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Liquid biopsy (LB) ipfungwa iri kukurumidza kuwana mukurumbira mumunda webiomedical.Pfungwa iyi inonyanya kubva pakuonekwa kwezvimedu zvekutenderera extracellular DNA (ccfDNA), izvo zvinonyanya kuburitswa sezvimedu zvidiki mushure mekufa kwesero mumatishu akasiyana.Chikamu chidiki chezvimedu izvi chinobva kune kunze (kunze) tishu kana zvipenyu.Mubasa razvino, takashandisa pfungwa iyi kune mussels, mhando yemurindi inozivikanwa nekukwira kwayo kwesefa yemvura yegungwa.Isu tinoshandisa kugona kwemussels kuita semasefa echisikigo kutora zvidimbu zveDNA zvezvakatipoteredza kubva kwakasiyana-siyana kuti tipe ruzivo rwekusiyana-siyana kwezvipenyu zvegungwa zvegungwa.Zvatinowana zvinoratidza kuti mussel hemolymph ine zvidimbu zveDNA zvinosiyana zvakanyanya muhukuru, kubva pa1 kusvika pa5 kb.Shotgun sequencing yakaratidza kuti nhamba huru yezvimedu zveDNA ndezvekune dzimwe nyika hutachiona.Pakati pavo, takawana zvidimbu zveDNA kubva kubhakitiriya, archaea, uye mavhairasi, kusanganisira mavhairasi anozivikanwa kutapurira mauto akasiyana-siyana anowanikwa mumahombekombe egungwa ecosystems.Mukupedzisa, kudzidza kwedu kunoratidza kuti pfungwa yeLB inoshandiswa kune mussels inomiririra hupfumi asi husati hwaongororwa ruzivo pamusoro pekusiyana kwehutachiona mumakungwa egungwa ecosystems.
Kukanganisa kwekushanduka kwemamiriro ekunze (CC) pane zvakasiyana-siyana zvezvipenyu zvegungwa inzvimbo iri kukurumidza kukura yetsvagiridzo.Kupisa kwepasi hakungokonzereri kushushikana kwemuviri, asi kunosundira miganho yekushanduka-shanduka kwekugadzikana kwekupisa kwezvipenyu zvemugungwa, zvichikanganisa hugaro hwemarudzi akati wandei, zvichiita kuti vatsvage mamwe mamiriro akanaka [1, 2].Pamusoro pekukanganisa kusiyana-siyana kwezvipenyu zvemetazoan, CC inovhiringa chiyero chakapfava chekudyidzana kweanotambira utachiona.Iyi microbial dysbacteriosis inotyisidzira zvakanyanya kune zvipenyu zvegungwa sezvo ichiita kuti zvipenyu zvemugungwa zvive nyore kutapukirwa nehutachiona [3, 4].Zvinotendwa kuti SS inoita basa rakakosha mukufa kwevazhinji, iro idambudziko rakakura kune manejimendi epasi rose ecosystems [5, 6].Iyi inyaya yakakosha yakapihwa kukonzeresa kwehupfumi, kwezvakatipoteredza uye chikafu chemarudzi mazhinji emugungwa.Izvi zvinonyanya kuitika kune bivalves inogara munzvimbo dzepolar, uko migumisiro yeCK inokurumidza uye yakaoma [6, 7].Muchokwadi, bivalves senge Mytilus spp.anoshandiswa zvakanyanya kutarisa mhedzisiro yeCC pane zvipenyu zvegungwa.Hazvishamise kuti nhamba yakakura kwazvo yezvinyorwa zvakagadziridzwa kuti zvitarise hutano hwavo, kazhinji vachishandisa nzira mbiri-tier inosanganisira zvinoshanda biomarkers zvichibva pane enzymatic chiitiko kana cellular mabasa senge cell viability uye phagocytic chiitiko [8].Nzira idzi dzinosanganisirawo kuyerwa kwehuwandu hwezviratidzo zvekumanikidzika zvinoungana mumasumbu akapfava mushure mekutorwa kwemvura zhinji yegungwa.Nekudaro, iyo yakanyanya kusefa simba uye semi-yakavhurika circulatory system yebivalves inopa mukana wekugadzira mitsva yehemolymph biomarkers uchishandisa iyo pfungwa yemvura biopsy (LB), nzira iri nyore uye shoma inopindira kune murwere manejimendi.sampuli dzeropa [9, 10].Kunyangwe mhando dzinoverengeka dzemamorekuru anotenderera achigona kuwanikwa muLB yemunhu, pfungwa iyi inonyanya kuenderana neDNA sequencing ongororo yekutenderera extracellular DNA (ccfDNA) zvimedu muplasma.Kutaura zvazviri, kuvapo kwekutenderera kweDNA muplasma yevanhu kwave kuchizivikanwa kubva pakati pezana remakore rechi20 [11], asi ingori mumakore achangopfuura apo kuuya kwepamusoro-soro nzira dzekugadzirisa zvakakonzera kuongororwa kwekliniki kubva paccfDNA.Kuvapo kwezvimedu zveDNA zvinotenderera izvi kunokonzerwa nechikamu chekuburitswa kwegenomic DNA (nuclear uye mitochondrial) mushure mekufa kwesero. Muvanhu vane utano hwakanaka, kuwanda kweccfDNA kunowanzova kwakaderera (<10 ng/mL) asi kunogona kuwedzerwa ne5-10 nguva kune varwere vanotambura nezvirwere zvakasiyana-siyana kana kuti vanomanikidzwa, zvichikonzera kukanganisa kwenyama. Muvanhu vane utano hwakanaka, kuwanda kweccfDNA kunowanzova kwakaderera (<10 ng/mL) asi kunogona kuwedzerwa ne5-10 nguva kune varwere vanotambura nezvirwere zvakasiyana-siyana kana kuti vanomanikidzwa, zvichikonzera kukanganisa kwenyama. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться 5–10 стрессу, приводящему к повреждению тканей. Muvanhu vane utano hwakanaka, kusanganiswa kwecccDNA kunowanzove kwakaderera (<10 ng / mL), asi inogona kuwedzera ne5-10 nguva kune varwere vane zvirwere zvakasiyana-siyana kana pasi pekunetseka kunotungamirira kukukuvara kwenyama.在健康个体中，ccfDNA的浓度通常较低（<10 ng/mL)致组织损伤。在 健康 个体 中 , ccfdna 的 浓度 较 低 （<10 ng/ml） 但 在 各 种 病理 或 承叅 压 承叅 压 1损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损伤 损Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут быть увеличены в 5-10 что приводит к повреждению тканей. ccfDNA concentrations inowanzove yakaderera (<10 ng/ml) muvanhu vane utano hwakanaka, asi inogona kuwedzerwa 5-10-fold kune varwere vane zvirwere zvakasiyana-siyana kana kushungurudzika, zvichiita kuti kukuvara kwenyama.Saizi yezvimedu zveccfDNA inosiyana zvakanyanya, asi kazhinji inotangira pa150 kusvika 200 bp.[12].Kuongororwa kweccfDNA yakazvimiririra, kureva, ccfDNA kubva kumasero akajairwa kana akashandurwa, anogona kushandiswa kuona genetic uye epigenetic shanduko iripo mune yenyukireya uye / kana mitochondrial genome, nokudaro kubatsira vanachiremba kuti vasarudze chaiwo molecular-targeted therapies [13].Zvisinei, ccfDNA inogona kuwanikwa kubva kune dzimwe nyika dzakadai seccfDNA kubva kumasero e fetus panguva yekuzvitakura kana kubva kune nhengo dzakasimwa [14,15,16,17].ccfDNA inzvimbo yakakosha yeruzivo rwekuona kuvepo kweiyo nucleic acids yeanotapukira agent (yekune imwe nyika), iyo inobvumira kusingaonekwe kwehutachiona hwehutachiona hwakapararira husingaonekwe netsika dzeropa, kudzivirira invasive biopsy yehutachiona hutachiwana [18].Zvidzidzo zvenguva pfupi yapfuura zvakaratidza kuti ropa remunhu rine ruzivo rwakapfuma runogona kushandiswa kuona hutachiona hwehutachiona uye hwehutachiona, uye kuti inenge 1% yeccfDNA inowanikwa muplasma yevanhu ndeyekune dzimwe nyika [19].Zvidzidzo izvi zvinoratidza kuti mutsauko wezvipenyu wekutenderera microbiome unogona kuongororwa uchishandisa ccfDNA ongororo.Zvisinei, kusvika munguva pfupi yapfuura, pfungwa iyi yakashandiswa chete muvanhu uye, kusvika pamwero muduku, mune mamwe vertebrates [20, 21].
Mubepa razvino, tinoshandisa iyo LB inogoneka kuongorora ccfDNA yeAulacomya atra, rudzi rwekumaodzanyemba runowanzo kuwanikwa muSuantarctic Kerguelen Islands, boka rezvitsuwa pamusoro pebani guru rakaumbwa makore 35 miriyoni apfuura.kuputika kwegomo.Tichishandisa in vitro experimental system, takaona kuti zvidimbu zveDNA mumvura yegungwa zvinokurumidza kutorwa nemhashu zvopinda muchikamu chehemolymph.Shotgun sequencing yakaratidza kuti mussel hemolymph ccfDNA ine zvimedu zveDNA zvayo uye zvisiri zvega, zvinosanganisira symbiotic bacteria uye zvidimbu zveDNA kubva kune biomes inotonhora yegungwa regungwa ecosystems.Hemolymph ccfDNA inewo mavhairasi sequences anotorwa kubva kumavhairasi ane akasiyana siyana.Takawanawo zvimedu zveDNA kubva kumhuka dzakawanda dzakaita sehove dzebhonzo, anemones emugungwa, algae uye tumbuyu.Mukupedzisa, kudzidza kwedu kunoratidza kuti iyo LB pfungwa inogona kushandiswa zvinobudirira kune zvipembenene zvemugungwa kuti ibudise yakapfuma genomic repertoire mumakungwa ecosystems.
Vakuru (55-70 mm kureba) Mytilus platensis (M. platensis) neAulacomya atra (A. atra) vakaunganidzwa kubva kumahombekombe ane matombo ePort-au-France (049°21.235 S, 070°13.490 E.).Zvitsuwa zveKerguelen munaDecember 2018. Mamwe mahombe ebhuruu evanhu vakuru (Mytilus spp.) akawanikwa kubva kune mutengesi wekutengesa (PEI Mussel King Inc., Prince Edward Island, Canada) uye akaiswa mumhepo inodzorwa tembiricha (4 ° C) aerated tank ine 10-20 L ye32 ‰ artificial brine.(artificial sea salt Reef Crystal, Instant Ocean, Virginia, USA).Pakuedza kwega kwega, kureba nekurema kwemateko ega ega zvakayerwa.
Protocol yemahara yakavhurika yechirongwa ichi inowanikwa online (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Nenguva pfupi, LB hemolymph yakaunganidzwa kubva kune abductor tsandanyama sezvakatsanangurwa [22].Iyo hemolymph yakajekeswa ne centrifugation pa 1200 × g kwemaminetsi matatu, iyo supernatant yakanga yakaomeswa nechando (-20 ° C) kusvikira yashandiswa.Kuzviparadzanisa uye kucheneswa kwecfDNA, masampuli (1.5-2.0 ml) akanyungudutswa uye akagadziriswa achishandisa NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) maererano nemirairo yemugadziri.ccfDNA yakachengetwa pa -80 ° C kusvikira kumwe kuongorora.Mune zvimwe zviedzo, ccfDNA yakaparadzaniswa uye yakacheneswa uchishandisa QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada).Yakanatswa DNA yakaverengerwa uchishandisa yakajairwa PicoGreen assay.Kugoverwa kwechidimbu checcfDNA yakaparadzaniswa yakaongororwa necapillary electrophoresis vachishandisa Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) vachishandisa High Sensitivity DNA Kit.Muedzo wakaitwa uchishandisa 1 µl yeccfDNA sampuro zvinoenderana nemirairo yemugadziri.
Nekutevedzana kwehemolymph ccfDNA zvidimbu, Génome Québec (Montreal, Quebec, Canada) akagadzira maraibhurari epfuti achishandisa Illumina DNA Mix kit yeIllumina MiSeq PE75 kit.A standard adapter (BioO) yakashandiswa.Mafaera e data mbishi anowanikwa kubva kuNCBI Sequence Read Archive (SRR8924808 uye SRR8924809).Hunhu hwekuverenga hwekutanga hwakaongororwa uchishandisa FastQC [23].Trimmomatic [24] yakashandiswa kuchekerera maadapter uye kuverengeka kwakashata.Shotgun inoverenga ine peya magumo yaive FLASH yakabatanidzwa mune yakareba kuverenga imwe chete ine zvishoma kupindirana kwe20 bp kudzivirira kusawirirana [25]. Kuverengerwa kwakaverengerwa kwakatsanangurwa neBLASTN vachishandisa bivalve NCBI Taxonomy dhatabhesi (e value <1e-3 uye 90% homology), uye masking yeyakaderera-yakaoma sequences yakaitwa uchishandisa DUST [26]. Kuverengerwa kwakaverengerwa kwakatsanangurwa neBLASTN vachishandisa bivalve NCBI Taxonomy dhatabhesi (e value <1e-3 uye 90% homology), uye masking yeyakaderera-yakaoma sequences yakaitwa uchishandisa DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN с использованием базы данных таксономии двустворчатых моллюсков NCBI-3 <br><br> маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Kuverengera kwakaverengerwa kwakatsanangurwa neBLASTN vachishandisa iyo NCBI bivalve taxonomy dhatabhesi (e kukosha <1e-3 uye 90% homology), uye yakaderera kuomarara kutevedzana 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，并使坏木DUST [26]掩蔽.使用 双 壳类 ncbi 分类（（（<1e-3 和 90% 同源） 用 用 注释 合并读数 , 并 使用如 列国 [26]掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩Объединенные чтения были аннотированы с помощью BLASTN с использованием таксономической базы даных двустворчатых моллюсков-NCBI а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Kuverengera kwakaverengerwa kwakatsanangurwa neBLASTN vachishandisa iyo NCBI bivalve taxonomic dhatabhesi (e kukosha <1e-3 uye 90% homology), uye yakaderera kuomarara kutevedzana masking yakaitwa uchishandisa DUST [26].Kuverenga kwakakamurwa kuita mapoka maviri: ane chekuita nenhevedzano yebivalve (pano inonzi kuzviverengera) uye isina hukama (isina kuzviverengera).Mapoka maviri akaungana akaparadzana achishandisa MEGAHIT kugadzira contigs [27].Zvichakadaro, kugoverwa kwetaxonomic yeaen microbiome kuverenga kwakarongedzwa uchishandisa Kraken2 [28] uye zvakajeka inomiririrwa neKrona pie chati paGirazi [29, 30].Iwo akanakisa kmers akatsunga kuve kmers-59 kubva kune yedu yekutanga miedzo. Self contigs yakabva yaonekwa nekuenderana neBLASTN (bivalve NCBI dhatabhesi, e value <1e-10 uye 60% homology) kune yekupedzisira dudziro. Self contigs yakabva yaonekwa nekuenderana neBLASTN (bivalve NCBI dhatabhesi, e value <1e-10 uye 60% homology) kune yekupedzisira dudziro. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, зная-010 e) нчательной аннотации. Self-contigs akazoonekwa nekufananidza neBLASTN (NCBI bivalve dhatabhesi, e value <1e-10 uye 60% homology) yekupedzisira rondedzero.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% 同源性）对齐來识别自身里行。然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% Затем были идентифицированы собственные контиги для окончательной аннотации путем сопоставления с BLASTN (база данных NCBI NCBI). е e <1e-10 uye гомология 60%). Self-contigs zvakazoonekwa kuti zviziviso zvekupedzisira nekufananidza neBLASTN (NCBI bivalve database, e value <1e-10 uye 60% homology). Mukufanana, nonself group contigs akatsanangurwa neBLASTN (nt NCBI dhatabhesi, e value <1e-10 uye 60% homology). Mukufanana, nonself group contigs akatsanangurwa neBLASTN (nt NCBI dhatabhesi, e value <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%). Mukufanana, contigs yeboka rekunze yakatsanangurwa neBLASTN (NT NCBI dhatabhesi, e kukosha <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%). Mukufanana, ma contigs asiri ega ega akatsanangurwa neBLASTN (nt NCBI dhatabhesi, e kukosha <1e-10 uye 60% homology). BLASTX yakaitwawo pane nonself contigs vachishandisa nr uye RefSeq protein NCBI dhatabhesi (e value <1e-10 uye 60% homology). BLASTX yakaitwawo pane nonself contigs vachishandisa nr uye RefSeq protein NCBI dhatabhesi (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 kune vasiri vega contigs vachishandisa nr uye RefSeq NCBI mapuroteni dhatabhesi (e kukosha <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 kune vasiri-self contigs vachishandisa nr uye RefSeq NCBI mapuroteni dhatabhesi (e kukosha <1e-10 uye 60% homology).Iwo BLASTN neBLASTX madziva evasiri-self-contigs anomiririra ekupedzisira contigs (ona Supplementary faira).
Iwo maprimers anoshandiswa kuPCR akanyorwa muTable S1.Taq DNA polymerase (Bio Basic Canada, Markham, ON) yakashandiswa kukwidziridza ccfDNA yakananga majini.Mamiriro ezvinhu anotevera akashandiswa: denaturation pa 95 ° C kwemaminitsi matatu, 95 ° C kweminiti imwe, kuisa annealing tembiricha kweminiti 1, elongation pa 72 ° C kweminiti 1, 35 kutenderera, uye pakupedzisira 72 ° C mukati memaminitsi gumi..PCR zvigadzirwa zvakapatsanurwa ne electrophoresis mune agarose gels (1.5%) ine SYBRTM Yakachengeteka DNA Gel Stain (Invitrogen, Burlington, ON, Canada) pa95 V.
Mussels (Mytilus spp.) akagadziridzwa mu500 ml yemvura yegungwa ine okisijeni (32 PSU) kwemaawa makumi maviri nemana pa4 ° C.Plasmid DNA ine inoisa encoding yemunhu galectin-7 cDNA sequence (NCBI accession number L07769) yakawedzerwa kune vial pakusangana kwekupedzisira kwe190 μg/μl.Mussels akaiswa pasi pemamiriro akafanana pasina DNA kuwedzera aive kutonga.Tangi yechitatu yekudzora yaive neDNA isina mussels.Kuti utarise kunaka kweDNA mumvura yegungwa, sampuli dzemvura yegungwa (20 μl; kudzokorora katatu) dzakatorwa kubva patangi rimwe nerimwe panguva yakatarwa.Nezve plasmid DNA traceability, LB mussels yakakohwewa panguva dzakatarwa uye yakaongororwa neqPCR uye ddPCR.Nekuda kwehuwandu hwemunyu hwemvura yegungwa, maaliquots akaderedzwa muPCR yemhando yemvura (1:10) isati yasvika yese PCR assays.
Digital droplet PCR (ddPCR) yakaitwa pachishandiswa BioRad QX200 protocol (Mississauga, Ontario, Canada).Shandisa tembiricha mbiri kuti uone tembiricha yakakwana (Table S1).Madonhwe akagadzirwa uchishandisa QX200 donhwe jenareta (BioRad).ddPCR yakaitwa sezvizvi: 95°C kwemaminetsi mashanu, 50 kutenderera kwe95°C kwe30s uye yakapihwa annealing tembiricha kwemaminiti 1 uye 72°C kwe30s, 4°C kwemaminetsi mashanu uye 90°C mukati memaminitsi mashanu.Huwandu hwemadonhwe uye maitiro akanaka (nhamba yemakopi / µl) akayerwa uchishandisa QX200 yekudonha kuverenga (BioRad).Samples ane madonhwe asingasviki gumi akarambwa.Patani kutonga hakuna kuitwa pese paiitwa ddPCR.
qPCR yakaitwa pachishandiswa Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) uye LGALS7 maprimers chaiwo.Ese maPCR ehuwandu akaitwa mu20 µl uchishandisa iyo QuantiFast SYBR Green PCR Kit (QIAGEN).qPCR yakatangwa ne 15 min incubation pa 95 ° C ichiteverwa ne40 cycles pa 95 ° C kwemasekonzi gumi uye pa 60 ° C kwemasekonzi makumi matanhatu nekuunganidzwa kwe data.Kunyunguduka kwakagadzirwa uchishandisa zviyero zvakatevedzana pa95 ° C ye5 s, 65 ° C ye60 s, uye 97 ° C pakupera kweqPCR.Imwe neimwe qPCR yakaitwa mumatatu, kunze kwemasampuli ekudzora.
Sezvo mbira dzichizivikanwa nekusefa kwakanyanya, takatanga taongorora kana dzichikwanisa kusefa nekuchengetedza zvidimbu zveDNA zviripo mumvura yegungwa.Taifarirawo kuti zvimedu izvi zvinoungana here musemi-open lymphatic system yavo.Isu takagadzirisa nyaya iyi tichiyedza nekutsvaga magumo ezvimedu zveDNA zvinonyungudika zvakawedzerwa kumatangi eblue mussel.Kufambisa kurondwa kwezvimedu zveDNA, takashandisa kunze (kwete pachedu) plasmid DNA ine munhu galectin-7 gene.ddPCR inoteedzera plasmid DNA zvimedu mumvura yegungwa uye mussels.Zvigumisiro zvedu zvinoratidza kuti kana uwandu hwezvidimbu zveDNA mumvura yegungwa hwakaramba huchienderana nekufamba kwenguva (kusvika kumazuva manomwe) mukushayikwa kwemussels, ipapo pamberi pemussels iyi nhanho inenge yakanyangarika zvachose mukati maawa masere (Fig. 1a, b).Zvimedu zveDNA exogenous zvakaonekwa zviri nyore mukati me15 min mu intravalvular fluid uye hemolymph (Fig. 1c).Izvi zvidimbu zvinogona kuramba zvichionekwa kusvika maawa mana mushure mekuratidzwa.Ichi chiitiko chekusefa maererano nezvidimbu zveDNA chinofananidzwa nebasa rekusefa kwebhakitiriya uye algae [31].Mhedzisiro iyi inoratidza kuti mussels anogona kusefa uye kuunganidza DNA yekunze mumakamuri avo emvura.
Hukama hwehuwandu hweplasmid DNA mumvura yegungwa muhupo (A) kana kusavapo (B) kwemhashu, kuyerwa neddPCR.MuA, mhedzisiro inoratidzwa sezvikamu, nemiganhu yemabhokisi anomiririra makumi manomwe neshanu uye makumi maviri neshanu muzana.Iyo yakashongedzwa logarithmic curve inoratidzwa mutsvuku, uye nzvimbo ine grey inomiririra iyo 95% yekuvimba nguva.Muna B, mutsara mutsvuku unomiririra zvinoreva uye mutsara webhuruu unomiririra 95% nguva yekuvimba yehutano.C Kuunganidzwa kweplasmid DNA muhemolymph uye valvular fluid ye mussels panguva dzakasiyana mushure mekuwedzerwa kweplasmid DNA.Mhedzisiro inounzwa semakopi akakwana akaonekwa/mL (±SE).
Tevere, takaongorora kwakabva ccfDNA mumhashu dzakaunganidzwa kubva pamibhedha yemhashu paKerguelen Islands, boka riri kure rezvitsuwa zvine simba shoma anthropogenic.Nechinangwa ichi, cccDNA kubva mussel hemolymphs yakaparadzaniswa uye yakacheneswa nenzira dzinowanzoshandiswa kuchenesa cccDNA yevanhu [32, 33].Takaona kuti avhareji hemolymph ccfDNA concentrations mumussels iri mu low micrograms per ml hemolymph range (ona Table S2, Supplementary Information).Urwu huwandu hwehuwandu hwakakura kupfuura muvanhu vane hutano (pasi nanograms pa milliliter), asi muzviitiko zvisingawanzoitiki, muvarwere vekenza, chiyero checcfDNA chinogona kusvika ma micrograms pa milliliter [34, 35].Ongororo yehukuru hwekugoverwa kwehemolymph ccfDNA yakaratidza kuti zvidimbu izvi zvinosiyana zvakanyanya muhukuru, kubva pa1000 bp kusvika 1000 bp.kusvika ku5000 bp (Fig. 2).Migumisiro yakafanana yakawanikwa uchishandisa silica-based QIAamp Investigator Kit, nzira inowanzoshandiswa musayenzi yezvesayenzi kuti ikurumidze kuparadzanisa uye kuchenesa DNA yejenomic kubva kune yakaderera DNA samples, kusanganisira ccfDNA [36].
Mumiririri ccfDNA electrophoregram yemussel hemolymph.Yakatorwa neNucleoSnap Plasma Kit (yepamusoro) uye QIAamp DNA Investigator Kit.B Violin plot inoratidza kugoverwa kwehemolymph ccfDNA concentrations (±SE) mumhashu.Mitsetse yakasviba uye tsvuku inomiririra yepakati uye yekutanga neyechitatu quartiles, zvichiteerana.
Inenge 1% yeccfDNA muvanhu uye primates ine kunze kwenyika [21, 37].Tichifunga nezve semi-yakavhurika circulatory system yemabivalves, microbial-rich-emvura yegungwa, uye saizi yekugoverwa kwemussel ccfDNA, takafungidzira kuti mussel hemolymph ccfDNA inogona kunge iine dziva rakapfuma uye rakasiyana remicrobial DNA.Kuti tiedze iyi hypothesis, takateedzera hemolymph ccfDNA kubva kuAulacomya atra samples yakaunganidzwa kubva kuKerguelen Islands, ichipa anopfuura 10 miriyoni kuverenga, 97.6% iyo yakapfuura kunaka kutonga.Kuverenga kwacho kwakazorongedzerwa zvichienderana neyako uye isiri-yega masosi uchishandisa iyo BLASTN uye NCBI bivalve dhatabhesi (Fig. S1, Supplementary Information).
Muvanhu, DNA yenyukireya uye mitochondrial inogona kuburitswa muropa [38].Zvisinei, muchidzidzo chezvino, hazvina kukwanisa kutsanangura zvakadzama nuclear yenyu genomic DNA yemamussels, zvichipiwa kuti A. atra genome haina kutevedzana kana kutsanangurwa.Zvisinei, takakwanisa kuziva zvimedu zveccfDNA zvekwakabva pachedu tichishandisa bivalve library (Fig. S2, Supplementary Information).Isu takasimbisawo kuvapo kwezvimedu zveDNA zvemabviro edu nekwakananga PCR kukwidziridzwa kweiyo A. atra majini akatevedzana (Fig. 3).Zvakangofanana, tichifunga kuti mitochondrial genome yeA. atra inowanikwa mudura reruzhinji, munhu anogona kuwana humbowo hwekuvapo kwemitochondrial ccfDNA zvidimbu muhemolymph yeA. atra.Kuvapo kwemitochondrial DNA zvidimbu zvakasimbiswa nePCR amplification (Fig. 3).
Mhando dzakasiyana-siyana dzemitochondrial dzakanga dziripo muhemolymph yeA. atra (mavara matsvuku - nhamba yemasheya: SRX5705969) uye M. platensis (blue dots - stock number: SRX5705968) yakasimbiswa nePCR.Mufananidzo wakatorwa kubva kuBreton et al., 2011 B Amplification yehemolymph supernatant kubva kuA. atra Yakachengetwa papepa reFTA.Shandisa 3 mm punch kuwedzera zvakananga kuPCR chubhu ine PCR musanganiswa.
Tichifunga nezvehuwandu hwehutachiona hwemukati mumvura yegungwa, isu takatanga takanangana nehunhu hwehutachiona hweDNA kutevedzana muhemolymph.Kuti tiite izvi, tinoshandisa nzira mbiri dzakasiyana.Nzira yekutanga yakashandisa Kraken2, algorithm-based sequence classification program inogona kuona microbial sequences nekurongeka kunoenzaniswa neBLAST nemamwe maturusi [28].Kupfuura 6719 kuverenga kwakatemwa kuva kwebhakitiriya yakabva, nepo 124 uye 64 yakabva kune archaea uye mavhairasi, maererano (Fig. 4).Zvimedu zvebhakteria zvakawanda zveDNA zvaive Firmicutes (46%), Proteobacteria (27%), uye Bacteroidetes (17%) (Fig. 4a).Uku kugoverwa kunopindirana nezvidzidzo zvekare zvegungwa rebhuruu mussel microbiome [39, 40].Gammaproteobacteria yaiva kirasi huru yeProteobacteria (44%), kusanganisira Vibrionales yakawanda (Fig. 4b).Nzira yeddPCR yakasimbisa kuvapo kweVibrio DNA zvidimbu muccfDNA yeA. atra hemolymph (Fig. 4c) [41].Kuti uwane mamwe mashoko pamusoro pebhakitiriya yakabva ccfDNA, imwe nzira yakatorwa (Fig. S2, Supplementary Information). Panyaya iyi, zvinoverengwa zvakaputirana zvakaunganidzwa sezviverengero zvepaviri-yekupedzisira uye zvakaiswa seyekuzvimiririra (bivalves) kana asiri wega mabviro uchishandisa BLASTN uye kukosha kwe1e-3 uye cutoff ne> 90% homology. Panyaya iyi, zvinoverengwa zvakaputirana zvakaunganidzwa sezviverengero zvepaviri-yekupedzisira uye zvakaiswa seyekuzvimiririra (bivalves) kana asiri wega mabviro uchishandisa BLASTN uye kukosha kwe1e-3 uye cutoff ne> 90% homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как были собраны как чтения с парными концами и были классифицированы как собственные безплатные происхождению с использованием BLASTN uye значения e 1e-3 uye отсечения с гомологией> 90%. Muchiitiko ichi, kuverenga kunopindirana kwakaunganidzwa sekuverenga-kwapera uye kwakarongedzerwa seyakaberekerwa (bivalve) kana isiri yepakutanga uchishandisa BLASTN uye kukosha kwe1e-3 uye cutoff ne> 90% homology.在這种情况下，重叠的話数组装為配对末端話数，并使用BLASTN 和1e-3 yako pamwe>90%类）或非自身來源.在 這 种 情况 下 , 重叠 读数 组装 為配 末端 读数 , 使用 使用 blastn 1e-3 自值% ne 90 （双 壳类） 非 自身。。。。。. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицированы как собственый как были собраны как чтения с парными концами и классифицированы как собственные (двуствонство) происхождению с использованием значений e BLASTN uye 1e-3 и порога гомологии> 90%. Muchiitiko ichi, kuverenga kunopindirana kwakaunganidzwa sekuverenga-kwapera uye kurongerwa seyako (bivalves) kana isiri-yekutanga uchishandisa e BLASTN uye 1e-3 kukosha uye chikumbaridzo chehomology> 90%.Sezvo iyo A. atra genome haisati yatevedzana, takashandisa de novo assembly strategy yeMEGAHIT Next Generation Sequencing (NGS) assembler.Huwandu hwe147,188 contigs vakaonekwa sevanovimba (bivalves) kwekwakabva.Aya ma contigs akabva aputika ne e-values ​​ye1e-10 vachishandisa BLASTN uye BLASTX.Iri zano rakaita kuti tikwanise kuziva 482 non-bivalve zvidimbu zviripo muA. atra ccfDNA.Inopfuura hafu (57%) yezvimedu zveDNA izvi zvakawanikwa kubva kubhakitiriya, kunyanya kubva kune gill symbionts, kusanganisira sulfotrophic symbionts, uye kubva kune gill symbionts Solemya velum (Fig. 5).
Kuwanda kwehukama pamhando yemhando.B Kusiyana-siyana kweMicrobial maviri makuru phyla (Firmicutes uye Proteobacteria).Mumiriri wekuwedzera kweddPCR C Vibrio spp.A. Zvimedu zve16S rRNA gene (blue) mumatatu atra hemolymphs.
Huwandu hwe482 hwakaunganidzwa contigs hwakaongororwa.Yakazara nhoroondo yekugovera taxonomic yemetagenomic contig annotations (prokaryotes uye eukaryotes).B Yakadzama kugovera kwebhakitiriya eDNA zvimedu zvakaonekwa neBLASTN neBLASTX.
Kraken2 analysis yakaratidzawo kuti mussel ccfDNA ine archaeal DNA fragments, kusanganisira DNA zvidimbu zveEuryarchaeota (65%), Crenarchaeota (24%), uye Thaurmarcheota (11%) (Fig. 6a).Kuvapo kwezvimedu zveDNA zvakatorwa kubva kuEuryarchaeota neCrenarchaeota, zvakambowanikwa munharaunda yemicrobial yeCalifornian mussels, hazvifanirwe kuuya sechishamiso [42].Kunyange zvazvo Euryarchaeota inowanzobatanidzwa nemamiriro ezvinhu akaipisisa, ikozvino yave kuzivikanwa kuti zvose Euryarchaeota neCrenarcheota ndezvimwe zveprokaryotes dzakawanda munzvimbo yegungwa cryogenic [43, 44].Kuvapo kwemethanogenic microorganisms mumhashu hazvishamise, zvichipihwa mishumo ichangoburwa yekudonha kwakawanda kwemethane kubva pasi pemvura paKerguelen Plateau [45] uye zvinogona kuitika kugadzirwa kwemethane methane kunoonekwa kubva kumahombekombe eKerguelen Islands [46].
Kutarisisa kwedu kwakabva kwachinja kuverenga kubva kuDNA virus.Sekuziva kwedu, iyi ndiyo yekutanga-isina chinangwa kudzidza yehutachiona hwemukati memussels.Sezvaitarisirwa, takawana zvidimbu zveDNA zve bacteriophages (Caudovirales) (Fig. 6b).Zvakadaro, hutachiona hweDNA huzhinji hunobva ku phylum ye nucleocytoviruses, inozivikanwawo senuclear cytoplasmic hombe DNA virus (NCLDV), iyo ine genome yakakura pane chero hutachiona.Mukati meiyi phylum, kutevedzana kweDNA kwakawanda ndekwemhuri Mimimidoviridae (58%) uye Poxviridae (21%), ane mauto echisikigo anosanganisira vertebrates uye arthropods, nepo chikamu chidiki cheizvi DNA sequences ndechezvinozivikanwa virological algae.Inokonzera marine eukaryotic algae.Kutevedzana kwacho kwakawanikwawo kubva kuhutachiona hwePandora, hutachiona hofori hune hukuru hukuru hwegenome yechero genera inozivikanwa yehutachiona.Sezvineiwo, huwandu hwemauto anozivikanwa kuti ane hutachiona, sekutsanangurwa kwehemolymph ccfDNA kutevedzana, yaive yakakura (Mufananidzo S3, Supplementary Information).Inosanganisira mavhairasi anotapurira zvipembenene zvakaita seBaculoviridae neIridoviridae, pamwe nemavhairasi anokanganisa amoeba, algae uye vertebrates.Isu takawanawo kutevedzana kunoenderana nePithovirus sibericum genome.Pitoviruses (inozivikanwawo se "zombie viruses") yakatanga kuparadzaniswa kubva ku30,000 year old permafrost muSiberia [47].Nekudaro, mhedzisiro yedu inopindirana nemishumo yapfuura inoratidza kuti hadzisi dzese marudzi emazuva ano evhairasi aya akatsakatika [48] uye kuti mavhairasi aya anogona kunge aripo munzvimbo dziri kure dzepasi pegungwa.
Pakupedzisira, takaedza kuona kana taigona kuwana zvidimbu zveDNA kubva kune dzimwe mhuka dzakawanda.Huwandu hwe482 ekunze contigs akaonekwa neBLASTN neBLASTX ine nt, nr uye RefSeq maraibhurari (genomic uye protein).Zvigumisiro zvedu zvinoratidza kuti pakati pezvimedu zvekune dzimwe nyika zveccfDNA yemhuka dzakasiyana-siyana DNA yemapfupa emapfupa inotangira (Fig. 5).Zvimedu zveDNA zvezvipembenene nemamwe marudzi zvakawanikwawo.Chikamu chikuru chezvimedu zveDNA hachina kuonekwa, zvichida nekuda kwekufananidzirwa kwenhamba yakawanda yezvipenyu zvegungwa muzvinyorwa zvejenomic zvichienzaniswa nemarudzi epasi [49].
Mune bepa razvino, isu tinoshandisa iyo LB pfungwa kune mussels, tichipokana kuti hemolymph ccfDNA pfuti sequencing inogona kupa nzwisiso mukuumbwa kwemakungwa egungwa ecosystem.Kunyanya, takaona kuti 1) mussel hemolymph ine huwandu hwepamusoro (microgram mazinga) ehukuru (~ 1-5 kb) inotenderera zvidimbu zveDNA;2) zvidimbu zveDNA izvi zvose zvakazvimiririra uye zvisina kusununguka 3) Pakati pemamwe marudzi ezvimedu zveDNA izvi, takawana mabhakitiriya, archaeal uye viral DNA, uyewo DNA yemamwe mhuka dzakawanda;4) Kuunganidzwa kwezvimedu zvekunze zveccfDNA muhemolymph kunoitika nekukurumidza uye kunobatsira mukusefa kwemukati kwemussels.Mukupedzisa, chidzidzo chedu chinoratidza kuti pfungwa yeLB, iyo yave ichishandiswa zvakanyanya mumunda webiomedicine, inoisa ruzivo rwakapfuma asi rusina kuongororwa runogona kushandiswa kunzwisisa zviri nani kuwirirana pakati pevarindi vemarudzi uye nharaunda yavo.
Kuwedzera kune primates, ccfDNA yekuzviparadzanisa yakataurwa mumhuka dzinoyamwisa, kusanganisira mbeva, imbwa, katsi, uye mabhiza [50, 51, 52].Nekudaro, kuruzivo rwedu, kudzidza kwedu ndiko kwekutanga kuzivisa kuwanikwa uye kutevedzana kweccfDNA mumhando dzemugungwa ine yakavhurika kutenderera system.Ichi cheanatomical chimiro uye kusefa kugona kwemussels kunogona, zvishoma muchikamu, kutsanangura akasiyana saizi maitiro ekutenderera zvidimbu zveDNA zvichienzaniswa nemamwe marudzi.Muvanhu, zvidimbu zveDNA zvakawanda zvinotenderera muropa zvimedu zvidiki zvinotangira pa150 kusvika pa200 bp.nepamusoro pepamusoro pe167 bp [34, 53].Chikamu chiduku asi chakakosha chezvidimbu zveDNA chiri pakati pe300 ne500 bp muhukuru, uye inenge 5% yakareba kudarika 900 bp.[54].Chikonzero chekugoverwa uku kwekukura ndechekuti iyo huru sosi yeccfDNA muplasma inoitika nekuda kwekufa kwesero, kungave nekuda kwekufa kwesero kana nekuda kwenecrosis yekutenderera hematopoietic masero muvanhu vane hutano kana nekuda kweapoptosis yebundu masero muvarwere vekenza (inozivikanwa sekutenderera bundu reDNA)., ctDNA).Saizi yekugoverwa kwehemolymph ccfDNA yatakawana mumasel kubva pa1000 kusvika pa5000 bp, zvichiratidza kuti mussel ccfDNA ine kwakabva kwakasiyana.Iyi ifungidziro ine musoro, sezvo mhashu dzine semi-yakavhurika vascular system uye inogara mugungwa remvura nharaunda ine yakanyanya kuwanda kweiyo microbial genomic DNA.Muchokwadi, kuedza kwedu murabhoritari tichishandisa exogenous DNA kwakaratidza kuti mussels anounganidza zvimedu zveDNA mumvura yegungwa, kanenge mushure memaawa mashoma anoderedzwa mushure mekutora masero uye / kana kuburitswa uye / kana kuchengetwa mumasangano akasiyana.Tichifunga nezve kushomeka kwemaseru (ese ari maviri eprokaryotic uye eukaryotic), kushandiswa kweiyo intravalvular compartments kunoderedza huwandu hweccfDNA kubva kuzvinobva uye kubva kune dzimwe nyika.Tichifunga nezvekukosha kwebivalve innate immune immune uye huwandu hukuru hwekutenderera phagocytes, isu tinoenderera mberi tichifungidzira kuti kunyangwe ccfDNA yekune dzimwe nyika inopfumiswa mukutenderera phagocytes inounganidza DNA yekune dzimwe nyika pakumedza tupukanana uye / kana cellular tsvina.Takatorwa pamwechete, mhedzisiro yedu inoratidza kuti bivalve hemolymph ccfDNA inzvimbo yakasarudzika yeruzivo rwemamorekuru uye inosimbisa chimiro chavo semhando yevarindi.
Yedu data inoratidza kuti kutevedzana uye kuongororwa kwebhakitiriya-inotorwa hemolymph ccfDNA zvimedu zvinogona kupa ruzivo rwakakosha nezve inotambira bhakitiriya maruva uye mabhakitiriya aripo mune yakatenderedza marine ecosystem.Mashot sequencing matekiniki akaburitsa kutevedzana kwecommensal bacteria A. atra gill ingadai yakapotswa dai nzira dzakajairwa dze16S rRNA dzakashandiswa, nekuda kwechikamu chereferensi raibhurari.Kutaura zvazviri, kushandiswa kwedu kweLB data yakaunganidzwa kubva kuM. platensis mune imwechete mussel layer paKerguelen yakaratidza kuti kuumbwa kwegill-associated bacterial symbionts kwakafanana kune zvose zviri zviviri mussels (Fig. S4, Supplementary Information).Kufanana kwemhando mbiri dzakasiyana dzemussels dzinogona kuratidza kuumbwa kwenharaunda dzebhakitiriya munzvimbo inotonhora, sulfurous, uye volcanic deposits yeKerguelen [55, 56, 57, 58].Hurefu hwema microorganisms anoderedza sarufa akatsanangurwa zvakanaka pakukohwa mhashu kubva munzvimbo dzine bioturbated dzemahombekombe [59], dzakadai semahombekombe ePort-au-France.Imwe mukana ndeyekuti iyo commensal mussel flora inogona kukanganiswa neyakachinjika kutapurirana [60, 61].Kutsvakurudza kwakawanda kunodiwa kuona kuwirirana pakati penzvimbo yegungwa, pasi pegungwa, uye kuumbwa kwemabhakitiriya esymbiotic mumamussels.Zvidzidzo izvi zvirikuenderera mberi.
Hurefu uye kusungirirwa kwehemolymph ccfDNA, kureruka kwayo kwekucheneswa, uye mhando yepamusoro kubvumira kukurumidza kutevedzana kwepfuti ndezvimwe zvezvakanakira kushandisa mussel ccfDNA kuongorora kusiyana-siyana kwezvipenyu mugungwa regungwa.Iyi nzira inonyanya kushanda pakuratidza nharaunda dzehutachiona (viromes) mune yakapihwa ecosystem [62, 63].Kusiyana nemabhakitiriya, archaea, uye eukaryotes, majenomes ehutachiona haana phylogenetically akachengetedzwa majini akadai se 16S sequences.Mhedzisiro yedu inoratidza kuti mvura biopsies kubva kumhando yechiratidzo senge mussels inogona kushandiswa kuona huwandu hukuru hwezvimedu zvehutachiona hweccfDNA zvinozivikanwa kutapurira mauto anogara mumahombekombe egungwa ecosystem.Izvi zvinosanganisira mavhairasi anozivikanwa kutapurira protozoa, arthropods, tumbuyu, zvirimwa, uye mavhairasi ebhakitiriya (semuenzaniso, bacteriophages).Kugoverwa kwakafanana kwakawanikwa patakaongorora hemolymph ccfDNA virome yeblue mussels (M. platensis) yakaunganidzwa mumutsara wakafanana wemussel paKerguelen (Table S2, Supplementary Information).Shotgun sequencing yeccfDNA zvirokwazvo inzira nyowani inowedzera mukudzidza kwe virome yevanhu kana mamwe marudzi [21, 37, 64].Iyi nzira inonyanya kubatsira pakudzidza mavhairasi eDNA ane tambo mbiri, sezvo pasina jena rimwe chete rinochengetedzwa pakati pemavhairasi eDNA ane tambo mbiri, anomiririra kirasi yakasiyana-siyana uye yakakura yemavhairasi muBaltimore [65].Kunyangwe mazhinji emavhairasi aya achiramba asina kuverengerwa uye anogona kusanganisira mavhairasi kubva kuchikamu chisingazivikanwe zvachose chenyika yehutachiona [66], takaona kuti maviromes uye mauto emussels A. atra neM. platensis anowira pakati pemhando mbiri idzi.zvakafanana (ona mufananidzo S3, rumwe ruzivo).Izvi zvakafanana hazvishamisi, sezvo zvingaratidza kushayikwa kwekusarudza mukutora DNA iripo munharaunda.Zvidzidzo zvenguva yemberi uchishandisa RNA yakacheneswa ikozvino inodiwa kuratidza iyo RNA virome.
Muchidzidzo chedu, takashandisa pombi yakaomesesa yakagadziridzwa kubva kubasa raKowarski nevamwe vaaishanda navo [37], avo vakashandisa nhanho mbiri kudzima kuverenga kwakabatanidzwa uye contigs pamberi uye mushure mekusangana kweiyo ccfDNA, zvichikonzera chikamu chepamusoro chekuverenga kusingaverengeki.Naizvozvo, isu hatigone kutonga kunze kuti mamwe eaya asina kuverengerwa kuverenga angangove achine mabviro azvo, zvakanyanya nekuti isu hatina referensi genome yerudzi urwu rwemussel.Isu takashandisawo pombi iyi nekuti tainetsekana nezve chimeras pakati pekuzviverengera uye kwete-yega kuverenga uye kureba kwekuverenga kwakagadzirwa neIllumina MiSeq PE75.Chimwe chikonzero chehuwandu hwekuverenga kusingaverengeki ndechekuti mazhinji ehutachiona hwemugungwa, kunyanya munzvimbo dziri kure seKerguelen, hazvina kunyorwa.Isu takashandisa Illumina MiSeq PE75, tichifunga kuti ccfDNA chidimbu chakareba chakafanana neccfDNA yevanhu.Zvezvidzidzo zvenguva yemberi, tichipihwa mhedzisiro yedu inoratidza kuti hemolymph ccfDNA ine kuverenga kwenguva refu kupfuura vanhu uye / kana mhuka dzinoyamwisa, tinokurudzira kushandisa sequencing chikuva chakakodzera kune yakareba ccfDNA zvimedu.Iyi tsika ichaita kuti zvive nyore kuziva zvimwe zviratidzo zvekuongorora kwakadzama.Kuwana iyo parizvino isingawanikwe yakakwana A. atra nuclear genome sequence kunofambisawo zvakanyanya rusarura rwe ccfDNA kubva kune yako uye isiri yako.Tichifunga kuti tsvakiridzo yedu yakanangana nekugona kushandisa pfungwa yemvura biopsy kune mussels, isu tinovimba kuti sezvo pfungwa iyi ichishandiswa mukutsvagisa mune ramangwana, maturusi matsva nemapaipi achagadzirwa kuwedzera mukana weiyi nzira yekudzidza hutachiona hwakasiyana-siyana hwema mussels.Marine ecosystem.
Sezvo isiri-invasive kliniki biomarker, yakakwirira yeplasma yevanhu mazinga eccfDNA anobatanidzwa nezvirwere zvakasiyana-siyana, kukuvara kwenyama, uye mamiriro ekunetseka [67,68,69].Kuwedzera uku kwakabatana nekuburitswa kwezvimedu zveDNA zvekwakabva mushure mekukuvara kwetishu.Isu takagadzirisa nyaya iyi tichishandisa acute heat stress, umo ma mussels akaratidzirwa kwenguva pfupi kune tembiricha ye30 °C.Isu takaita ongororo iyi pamhando nhatu dzakasiyana dzemussels mumayedzo matatu akazvimirira.Nekudaro, isu hatina kuwana chero shanduko muccfDNA mazinga mushure mekunyanya kupisa kunetseka (ona Mufananidzo S5, rumwe ruzivo).Kuwanikwa uku kunogona kutsanangura, zvirinani muchikamu, chokwadi chekuti mussels ane semi-yakavhurika circulatory system uye inounganidza yakakura yakawanda yekunze DNA nekuda kwekusefa kwavo kwakanyanya.Kune rumwe rutivi, mussels, kufanana neakawanda invertebrates, angave akanyanya kurwisa kushushikana-kunokonzerwa nekukuvara kwetishu, nekudaro kumisa kuburitswa kweccfDNA muhemolymph yavo [70, 71].
Parizvino, kuongororwa kweDNA kwezvipenyu zvakasiyana-siyana muzvisikwa zvemumvura zvakanyanya kutarisisa nezvezvakatipoteredza DNA (eDNA) metabarcoding.Zvisinei, nzira iyi inowanzoganhurirwa mukuongororwa kwezvipenyu zvakasiyana-siyana kana maprimers ashandiswa.Iko kushandiswa kwepfuti kutevedzana kunotenderedza zvipimo zvePCR uye kusarudzwa kwakarerekera kwema primer seti.Nokudaro, neimwe nzira, nzira yedu iri pedyo neyo ichangobva kushandiswa yepamusoro-eDNA Shotgun sequencing nzira, iyo inokwanisa kurongedza zvakananga DNA yakatsemuka uye kuongorora zvinenge zvipenyu zvose [72, 73].Nekudaro, pane akati wandei akakosha nyaya dzinosiyanisa LB kubva kune yakajairwa eDNA nzira.Ehe, musiyano mukuru pakati peDNA neLB ndiko kushandiswa kweakasikwa mafirita mauto.Kushandiswa kwezvipenyu zvemugungwa zvakadai semaponji uye bivalves (Dresseina spp.) sesefa yepanyama yekudzidza eDNA yakashumwa [74, 75].Nekudaro, chidzidzo chaDreissena chakashandisa mabiopsies ematishu umo DNA yakatorwa.Ongororo yeccfDNA kubva kuLB haidi tishu biopsy, yakasarudzika uye dzimwe nguva inodhura michina uye zvinhu zvine chekuita neDNA kana tishu biopsy.Kutaura zvazviri, isu munguva pfupi yapfuura takashuma kuti ccfDNA kubva kuLB inogona kuchengetwa uye kuongororwa neFTA kutsigirwa pasina kuchengetedza ketani inotonhora, iyo idambudziko guru rekutsvakurudza munzvimbo dziri kure [76].Kubviswa kweccfDNA kubva mumvura biopsies zvakare iri nyore uye inopa yakakwira mhando DNA yekuteedzera pfuti uye PCR kuongororwa.Iyi ibhenefiti yakakura yakapihwa mamwe emagetsi ekupedzisira anoenderana ne eDNA kuongororwa [77].Iyo iri nyore uye yakaderera mutengo weiyo sampling nzira inonyanya kukodzera kune-refu-yekutarisa zvirongwa.Pamusoro pekugona kwavo kusefa kwepamusoro, chimwe chinozivikanwa chebivalves ikemikari mucopolysaccharide kuumbwa kwemukosi yavo, iyo inokurudzira kubatwa kwemavhairasi [78, 79].Izvi zvinoita kuti bivalves ive sefa yakaringana yechisikigo yekuratidza kusiyana-siyana kwezvipenyu uye kukanganisa kwekushanduka kwemamiriro ekunze mune yakapihwa ecosystem yemvura.Kunyange zvazvo kuvapo kwezvimedu zveDNA zvakatorwa nemauto zvinogona kuonekwa sechidziviriro chenzira inofananidzwa neDNA, mari inobatanidza nekuva neccfDNA yakadaro yakaenzaniswa neDNA inonzwisisika panguva imwe chete yehuwandu hwemashoko anowanikwa kune zvidzidzo zvehutano.offset host.Izvi zvinosanganisira kuvapo kwehutachiwana hwehutachiwana hwakabatanidzwa mujenome yemuiti.Izvi zvinonyanya kukosha kune mussels, zvichipihwa kuvepo kweiyo horizontally transmitted leukemic retroviruses mu bivalves [80, 81].Imwe mukana weLB pamusoro peDNA ndeyekuti inoshandisa phagocytic basa rekutenderera masero eropa muhemolymph, iyo inomedza ma microorganisms (uye genomes avo).Phagocytosis ndiro basa guru remasero eropa mu bivalves [82].Pakupedzisira, iyo nzira inotora mukana wepamusoro wekusefa kwema mussels (avhareji 1.5 l / h yemvura yegungwa) uye kutenderera kwemazuva maviri, izvo zvinowedzera kusanganiswa kwezvikamu zvakasiyana zvemvura yegungwa, zvichibvumira kutora heterologous eDNA.[83, 84].Nekudaro, mussel ccfDNA ongororo inzira inonakidza yakapihwa hutano, hupfumi, uye nharaunda kukanganisa kwemhashu.Zvakafanana nekuongororwa kweLB yakaunganidzwa kubva kuvanhu, nzira iyi zvakare inovhura mukana wekuyera genetic uye epigenetic shanduko muhost DNA mukupindura kune exogenous zvinhu.Semuyenzaniso, yechitatu-chizvarwa kutekinoroji tekinoroji inogona kufungirwa kuita genome-wide methylation ongororo mune yemuno ccfDNA uchishandisa nanopore sequencing.Iyi nzira inofanira kufambiswa nechokwadi chokuti kureba kwezvimedu zvemussel ccfDNA zvinonyatsoenderana nemapuratifomu akaverengwa kwenguva refu anobvumira genome-wide DNA methylation kuongorora kubva kune imwechete sequencing inomhanya pasina kudiwa kwekuchinja kwemakemikari.85,86] Ichi ndicho chiitiko chinonakidza, sezvakaratidzwa kuti DNA mhinduro kune methylation yakawanda inoshungurudza maitiro uye maitiro ezvakatipoteredza.Nokudaro, inogona kupa ruzivo rwakakosha mumatanho ari pasi anotonga mhinduro mushure mekutarisana nekushanduka kwemamiriro ekunze kana zvinosvibisa [87].Nekudaro, kushandiswa kweLB hakuna miganhu.Hazvina maturo kutaura, izvi zvinoda kuvapo kwechiratidzo chemhando mu ecosystem.Sezvambotaurwa pamusoro, kushandisa LB kuongorora kusiyana-siyana kweiyo yakapihwa ecosystem inodawo yakasimba bioinformatics pombi inorangarira kuvapo kwezvimedu zveDNA kubva kwakabva.Rimwe dambudziko guru nderekuwanikwa kwereferensi genome yezvipenyu zvemugungwa.Zvinotarisirwa kuti zvirongwa zvakadai seMarine Mammal Genomes Project uye chirongwa cheFish10k chichangobva kugadzwa [88] chichafambisa kuongorora kwakadaro mune ramangwana.Iko kushandiswa kweiyo pfungwa yeLB kune zvipenyu zvemugungwa zvinodyisa zvipenyu zvinowiriranawo nekufambira mberi kwazvino mukutevedzana tekinoroji, zvichiita kuti inyatsokodzera kuvandudzwa kwe-multi-ohm biomarkers kuti ipe ruzivo rwakakosha nezvehutano hwenzvimbo dzemugungwa mukupindura kushushikana kwezvakatipoteredza.
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