muvarwere vane zvirwere zvakasiyana-siyana kana kuti vari pasi pekushushikana, zvichikonzera kukuvara kwenyama. Muvanhu vane hutano hwakanaka, huwandu hweccfDNA hunowanzova hwakaderera (<10 ng/mL) asi hunogona kuwedzerwa ne5-10 nguva muvarwere vane zvirwere zvakasiyana-siyana kana kuti vari pasi pekushushikana, zvichikonzera kukuvara kwenyama. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 подвергающихся стрессу, приводящему к повреждению тканей. Muvanhu vane hutano hwakanaka, huwandu hwe cccDNA hunowanzova hwakaderera (<10 ng/mL), asi hunogona kuwedzera ne5-10 nguva muvarwere vane zvirwere zvakasiyana-siyana kana vari pasi pekushushikana kunokonzera kukuvara kwenyama.在健康个体中，ccfDNA的浓度通常较低（<10 ng/mL），但在患有各种病理或承受压力的悢1者倍，从而导致组织损伤。在 健康 个体 中 , ccfdna 的 浓度 较 低 （<10 ng/ml） 在 各 种 病理 或 承叅 压叅5-10 倍 , 从而 组织。。。Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут быть увеличены в 5-10 раз у пациентонтовый стрессом, что приводит к повреждению тканей. Kuwanda kweCCFDNA kunowanzova kwakaderera (<10 ng/ml) muvanhu vane hutano hwakanaka, asi kunogona kuwedzerwa ka5-10 muvarwere vane zvirwere zvakasiyana-siyana kana kushushikana, zvichikonzera kukuvara kwenyama.Saizi yezvidimbu zveccfDNA inosiyana zvakanyanya, asi kazhinji inotangira pa150 kusvika 200 bp. [12]. Kuongororwa kweccfDNA inotorwa yega, kureva, ccfDNA kubva kumasero akajairwa kana akachinjwa, kunogona kushandiswa kuona shanduko dzemajini uye epigenetic dziripo mugenome yenyukireya uye/kana mitochondrial, nokudaro zvichibatsira vanachiremba kusarudza mishonga chaiyo inotarirwa nemamorekuru [13]. Zvisinei, ccfDNA inogona kuwanikwa kubva kune dzimwe nyika dzakadai seccfDNA kubva kumasero emwana ari mudumbu panguva yekuzvitakura kana kubva kunhengo dzakasimwa [14,15,16,17]. ccfDNA inzvimbo yakakosha yeruzivo rwekuona kuvapo kwenucleic acids yemushonga unotapukira (wekunze), izvo zvinobvumira kuonekwa kwehutachiona hwakapararira husingazivikanwe netsika dzeropa, zvichidzivirira biopsy inopararira yetishu ine hutachiona [18]. Zvidzidzo zvechangobva kuitika zvakaratidza kuti ropa revanhu rine ruzivo rwakakura runogona kushandiswa kuona hutachiona hwehutachiona nebhakitiriya, uye kuti inenge 1% yeccfDNA inowanikwa muplasma yevanhu inobva kune dzimwe nyika [19]. Zvidzidzo izvi zvinoratidza kuti kusiyana-siyana kwehutachiona hwe microbiome inotenderera muchisikwa kunogona kuongororwa uchishandisa ccfDNA analysis. Zvisinei, kusvika munguva pfupi yapfuura, pfungwa iyi yaishandiswa chete muvanhu uye, zvishoma, mune dzimwe mhuka dzine mapfupa anotenderera [20, 21].
Mupepa razvino, tinoshandisa mukana weLB kuongorora ccfDNA yeAulacomya atra, rudzi rwekumaodzanyemba runowanzo kuwanikwa muKerguelen Islands iri pasi peAntarctic, boka rezvitsuwa zviri pamusoro pegomo guru rakaumbwa makore mamiriyoni makumi matatu nemashanu apfuura. Tichishandisa sisitimu yekuedza ye in vitro, takaona kuti zvidimbu zveDNA mumvura yegungwa zvinotorwa nekukurumidza ne mussels uye zvinopinda mu hemolymph compartment. Kutevedzana kwepfuti kwakaratidza kuti mussel hemolymph ccfDNA ine zvidimbu zveDNA zvayo uye zvisiri zvega, kusanganisira mabhakitiriya e symbiotic uye zvidimbu zveDNA kubva ku biomes zvinowanzoonekwa mu cold volcanic marine coastline ecosystems. Hemolymph ccfDNA inewo hutachiona hunobva kumavhairasi ane mhando dzakasiyana dzemugadziri. Takawanawo zvidimbu zveDNA kubva kumhuka dzakawanda dzakadai se bony fish, sea anemones, algae nezvipembenene. Mukupedzisa, chidzidzo chedu chinoratidza kuti pfungwa yeLB inogona kushandiswa zvinobudirira kune veterine invertebrates kugadzira genomic repertoire yakapfuma mu marine ecosystems.
Hove dzakakura (55-70 mm kureba) Mytilus platensis (M. platensis) uye Aulacomya atra (A. atra) dzakatorwa kubva kumahombekombe ane matombo ePort-au-France (049°21.235 S, 070°13.490 E .). Kerguelen Islands muna Zvita 2018. Dzimwe hove dzebhuruu dzakakura (Mytilus spp.) dzakatorwa kubva kumutengesi wekutengesa (PEI Mussel King Inc., Prince Edward Island, Canada) ndokuiswa mutangi rine mhepo inodziya (4°C) ine 10–20 L ye32‰ artificial brine. (artificial sea salt Reef Crystal, Instant Ocean, Virginia, USA). Pakuyedza kwega kwega, kureba nehuremu hwemagoko ega ega zvakayerwa.
Nzira yemahara yekupinda pachena yechirongwa ichi inowanikwa online (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). Muchidimbu, LB hemolymph yakatorwa kubva mutsandanyama dzeabductor sezvakatsanangurwa [22]. Hemolymph yakajekeswa ne centrifugation pa 1200×g kwemaminitsi matatu, supernatant yakaomeswa nechando (-20°C) kusvika yashandiswa. Kuti cfDNA iparadzwe uye icheneswe, sampuro (1.5-2.0 ml) dzakanyungudutswa uye dzakagadziriswa uchishandisa NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) zvichienderana nemirairo yemugadziri. ccfDNA yakachengetwa pa -80°C kusvika yaongororwazve. Mune kumwe kuyedza, ccfDNA yakaparadzaniswa uye yakacheneswa uchishandisa QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada). DNA yakachena yakayerwa uchishandisa PicoGreen assay yakajairwa. Kugoverwa kwezvidimbu zveccfDNA yakaparadzaniswa kwakaongororwa ne capillary electrophoresis uchishandisa Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) uchishandisa High Sensitivity DNA Kit. Kuyedzwa kwakaitwa uchishandisa 1 µl yesampuli yeccfDNA zvichienderana nemirairo yemugadziri.
Pakutevedzana kwezvikamu zve hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Canada) akagadzira maraibhurari epfuti achishandisa Illumina DNA Mix kit yeIllumina MiSeq PE75 kit. Adapter yakajairika (BioO) yakashandiswa. Mafaira edata asina kurongeka anowanikwa kubva kuNCBI Sequence Read Archive (SRR8924808 uye SRR8924809). Hunhu hwekuverenga hwakakosha hwakaongororwa uchishandisa FastQC [23]. Trimmomatic [24] yakashandiswa pakucheka adapta uye kuverenga kusina kunaka. Kuverenga kwepfuti nemagumo maviri kwakabatanidzwa FLASH kuita kuverenga kurefu ne20 bp kudzivirira kusawirirana [25]. Kuverenga kwakabatanidzwa kwakanyorwa neBLASTN uchishandisa database yeNCBI Taxonomy ye bivalve (e value < 1e−3 uye 90% homology), uye kuvhara ma sequences asina kuoma kwakaitwa uchishandisa DUST [26]. Kuverenga kwakabatanidzwa kwakanyorwa neBLASTN uchishandisa database yeNCBI Taxonomy ye bivalve (e value < 1e−3 uye 90% homology), uye kuvhara ma sequences asina kuoma kwakaitwa uchishandisa DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN базы данных таксономии двустворчатых моллюсков NCBI -0 гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Kuverenga kwakabatana kwakanyorwa neBLASTN uchishandisa database yeNCBI bivalve taxonomy (e value < 1e-3 uye 90% homology), uye low complexity sequence masking yakaitwa uchishandisa 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 使用如 列祖 [26]的。。。бъединенные чтения были аннотированы с помощью BLASTN с использованием таксономической базы данных двустворчатых моллюс10 NCBI (9) гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Kuverenga kwakabatana kwakanyorwa neBLASTN uchishandisa NCBI bivalve taxonomic database (e value <1e-3 uye 90% homology), uye low complexity sequence masking yakaitwa uchishandisa DUST [26].MaRead akakamurwa kuita mapoka maviri: ane chekuita ne bivalve sequences (pano anonzi self-reads) uye asina hukama (non-self-reads). Mapoka maviri akaunganidzwa zvakasiyana achishandisa MEGAHIT kugadzira contigs [27]. Panguva iyi, taxonomic distribution ye alien microbiome reads yakarongwa uchishandisa Kraken2 [28] uye yakamiririrwa nemifananidzo ne Krona pie chart paGalaxy [29, 30]. Ma kmers akanakisa akaonekwa kuti aiva kmers-59 kubva mukuyedza kwedu kwekutanga. Zvikamu zvega zvakazoonekwa nekuenzanisa neBLASTN (bivalve NCBI database, e value < 1e−10 uye 60% homology) kuti zvitsanangurwe zvekupedzisira. Zvikamu zvega zvakazoonekwa nekuenzanisa neBLASTN (bivalve NCBI database, e value < 1e−10 uye 60% homology) kuti zvitsanangurwe zvekupedzisira. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, значени10 e) для окончательной аннотации. Zvikamu zvega zvakazoonekwa nekuenzanisa neBLASTN (NCBI bivalve database, e value <1e-10 uye 60% homology) kuti zvitsanangurwe zvekupedzisira.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). значение e <1e-10 и гомология 60%). Zvikamu zvega zvakazoonekwa kuti zvitsanangurwe zvekupedzisira nekuenzanisa neBLASTN (NCBI bivalve database, e value <1e-10 uye 60% homology). Zvakafanana, mapoka eboka risiri remunhu pachake akanyorwa neBLASTN (database yeNT NCBI, kukosha kwe e < 1e−10 uye 60% homology). Zvakafanana, mapoka eboka risiri remunhu pachake akanyorwa neBLASTN (database yeNT NCBI, kukosha kwe e < 1e−10 uye 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 и гомология 60%). Zvakafanana, mapoka emapoka ekunze akanyorwa neBLASTN (dhatabhesi reNT NCBI, kukosha kwe e <1e-10 uye 60% homology).平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群.平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群. Параллельно контиги, не относящиеся к собственной группе, были аннотированы с помощью BLASTN (база данных nt NCBI, значение e и 1e-1%). Zvakafanana, mapoka asiri ega akanyorwa neBLASTN (database yeNT NCBI, kukosha kwe e <1e-10 uye 60% homology). BLASTX yakaitwawo pazvikamu zvisiri zvemunhu pachake uchishandisa nr uye RefSeq protein NCBI databases (e value < 1e−10 uye 60% homology). BLASTX yakaitwawo pazvikamu zvisiri zvemunhu pachake uchishandisa nr uye RefSeq protein NCBI databases (e value < 1e−10 uye 60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 6%). BLASTX yakaitwawo pane zvisiri zvega uchishandisa nr uye RefSeq NCBI protein databases (e value < 1e-10 uye 60% homology).还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 同源性）.还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 同源性）. BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 и 60%). BLASTX yakaitwawo pane zvisiri zvega uchishandisa nr uye RefSeq NCBI protein databases (e value <1e-10 uye 60% homology).Mapombi eBLASTN neBLASTX evasiri ivo pachavo anomiririra zvikamu zvekupedzisira (ona faira rekuwedzera).
Ma primer anoshandiswa paPCR akanyorwa muTable S1. Taq DNA polymerase (Bio Basic Canada, Markham, ON) yakashandiswa kuwedzera majini eCCFDNA. Mamiriro anotevera ekuita akashandiswa: denaturation pa95°C kwemaminitsi matatu, 95°C kwemaminitsi 1, set annealing temperature kweminiti 1, elongation pa72°C kweminiti 1, 35 cycles, uye pakupedzisira 72°C mukati memaminitsi gumi. . Zvigadzirwa zvePCR zvakaparadzaniswa ne electrophoresis muma agarose gels (1.5%) ane SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Canada) pa95 V.
Hove (Mytilus spp.) dzakajairwa mumvura yegungwa ine oxygen ye500 ml (32 PSU) kwemaawa makumi maviri nemana pa4°C. Plasmid DNA ine chinhu chinoisa mucherechedzo we human galectin-7 cDNA sequence (NCBI accession number L07769) yakawedzerwa mubhodhoro pahuwandu hwekupedzisira hwe190 μg/μl. Hove dzakachengetwa mumamiriro akafanana pasina kuwedzerwa kweDNA ndidzo dzaive dzekutonga. Tangi rechitatu rekudzora raiva neDNA isina hove. Kuti vatarise mhando yeDNA mumvura yegungwa, samples dzemvura yegungwa (20 μl; katatu) dzakatorwa kubva mutangi rega rega panguva yakatarwa. Kuti DNA ionekwe seyakakodzera kuteverwa, hove dzeLB dzakatorwa panguva dzakatarwa uye dzakaongororwa neqPCR neddPCR. Nekuda kwehuwandu hwakawanda hwemunyu mumvura yegungwa, ma aliquots akasanganiswa mumvura yemhando yePCR (1:10) vasati vaongororwa PCR yese.
Digital droplet PCR (ddPCR) yakaitwa uchishandisa BioRad QX200 protocol (Mississauga, Ontario, Canada). Shandisa tembiricha kuti uone tembiricha yakakodzera (Tafura S1). Madonhwe akagadzirwa uchishandisa QX200 drop generator (BioRad). ddPCR yakaitwa seinotevera: 95°C kwemaminitsi mashanu, makumi mashanu e95°C cycles kwemaminitsi makumi matatu uye tembiricha yakapihwa ye annealing kwemaminitsi 1 uye 72°C kwemaminitsi makumi matatu, 4°C kwemaminitsi mashanu uye 90°C mukati memaminitsi mashanu. Huwandu hwemadonhwe uye positive reactions (nhamba yemakopi/µl) zvakayerwa uchishandisa QX200 drop reader (BioRad). Samples dzine madonhwe asingasviki 10,000 dzakarambwa. Kudzora mapatani hakuna kuitwa pese paishandiswa ddPCR.
qPCR yakaitwa uchishandisa Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) uye LGALS7 specific primers. MaPCR ese equantitative akaitwa mu20 µl uchishandisa QuantiFast SYBR Green PCR Kit (QIAGEN). qPCR yakatangwa ne15 min incubation pa95°C ichiteverwa ne40 cycles pa95°C kwemasekondi gumi uye pa60°C kwemasekondi makumi matanhatu nekuunganidza data kamwe chete. Melting curves yakagadzirwa uchishandisa zviyero zvinotevedzana pa95°C kwemasekondi mashanu, 65°C kwemasekondi makumi matanhatu, uye 97°C pakupera kweqPCR. qPCR yega yega yakaitwa katatu, kunze kwemasamples ekudzora.
Sezvo hove dzichizivikanwa nekuwanda kwazvo kwekusefa, takatanga taongorora kana dzichigona kusefa nekuchengetedza zvidimbu zveDNA zviri mumvura yegungwa. Taidawo kuziva kana zvidimbu izvi zvichiungana mu semi-open lymphatic system yadzo. Takagadzirisa nyaya iyi nekuyedza nekutsvaga magumo ezvidimbu zveDNA zvinonyungudika zvakawedzerwa kumatangi eblue mussel. Kuti zvive nyore kutevedzera zvidimbu zveDNA, takashandisa plasmid DNA yekune imwe nyika (kwete yega) ine human galectin-7 gene. ddPCR inotsvaga zvidimbu zveDNA zveplasmid mumvura yegungwa nemahombe. Zvatakawana zvinoratidza kuti kana huwandu hwezvidimbu zveDNA mumvura yegungwa hwakaramba hwakafanana nekufamba kwenguva (kusvika mazuva manomwe) pasina hove, saka kana hove dziripo, huwandu uhwu hwakapotsa hwanyangarika mukati memaawa masere (Mufananidzo 1a,b). Zvidimbu zveDNA yekunze zvakaonekwa zviri nyore mukati memaminitsi gumi nemashanu mu intravalvular fluid ne hemolymph (Mufananidzo 1c). Zvidimbu izvi zvinogona kuonekwa kusvika maawa mana mushure mekusangana nazvo. Basa iri rekusefa maererano nezvidimbu zveDNA rakafanana nebasa rekusefa remabhakitiriya ne algae [31]. Mhedzisiro iyi inoratidza kuti hozhwa dzinogona kusefa nekuunganidza DNA yekunze munzvimbo dzadzo dzemvura.
Kuwanda kweDNA yeplasmid mumvura yegungwa kana mussels dziripo (A) kana kuti dzisipo (B), zvichiyerwa neddPCR. MuA, mhedzisiro yacho inoratidzwa semapercentage, nemiganhu yemabhokisi ichimiririra 75th ne25th percentiles. Curve yelogarithmic inoratidzwa mutsvuku, uye nzvimbo ine mumvuri wegrey inomiririra 95% confidence interval. MuB, mutsetse mutsvuku unomiririra avhareji uye mutsetse webhuruu unomiririra 95% confidence interval ye concentration. C Kuunganidzwa kweDNA yeplasmid mu hemolymph uye valvular fluid ye mussels panguva dzakasiyana mushure mekuwedzerwa kweDNA yeplasmid. Mhedzisiro yacho inoratidzwa se absolute copies detected/mL (±SE).
Tevere, takaongorora kwakabva ccfDNA muhomba dzakaunganidzwa kubva mumibhedha yehomba paKerguelen Islands, boka rezvitsuwa zviri kure zvine simba shoma rekukonzera hunhu hwevanhu. Nechinangwa ichi, cccDNA kubva muhomba dzehomba dzakabviswa ndokucheneswa nenzira dzinowanzo shandiswa kuchenesa cccDNA yevanhu [32, 33]. Takaona kuti avhareji yehuwandu hwe hemolymph ccfDNA muhomba dziri mu micrograms shoma pa ml hemolymph (ona Tafura S2, Ruzivo Rwekuwedzera). Huwandu uhwu hwehuwandu hwakakura zvikuru kupfuura hwevanhu vane hutano (nanograms shoma pa milliliter), asi mune zvisingawanzoitiki, muvarwere vegomarara, huwandu hweccfDNA hunogona kusvika ma micrograms akati wandei pa milliliter [34, 35]. Kuongororwa kwekugoverwa kwehukuru hwe hemolymph ccfDNA kwakaratidza kuti zvidimbu izvi zvinosiyana zvikuru muhukuru, kubva pa1000 bp kusvika 1000 bp. kusvika 5000 bp (Mufananidzo 2). Zvakafanana zvakawanikwa uchishandisa silica-based QIAamp Investigator Kit, nzira inowanzo shandiswa musainzi ye forensic kupatsanura nekukurumidza nekuchenesa genomic DNA kubva kuma DNA samples eDNA ane huwandu hwakaderera, kusanganisira ccfDNA [36].
Electrophoregram yeccfDNA inomiririra hemolymph yemussel. Yakatorwa neNucleoSnap Plasma Kit (kumusoro) uye QIAamp DNA Investigator Kit. B Violin plot inoratidza kugoverwa kwehemolymph ccfDNA concentrations (±SE) muhassels. Mitsetse mitema netsvuku inomiririra medium uye first and third quartiles, zvichiteerana.
Inenge 1% yeccfDNA muvanhu nemhuka dzerudzi rweprimates ine kwainobva [21, 37]. Tichifunga nezve semi-open circulatory system ye bivalves, mvura yegungwa ine microbial-rich, uye kukura kwe mussel ccfDNA, takafungidzira kuti mussel hemolymph ccfDNA inogona kunge iine dziva re microbial DNA rakapfuma uye rakasiyana-siyana. Kuti tiedze fungidziro iyi, takaongorora hemolymph ccfDNA kubva kuAulacomya atra samples dzakatorwa kubva kuKerguelen Islands, zvikaburitsa kuverenga kunodarika mamiriyoni gumi, 97.6% avo vakapasa kudzorwa kwemhando yepamusoro. Kuverenga kwacho kwakarongwa zvichienderana nekwavo uye kwavasiri ivo pachavo vachishandisa BLASTN neNCBI bivalve databases (Mufananidzo S1, Supplementary Information).
Muvanhu, DNA yenyukireya neyemitochondrial inogona kuburitswa muropa [38]. Zvisinei, muchidzidzo chino, hazvina kukwanisa kutsanangura zvakadzama DNA yemutochondrial yemussels, tichifunga kuti genome yeA. atra haina kurongwa kana kutsanangurwa. Zvisinei, takakwanisa kuona zvidimbu zveccfDNA zvakati wandei zvatakabva tichishandisa raibhurari yebivalve (Mufananidzo S2, Ruzivo Rwekuwedzera). Takasimbisawo kuvapo kwezvidimbu zveDNA zvatakabva nazvo nekuwedzera PCR zvakananga kwemajini eA. atra akarongwa (Mufananidzo 3). Saizvozvowo, tichifunga kuti genome yemitochondrial yeA. atra inowanikwa mumadhatabhesi everuzhinji, munhu anogona kuwana humbowo hwekuvapo kwezvidimbu zveccfDNA zvemitochondrial muhemolymph yeA. atra. Kuvapo kwezvidimbu zveDNA zvemitochondrial kwakasimbiswa nekuwedzera kwePCR (Mufananidzo 3).
Majini akasiyana-siyana emumitochondrial aivepo muhemolymph yeA. atra (madotsi matsvuku - nhamba yemasheya: SRX5705969) uye M. platensis (madotsi ebhuruu - nhamba yemasheya: SRX5705968) akawedzerwa nePCR. Mufananidzo wakashandurwa kubva kuna Breton et al., 2011 B Kuwedzera kwehemolymph supernatant kubva kuA. atra Yakachengetwa pabepa reFTA. Shandisa punch ye3 mm kuwedzera zvakananga kuPCR tube ine musanganiswa wePCR.
Tichifunga nezvehuwandu hwakawanda hwemabhakitiriya ari mumvura yegungwa, pakutanga takatarisa pahunhu hwemaDNA emakemikari ari muropa. Kuti tiite izvi, tinoshandisa nzira mbiri dzakasiyana. Nzira yekutanga yakashandisa Kraken2, chirongwa chekupatsanura maDNA che algorithm chinogona kuona maDNA emakemikari nenzira chaiyo yakafanana neBLAST nezvimwe zvishandiso [28]. Kuverenga kunopfuura 6719 kwakaongororwa kuti kwakabva kumabhakitiriya, nepo 124 ne64 zvaibva kuarchaea nemavhairasi, zvichiteerana (Mufananidzo 4). Zvidimbu zveDNA zvebhakitiriya zvakawanda zvaive Firmicutes (46%), Proteobacteria (27%), uye Bacteroidetes (17%) (Mufananidzo 4a). Kugoverwa uku kunoenderana nezvidzidzo zvekare zve marine blue mussel microbiome [39, 40]. Gammaproteobacteria ndiyo yaive boka guru reProteobacteria (44%), kusanganisira maVibrionales mazhinji (Mufananidzo 4b). Nzira yeddPCR yakasimbisa kuvapo kwezvidimbu zveVibrio DNA muccfDNA yeA. atra hemolymph (Mufananidzo 4c) [41]. Kuti uwane rumwe ruzivo nezvekwakabva ccfDNA nemabhakitiriya, imwe nzira yakatorwa (Mufananidzo S2, Ruzivo Rwekuwedzera). Muchiitiko ichi, maverengero akapindirana akaunganidzwa semaverengero ekupedzisira maviri uye akarongwa seanobva pachawo (bivalve) kana kuti asiri ega uchishandisa BLASTN uye kukosha kwe e kwe1e−3 uye cutoff ine >90% homology. Muchiitiko ichi, maverengero akapindirana akaunganidzwa semaverengero ekupedzisira maviri uye akarongwa seanobva pachawo (bivalve) kana kuti asiri ega uchishandisa BLASTN uye kukosha kwe e kwe1e−3 uye cutoff ine >90% homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственные (двуста) чужие по происхождению с использованием BLASTN uye значения e 1e-3 uye отсечения с гомологией> 90%. Muchiitiko ichi, kuverenga kwakabatana kwakaunganidzwa sekuverenga kwepaired-end uye kwakarongwa sekwakabva (bivalve) kana kuti kwete kwepakutanga uchishandisa BLASTN uye e value ye1e-3 uye cutoff ine >90% homology.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 zvoupfu pamwe>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 blastn 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 (двуствор) несобственные по происхождению с использованием значений e BLASTN uye 1e-3 uye порога гомологии> 90%. Muchiitiko ichi, kuverenga kwakabatana kwakaunganidzwa sekuverenga kwakapetwa kaviri uye kwakarongwa sekwako (bivalve) kana kuti kwete kwepakutanga uchishandisa e BLASTN uye 1e-3 values ​​​​uye chiyero che homology >90%.Sezvo genome reA. atra risati rarongwa, takashandisa nzira yekubatanidza de novo yeMEGAHIT Next Generation Sequencing (NGS) assembler. Ma contig anosvika 147,188 akaonekwa seanobva (bivalve). Ma contig aya akabva aputirwa ne e-values ​​​​ye 1e-10 tichishandisa BLASTN neBLASTX. Nzira iyi yakatibvumira kuona 482 non-bivalve fragments dziri muA. atra ccfDNA. Zvinopfuura hafu (57%) yezvidimbu zveDNA izvi zvakatorwa kubva kumabhakitiriya, kunyanya kubva kuma gill symbionts, kusanganisira sulfotrophic symbionts, uye kubva kuma gill symbionts Solemya velum (Mufananidzo 5).
Kuwanda kwemhando dzakasiyana. B Kusiyana kwezvipembenene zve phyla mbiri huru (Firmicutes neProteobacteria). Kuwedzerwa kweddPCR C Vibrio spp. A. Zvidimbu zve 16S rRNA gene (bhuruu) muzvikamu zvitatu zve atra hemolymph.
Huwandu hwezvinhu zvakaunganidzwa zvinosvika 482 zvakaongororwa. Hunhu hwese hwekupararira kwetaxonomic kwe metagenomic contig annotations (prokaryotes ne eukaryotes). B Kugoverwa kwakadzama kwezvidimbu zveDNA rebhakitiriya zvakaonekwa neBLASTN neBLASTX.
Ongororo yeKraken2 yakaratidzawo kuti mussel ccfDNA yaive nezvidimbu zveDNA zvekare, kusanganisira zvidimbu zveDNA zveEuryarchaeota (65%), Crenarchaeota (24%), uye Thaurmarcheota (11%) (Mufananidzo 6a). Kuvapo kwezvidimbu zveDNA zvakatorwa kubva kuEuryarchaeota neCrenarchaeota, zvaimbowanikwa munharaunda yezvipembenene zvemuCalifornian mussels, hazvifanirwe kushamisa [42]. Kunyangwe Euryarchaeota ichiwanzobatanidzwa nemamiriro ezvinhu akaipisisa, ikozvino zvave kuzivikanwa kuti Euryarchaeota neCrenarcheota zviri pakati pemaprokaryotes akajairika munzvimbo yegungwa ine cryogenic [43, 44]. Kuvapo kwezvipembenene zvemethanogenic mu mussels hakushamisi, tichifunga nezvemishumo yazvino yekudonha kukuru kwemethane kubva pakudonha pasi paKerguelen Plateau [45] uye mukana wekugadzirwa kwemethane yemicrobial wakaonekwa pamhenderekedzo yeKerguelen Islands [46].
Takabva tatanga kufunga nezvemavhairasi eDNA. Sekuziva kwedu, iyi ndiyo yekutanga ongororo yehuwandu hwevhairasi iri mu mussels. Sezvataitarisira, takawana zvidimbu zveDNA zvebacteriophages (Caudovirales) (Mufananidzo 6b). Zvisinei, DNA yevhairasi inowanzo wanikwa inobva mu phylum ye nucleocytoviruses, inozivikanwawo se nuclear cytoplasmic large DNA virus (NCLDV), iyo ine genome yakakura kupfuura chero vhairasi. Mukati me phylum iyi, ma DNA sequences mazhinji ndeemhuri yeMimimidoviridae (58%) nePoxviridae (21%), ayo ane mhuka dzemusana dzinosanganisira vertebrates ne arthropods, nepo chikamu chidiki cheDNA sequences idzi chiri che virological algae inozivikanwa. Inokanganisa marine eukaryotic algae. Ma sequences aya akawanikwawo kubva kuPandora virus, vhairasi hombe ine genome yakakura kupfuura chero genera revhairasi rinozivikanwa. Zvinofadza kuti, huwandu hwevaya vanozivikanwa kuti vane hutachiona, sezvakatsanangurwa ne hemolymph ccfDNA sequencing, hwaive hwakakura (Mufananidzo S3, Supplementary Information). Hunosanganisira mavhairasi anokonzera zvipembenene zvakaita seBaculoviridae neIridoviridae, pamwe nemavhairasi anokonzera amoeba, algae ne vertebrates. Takawanawo ma sequences akafanana ne genome rePithovirus sibericum. Ma pitoviruses (anozivikanwawo se "zombie viruses") akatanga kuparadzaniswa kubva ku permafrost ine makore 30,000 muSiberia [47]. Saka, zvatakawana zvinoenderana nemishumo yapfuura inoratidza kuti haasi marudzi ese emazuva ano emavhairasi aya akatsakatika [48] uye kuti mavhairasi aya anogona kunge aripo munzvimbo dziri kure dzegungwa.
Pakupedzisira, takaedza kuti tione kana taigona kuwana zvidimbu zveDNA kubva kune dzimwe mhuka dzine masero akawanda. BLASTN neBLASTX zvakawanikwa neBLASTN neBLASTX nemabhuku e nt, nr neRefSeq (genomic neprotein). Zvakabuda muongororo yedu zvinoratidza kuti pakati pezvidimbu zvekunze zveccfDNA yemhuka dzine masero akawanda DNA yemapfupa emapfupa ndiyo inonyanya kuoneka (Mufananidzo 5). Zvidimbu zveDNA kubva kuzvipembenene nedzimwe mhando zvakawanikwawo. Chikamu chikuru chezvidimbu zveDNA hachina kuonekwa, zvichida nekuda kwekushaikwa kwehuwandu hwakawanda hwemarudzi emugungwa mumadatabase e genomic zvichienzaniswa nemarudzi epasi [49].
Mupepa razvino, tinoshandisa pfungwa yeLB kune mussels, tichiti hemolymph ccfDNA shot sequencing inogona kupa ruzivo pamusoro pekuumbwa kwezvakatipoteredza zvegungwa. Zvikuru, takaona kuti 1) mussel hemolymph ine huwandu hwakanyanya (microgram levels) yezvidimbu zveDNA zvakakura (~1-5 kb) zvinotenderera; 2) izvi zvidimbu zveDNA zvakazvimiririra uye hazvizvimiririre 3) Pakati pezvinhu zvekunze zvezvidimbu izvi zveDNA, takawana DNA yemabhakitiriya, yekare uye yehutachiona, pamwe neDNA yemamwe mhuka akawanda; 4) Kuunganidzwa kwezvidimbu izvi zveCCFDNA zvekunze muhemolymph kunoitika nekukurumidza uye kunobatsira mukusefa kwemukati kwemussels. Mukupedzisa, chidzidzo chedu chinoratidza kuti pfungwa yeLB, iyo kusvika pari zvino yakashandiswa zvakanyanya mumunda we biomedicine, inoburitsa ruzivo rwakapfuma asi rusina kuongororwa runogona kushandiswa kunzwisisa zviri nani kudyidzana pakati pemarudzi e sentinel nenzvimbo dzawo.
Pamusoro pemhuka dzerudzi rweprimates, ccfDNA isolation yakataurwa mumhuka dzinoyamwisa, kusanganisira mbeva, imbwa, katsi, nemabhiza [50, 51, 52]. Zvisinei, maererano neruzivo rwedu, chidzidzo chedu ndicho chekutanga kushuma kuonekwa nekutevedzana kweccfDNA mumarudzi egungwa ane system yekutenderera yakavhurika. Hunhu uhwu hwemuviri uye kugona kusefa kwemussels zvinogona, zvishoma nezvishoma, kutsanangura hunhu hwakasiyana hwezvidimbu zveDNA zvinotenderera zvichienzaniswa nezvimwe zvipenyu. Muvanhu, zvidimbu zvakawanda zveDNA zvinotenderera muropa zvidimbu zvidiki zvinotangira pa150 kusvika 200 bp. zvine peak yepamusoro ye167 bp [34, 53]. Chikamu chidiki asi chakakosha chezvidimbu zveDNA chiri pakati pe300 ne500 bp muhukuru, uye inenge 5% yakareba kupfuura 900 bp. [54]. Chikonzero chekupararira kwehukuru uhwu ndechekuti sosi huru yeccfDNA muplasma inokonzerwa nekufa kwemasero, kungave nekuda kwekufa kwemasero kana nekuda kwekufa kwemasero eropa anotenderera muvanhu vane hutano kana nekuda kweapoptosis yemasero egomarara muvarwere vegomarara (inozivikanwa secirculating tumor DNA). , ctDNA). Kupararira kwehukuru hwehemolymph ccfDNA yatakawana muhombs kwaitangira pa1000 kusvika 5000 bp, zvichiratidza kuti mussel ccfDNA ine mavambo akasiyana. Iyi ifungidziro ine musoro, sezvo hombs dzine semi-open vascular system uye dzinogara munzvimbo dzegungwa dzine huwandu hwakawanda hwe microbial genomic DNA. Kutaura zvazviri, kuedza kwedu kwerabhoritari tichishandisa exogenous DNA kwakaratidza kuti hombs dzinounganidza zvidimbu zveDNA mumvura yegungwa, mushure memaawa mashoma dzinoora mushure mekutorwa kwemasero uye/kana kuburitswa uye/kana kuchengetwa mumasangano akasiyana-siyana. Zvichienderana nekushaikwa kwemasero (ese ari maviri prokaryotic ne eukaryotic), kushandiswa kwezvikamu zvemukati mevhavha kuchaderedza huwandu hweccfDNA kubva kumasero ega pamwe nekubva kune dzimwe nyika. Tichifunga nezvekukosha kwekudzivirirwa kwemuviri kwevaviri uye huwandu hwakawanda hwemaphagocytes anotenderera, takafungidzira zvakare kuti kunyange ccfDNA yekune dzimwe nyika inowedzerwa mumaphagocytes anotenderera anounganidza DNA yekune dzimwe nyika kana apinda mutupukanana uye/kana tsvina yemasero. Kana zvatorwa pamwe chete, zvatakawana zvinoratidza kuti bivalve hemolymph ccfDNA inzvimbo yakasarudzika yeruzivo rwemamorekuru uye inosimbisa chinzvimbo chavo serudzi rwevarindi.
Data redu rinoratidza kuti kutevedzana uye kuongorora zvidimbu zve hemolymph ccfDNA zvinobva kubhakitiriya zvinogona kupa ruzivo rwakakosha nezve maruva ebhakitiriya aripo uye mabhakitiriya aripo munzvimbo dzakapoteredza dzegungwa. Maitiro ekutevera shot sequencing akaratidza kutevedzana kwe commensal bacteria A. atra gill ingadai isina kushaikwa dai nzira dzekuziva dze 16S rRNA dzakashandiswa, nekuda kwechikonzero chekusarura kwe library. Kutaura zvazviri, kushandisa kwedu data reLB rakaunganidzwa kubva kuM. platensis muchikamu chimwe chete che mussel paKerguelen kwakaratidza kuti kuumbwa kwe bacterial symbionts dzinobatanidzwa ne gill kwaive kwakafanana kune ese marudzi e mussel (Mufananidzo S4, Supplementary Information). Kufanana uku kwe mussels mbiri dzakasiyana majini kunogona kuratidza kuumbwa kwe bacterial communities mu Kerguelen inotonhora, ine sulfurous, uye ine volcanic [55, 56, 57, 58]. Mazinga akakwira e microorganisms anoderedza sulfur akatsanangurwa zvakanaka pakukohwa mussels kubva munzvimbo dzegungwa dzakabikwa [59], dzakadai semahombekombe ePort-au-France. Imwe mukana ndeyekuti zvirimwa zvemussel zvinogona kukanganiswa nekupararira kwehove [60, 61]. Kutsvagurudza kwakawanda kunodiwa kuti uone hukama huripo pakati penzvimbo yegungwa, pamusoro pegungwa, uye kuumbwa kwebhakitiriya rinobatana muhove. Zvidzidzo izvi zviri kuenderera mberi parizvino.
Kureba uye huwandu hwe hemolymph ccfDNA, nyore kwayo kucheneswa, uye mhando yepamusoro yekubvumira kukurumidza kutevedzana kwepfuti ndezvimwe zvezvakanakira zvakawanda zvekushandisa mussel ccfDNA kuongorora zvipenyu zvakasiyana-siyana mugungwa. Nzira iyi inonyanya kushanda pakuratidza nharaunda dzehutachiona (viromes) mune imwe ecosystem [62, 63]. Kusiyana nemabhakitiriya, archaea, uye eukaryotes, majenomu ehutachiona haana majini akachengetedzwa ne phylogenetically akadai se 16S sequences. Zvatakawana zvinoratidza kuti liquid biopsies kubva kumarudzi ezviratidzo akadai se mussels anogona kushandiswa kuona huwandu hwakawanda hwezvidimbu zve ccfDNA virus zvinozivikanwa kuti zvinotapukira vanhu vanogara mugungwa ecosystems. Izvi zvinosanganisira mavhairasi anozivikanwa kuti anotapukira protozoa, arthropods, zvipembenene, zvirimwa, uye mavhairasi ebhakitiriya (semuenzaniso, bacteriophages). Kupararira kwakafanana kwakawanikwa patakaongorora hemolymph ccfDNA virome ye blue mussels (M. platensis) yakaunganidzwa mu mussel layer imwe chete paKerguelen (Tafura S2, Ruzivo Rwekuwedzera). Kutevedzana kwepfuti dzeccfDNA inzira itsva iri kuwedzera simba mukudzidza nezve virome yevanhu kana mamwe marudzi [21, 37, 64]. Nzira iyi inonyanya kubatsira pakudzidza mavhairasi eDNA ane tambo mbiri, sezvo pasina jini rimwe chete rakachengetwa pakati pemavhairasi ese eDNA ane tambo mbiri, anomiririra boka remavhairasi akasiyana-siyana uye akafara muBaltimore [65]. Kunyangwe akawanda emavhairasi aya achiri asina kuiswa muzvikamu uye anogona kusanganisira mavhairasi anobva kuchikamu chisingazivikanwe zvachose chenyika yemavhairasi [66], takaona kuti maviromes nemarudzi emurwi wemussels A. atra neM. platensis ari pakati pemarudzi maviri aya. zvakafanana (ona mufananidzo S3, rumwe ruzivo). Kufanana uku hakushamisi, sezvo kunogona kuratidza kusasarudzwa kwekutora DNA iri munzvimbo. Zvidzidzo zvemangwana zvinoshandisa RNA yakacheneswa parizvino zvinodiwa kuti zvitsanangure RNA virome.
Mukudzidza kwedu, takashandisa nzira yakaoma zvikuru yekubvisa zvinhu zvakaunganidzwa pamwe chete uye zvakasanganiswa ne ccfDNA isati yaungana uye yapera, zvikaita kuti pave nehuwandu hwakawanda hwezvinhu zvisina kurongwa. Saka, hatigoni kuramba kuti zvimwe zvezvinhu izvi zvisina kurongwa zvinogona kunge zvichiri nekwazvakatangira, kunyanya nekuti hatina genome reference yemhando iyi ye mussel. Takashandisawo nzira iyi yekufambisa nekuti tainetseka nezve chimera pakati pezvinhu zvakazvimiririra nezvisiri zvemuviri uye urefu hwezvinhu zvakaverengwa hunogadzirwa ne Illumina MiSeq PE75. Chimwe chikonzero chehuwandu hwezvinhu zvisina kurongwa ndechekuti hutachiona hwakawanda hwemugungwa, kunyanya munzvimbo dziri kure dzakadai seKerguelen, hahuna kunyorwa. Takashandisa Illumina MiSeq PE75, tichifunga kuti urefu hwezvidimbu zveccfDNA hwakafanana neccfDNA yevanhu. Kune zvidzidzo zvemangwana, tichifunga nezvemhedzisiro yedu inoratidza kuti hemolymph ccfDNA ine kuverenga kwenguva yakareba kupfuura vanhu uye/kana mhuka dzinoyamwisa, tinokurudzira kushandisa sequencing platform yakakodzera zvidimbu zveccfDNA zvakareba. Maitiro aya achaita kuti zvive nyore kuona zviratidzo zvakawanda zvekuongorora kwakadzama. Kuwana A. atra nuclear genome sequence yakakwana iripo parizvino kwaizobatsirawo zvikuru kusiyanisa ccfDNA kubva kune yega uye isiri yega. Tichifunga kuti tsvakiridzo yedu yakatarisana nekukwanisa kushandisa pfungwa ye liquid biopsy kune mussels, tinotarisira kuti sezvo pfungwa iyi ichizoshandiswa mukutsvagisa mune ramangwana, maturusi matsva nemapipeline zvichagadzirwa kuti zviwedzere mukana weiyi nzira yekudzidza kusiyana kwema microbial emussels.
Sechiratidzo chekiriniki chisingapindike, huwandu hwakakwira hweCCFDNA muropa revanhu hunobatanidzwa nezvirwere zvakasiyana-siyana, kukuvara kwenyama, uye mamiriro ekushushikana [67,68,69]. Kuwedzera uku kwakabatana nekuburitswa kwezvidimbu zveDNA zvaro mushure mekukuvadzwa kwenyama. Takagadzirisa nyaya iyi tichishandisa kupisa kwakanyanya, umo mussels dzakaratidzwa kwenguva pfupi patembiricha ye30 °C. Takaita ongororo iyi pamhando nhatu dzakasiyana dzemussels mukuyedza katatu kwakazvimirira. Zvisinei, hatina kuwana shanduko mumazinga eCCFDNA mushure mekushushikana kwekupisa kwakanyanya (ona Mufananidzo S5, rumwe ruzivo). Kuwanikwa uku kunogona kutsanangura, kanenge muchikamu, chokwadi chekuti mussels dzine semi-open circulatory system uye dzinounganidza huwandu hwakawanda hweDNA yekune dzimwe nyika nekuda kwebasa radzo rekusefa zvakanyanya. Kune rumwe rutivi, mussels, sezvizhinji zvezvipenyu zvisina mapfupa, dzinogona kuramba zvakanyanya kukuvara kwenyama kunokonzerwa nekushushikana, nokudaro zvichideredza kuburitswa kweCCFDNA muhemolymph yadzo [70, 71].
Kusvika pari zvino, kuongororwa kweDNA yehupenyu hwemarudzi akasiyana-siyana muzvipenyu zvemumvura kwave kunonyanya kutarisa pakuongorora DNA yezvakatipoteredza (eDNA). Zvisinei, nzira iyi inowanzo shotgun mukuongorora zvipenyu zvakasiyana-siyana kana ma primer achishandiswa. Kushandiswa kwe shotgun sequencing kunodzivirira miganho yePCR uye kusarudzwa kwakasarudzika kwema primer sets. Saka, neimwe nzira, nzira yedu iri pedyo nenzira ichangobva kushandiswa ye high-throughput eDNA Shotgun sequencing, iyo inokwanisa kurongeka zvakananga DNA yakakamurwa uye kuongorora zvinenge zvipenyu zvese [72, 73]. Zvisinei, kune nyaya dzinoverengeka dzakakosha dzinosiyanisa LB kubva kune standard eDNA methods. Ehe, musiyano mukuru pakati pe eDNA neLB kushandiswa kwe natural filter hosts. Kushandiswa kwemhando dzegungwa dzakadai sesponges ne bivalves (Dresseina spp.) se natural filter yekudzidza eDNA kwakataurwa [74, 75]. Zvisinei, chidzidzo chaDreissena chakashandisa tissue biopsies iyo DNA yakabviswa mairi. Kuongororwa kweccfDNA kubva kuLB hakudi tissue biopsy, michina yakasarudzika uye dzimwe nguva inodhura uye logistics ine chekuita ne eDNA kana tissue biopsy. Kutaura zvazviri, munguva pfupi yapfuura takashuma kuti ccfDNA kubva kuLB inogona kuchengetwa uye kuongororwa nerutsigiro rweFTA pasina kuchengetedza cold chain, inova dambudziko guru rekutsvagisa munzvimbo dziri kure [76]. Kubviswa kweccfDNA kubva ku liquid biopsies kuri nyorewo uye kunopa DNA yepamusoro ye shotgun sequencing uye PCR analysis. Iyi mukana mukuru tichifunga nezvezvimwe zvipingamupinyi zvehunyanzvi zvine chekuita ne eDNA analysis [77]. Kureruka uye mutengo wakaderera wenzira ye sampling kwakakodzerawo zvakanyanya kune zvirongwa zvekutarisa kwenguva refu. Kuwedzera pakugona kwavo kusefa kwakanyanya, chimwe chinhu chinozivikanwa che bivalves ikemikari mucopolysaccharide composition yemucus yavo, iyo inokurudzira kunyudzwa kwemavhairasi [78, 79]. Izvi zvinoita kuti bivalves ive sefa yakanaka yechisikigo yekuratidza biodiversity uye kukanganisa kwekuchinja kwemamiriro ekunze mune imwe nzvimbo yemvura. Kunyangwe kuvapo kwezvidimbu zveDNA zvinobva kuhost kuchigona kuonekwa sekuganhurirwa kwenzira iyi zvichienzaniswa ne eDNA, mutengo unobatanidzwa nekuva ne ccfDNA yemuno yakadaro uchienzaniswa ne eDNA unonzwisisika panguva imwe chete nekuda kweruzivo rwakakura rwuripo rwezvidzidzo zvehutano. Izvi zvinosanganisira kuvapo kwehutachiona hwakatevedzana hwakabatanidzwa mu genome yehost host. Izvi zvakakosha zvikuru kune mussels, tichifunga nezvekuvapo kwe leukemic retroviruses dzakatapuriranwa dzakatarisana mu bivalves [80, 81]. Imwe mukana we LB kupfuura eDNA ndeyekuti inoshandisa phagocytic activity yemasero eropa anotenderera mu hemolymph, iyo inonyudza tupukanana (uye majini avo). Phagocytosis ndiyo basa guru remasero eropa mu bivalves [82]. Chekupedzisira, nzira iyi inoshandisa mukana wepamusoro wekusefa kwe mussels (avhareji 1.5 l/h yemvura yegungwa) uye kutenderera kwemazuva maviri, izvo zvinowedzera kusanganiswa kwezvikamu zvakasiyana zvemvura yegungwa, zvichibvumira kubatwa kwe heterologous eDNA. [83, 84]. Saka, kuongorora kwemussel ccfDNA inzira inonakidza tichifunga nezvekudya, hupfumi, uye nharaunda zvinokanganisa mussels. Kufanana nekuongorora kweLB yakatorwa kubva kuvanhu, nzira iyi inovhurawo mukana wekuyera shanduko dzemajini uye epigenetic muDNA yemugari mukupindura kune zvinhu zvekunze. Semuenzaniso, matekinoroji ekutevera echizvarwa chechitatu anogona kufungidzirwa kuita ongororo ye methylation ye genome-wide muccfDNA yemuno uchishandisa nanopore sequencing. Maitiro aya anofanira kufambiswa nekuti kureba kwezvidimbu zvemussel ccfDNA kunoenderana zvakanaka nemapuratifomu ekutevera akareba anobvumira ongororo yeDNA methylation ye genome-wide kubva pakutevedzana kumwe chete pasina chikonzero chekushandurwa kwemakemikari.85,86] Iyi mukana unonakidza, sezvo zvakaratidzwa kuti mapatani eDNA methylation anoratidza mhinduro yekushushikana kwezvakatipoteredza uye anogara kwezvizvarwa zvakawanda. Nokudaro, inogona kupa ruzivo rwakakosha pamusoro penzira dziri pasi dzinotonga mhinduro mushure mekusangana nekuchinja kwemamiriro ekunze kana kusvibiswa [87]. Zvisinei, kushandiswa kweLB hakusi kusina miganhu. Hazvina basa kutaura, izvi zvinoda kuvapo kwemarudzi ezviratidzo mu ecosystem. Sezvambotaurwa pamusoro apa, kushandisa LB kuongorora kusiyana-siyana kwenzvimbo yakapihwa kunodawo nzira yakasimba ye bioinformatics inotarisa kuvapo kwezvidimbu zveDNA kubva kunobva. Chimwe chinetso chikuru kuwanikwa kwema genomes emhando dzegungwa. Zvinotarisirwa kuti zvirongwa zvakaita seMarine Mammal Genomes Project neFish10k project ichangobva kutangwa [88] zvichabatsira ongororo yakadaro mune ramangwana. Kushandiswa kwepfungwa yeLB kune zvipenyu zvinopa firita kunoenderanawo nekufambira mberi kwazvino mu sequencing technology, zvichiita kuti ive yakakodzera kugadzirwa kwe multi-ohm biomarkers kupa ruzivo rwakakosha nezve hutano hwenzvimbo dzegungwa mukupindura kushushikana kwezvakatipoteredza.
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