Enkosi ngokundwendwela i-Nature.com.Inguqulelo yesikhangeli oyisebenzisayo inenkxaso enyiniweyo yeCSS.Ngowona mava angcono, sicebisa ukuba usebenzise isikhangeli esihlaziyiweyo (okanye uvale iModi yokuThelela kwi-Internet Explorer).Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siya kunika isayithi ngaphandle kwezitayela kunye neJavaScript.
I-Liquid biopsy (LB) ngumbono ofumana ngokukhawuleza ukuthandwa kwindawo yebhayoloji.Le ngcamango isekelwe ikakhulu ekubhaqweni kwamaqhekeza ajikelezayo e-extracellular DNA (ccfDNA), ekhutshwa ngokukodwa njengamaqhekeza amancinci emva kokufa kweeseli kwiithishu ezahlukeneyo.Inxalenye encinci yezi ziqwenga zivela kwizicubu zangaphandle (zangaphandle) okanye izinto eziphilayo.Kumsebenzi okhoyo ngoku, sisebenzise le ngcamango kwiimbaza, uhlobo lomkhosi owaziwayo ngomthamo wazo omkhulu wokuhluza amanzi olwandle.Sisebenzisa isakhono sembaza ukwenza njengezihluzi zendalo ukubamba amaqhekeza e-DNA yokusingqongileyo ukusuka kwimithombo eyahlukeneyo ukunika ulwazi malunga neentlobo ngeentlobo zezinto eziphilayo zolwandle lwaselwandle.Iziphumo zethu zibonisa ukuba i-mussel hemolymph iqulethe iziqwenga ze-DNA ezihluka kakhulu ngobukhulu, ukusuka kwi-1 ukuya kwi-5 kb.Ukulandelelana kwe-shotgun kubonise ukuba inani elikhulu lamaqhekeza e-DNA avela kwimvelaphi ye-microbial yangaphandle.Phakathi kwazo, sifumene amaqhekeza e-DNA asuka kwiibhaktheriya, i-archaea, kunye neentsholongwane, kubandakanywa iintsholongwane ezaziwa ngokusulela iindidi zemikhosi exhaphakileyo efumaneka kwi-ecosystems yaselwandle.Ukuqukumbela, uphononongo lwethu lubonisa ukuba ingqikelelo ye-LB esetyenziswa kwiimbaza imele umthombo osisityebi kodwa ongekaphononongwa wolwazi malunga neyantlukwano ye-microbial kwi-ecosystem yonxweme yolwandle.
Impembelelo yokutshintsha kwemozulu (CC) kwintlobo-ntlobo zezinto eziphilayo zaselwandle yindawo ekhula ngokukhawuleza yophando.Ukufudumala kwehlabathi akubangeli kuphela uxinzelelo olubalulekileyo lwe-physiological, kodwa lutyhala imida yendalo yokuzinza kwe-thermal yezinto eziphila elwandle, echaphazela indawo yokuhlala yeentlobo ezininzi, ezibangela ukuba zikhangele iimeko ezilungele ngakumbi [1, 2].Ukongeza kokuchaphazela ukuhlukahluka kwebhayoloji yeemetazoans, iCC iphazamisa ulungelelwaniso oluthambileyo lwentsebenziswano ye-host-microbial.Le dysbacteriosis ye-microbial ibeka isoyikiso esibi kwi-ecosystems yaselwandle njengoko yenza izidalwa zaselwandle zichaphazeleke ngakumbi kwiintsholongwane ezosulelayo [3, 4].Kukholelwa ukuba i-SS idlala indima ebalulekileyo ekufeni kwabantu abaninzi, okuyingxaki enkulu kulawulo lwe-ecosystems yaselwandle [5, 6].Lo ngumba obalulekileyo xa kujongwa iimpembelelo zoqoqosho, i-ikholoji kunye nesondlo seentlobo ezininzi zaselwandle.Oku kuyinyani ngokukodwa kwi-bivalves ehlala kwimimandla ye-polar, apho iziphumo ze-CK zikhawuleza kwaye zinzima [6, 7].Ngapha koko, ii-bivalves ezifana neMytilus spp.zisetyenziswa ngokubanzi ukujonga iziphumo ze-CC kwi-ecosystem yolwandle.Akumangalisi ukuba, inani elikhulu lee-biomarkers ziye zaphuhliswa ukujonga impilo yazo, zihlala zisebenzisa indlela enemigangatho emibini ebandakanya i-biomarkers esebenzayo esekwe kwimisebenzi ye-enzymatic okanye imisebenzi yeselula efana nokusebenza kweeseli kunye nomsebenzi we-phagocytic [8].Ezi ndlela zikwabandakanya ukulinganiswa koxinzelelo lwezikhombisi ezithile zoxinzelelo eziqokelela kwizicubu ezithambileyo emva kokufunxa ubuninzi bamanzi olwandle.Nangona kunjalo, umthamo ophezulu wokuhluza kunye ne-semi-open circulatory system ye-bivalves inika ithuba lokuphuhlisa i-hemolymph biomarkers usebenzisa ingcamango ye-liquid biopsy (LB), indlela elula kunye nencinci yokulawula isigulane.iisampuli zegazi [9, 10].Nangona iindidi ezininzi zeemolekyuli ezijikelezayo zinokufumaneka kwi-LB yomntu, le ngcamango isekelwe ngokuyinhloko kuhlalutyo lokulandelelana kwe-DNA yokujikeleza amaqhekeza e-extracellular DNA (ccfDNA) kwiplasma.Enyanisweni, ubukho be-DNA ejikelezayo kwi-plasma yabantu buye kwaziwa ukususela kwi-20 yenkulungwane ye-20 [11], kodwa kuphela kwiminyaka yamuva nje ukuba ukuvela kweendlela eziphezulu zokulandelelana kuye kwakhokelela ekuxilongweni kwekliniki ngokusekelwe kwi-ccfDNA.Ubukho bamaqhekeza e-DNA ajikelezayo ngenxa yenxalenye yokukhululwa kwe-DNA ye-genomic (i-nuclear kunye ne-mitochondrial) emva kokufa kweseli. Kubantu abaphilileyo, ukuxinwa kwe-ccfDNA ngokuqhelekileyo kuphantsi (<10 ng / mL) kodwa kunokunyuswa ngamaxesha angama-5-10 kwizigulane eziphethwe yi-pathologies ezahlukahlukeneyo okanye eziphantsi koxinzelelo, okubangela umonakalo wezicubu. Kubantu abaphilileyo, ukuxinwa kwe-ccfDNA ngokuqhelekileyo kuphantsi (<10 ng / mL) kodwa kunokunyuswa ngamaxesha angama-5-10 kwizigulane eziphethwe yi-pathologies ezahlukahlukeneyo okanye eziphantsi koxinzelelo, okubangela umonakalo wezicubu. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 стрессу, приводящему к повреждению тканей. Kubantu abaphilileyo, ukuxinwa kwe-cccDNA ngokuqhelekileyo kuphantsi (<10 ng / mL), kodwa kunokunyuka ngamaxesha angama-5-10 kwizigulane ezine-pathologies ezahlukahlukeneyo okanye phantsi koxinzelelo olukhokelela kumonakalo wezicubu.在健康个体中,ccfDNA的浓度通常较低(<10 ng/mL)致组织损伤。20倍, 从而 组织。。。 损伤 损伤 损伤 损伤 损伤 损Концентрации ccfDNA обычно низкие (<10 нг/мл) здоровых людей, но могут увеличены в 5-10 раз у пациентоный что приводит к повреждению тканей. Ugxininiso lwe-ccfDNA luhlala luphantsi (<10 ng / ml) kubantu abanempilo, kodwa inokunyuswa i-5-10-fold in izigulane ezine-pathologies ezahlukahlukeneyo okanye uxinzelelo, okubangela umonakalo wezicubu.Ubungakanani bamaqhekeza e-ccfDNA buyohluka kakhulu, kodwa buqhelekile ukusuka kwi-150 ukuya kwi-200 bp.[12].Uhlalutyo lwe-ccfDNA eyenziwe ngokwayo, oko kukuthi, i-ccfDNA evela kwiiseli eziqhelekileyo okanye eziguqulwayo, zingasetyenziselwa ukufumanisa utshintsho lwezofuzo kunye ne-epigenetic ezikhoyo kwi-genome yenyukliya kunye / okanye i-mitochondrial, ngaloo ndlela inceda oogqirha ukuba bakhethe unyango oluthile olujoliswe kwi-molecular [13].Nangona kunjalo, i-ccfDNA inokufumaneka kwimithombo yangaphandle njenge-ccfDNA kwiiseli ze-fetus ngexesha lokukhulelwa okanye kwizitho ezitshintshiweyo [14,15,16,17].I-ccfDNA ikwangumthombo obalulekileyo wolwazi wokubona ubukho be-nucleic acids ye-arhente eyosulelayo (yangaphandle), evumela ukubonwa okungahlaseliyo kosulelo oluxhaphakileyo olungachongwanga ziinkcubeko zegazi, ukuthintela i-biopsy ehlaselayo yezicubu ezosulelekileyo [18].Uphononongo lwakutsha nje lubonise ngokwenene ukuba igazi lomntu liqulethe umthombo otyebileyo wolwazi olunokusetyenziselwa ukuchonga iintsholongwane zentsholongwane kunye nebhaktiriya, kwaye malunga ne-1% ye-ccfDNA efunyenwe kwiplasma yomntu imvelaphi yangaphandle [19].Olu phononongo lubonisa ukuba i-biodiversity ye-microbiome ejikelezayo yezinto eziphilayo inokuvavanywa kusetyenziswa uhlalutyo lwe-ccfDNA.Nangona kunjalo, kude kube kutshanje, le ngcamango yayisetyenziswa kuphela ebantwini kwaye, kwinqanaba elingaphantsi, kwezinye izilwanyana ezinomqolo [20, 21].
Kwiphepha langoku, sisebenzisa i-LB enokubakho ukuhlalutya i-ccfDNA ye-Aulacomya atra, uhlobo olusezantsi oluqhelekileyo olufumaneka kwi-subantarctic ye-Kerguelen Islands, iqela leziqithi ezikwi-plateau enkulu eyenza i-35 yezigidi zeminyaka edlulileyo.Ukuphuphuma kwevalkhano.Sisebenzisa inkqubo yovavanyo ye-in vitro, safumanisa ukuba amaqhekeza e-DNA kumanzi olwandle athatyathwa ngokukhawuleza ziimbaza aze angene kwi-hemolymph compartment.Ulandelelwano lwe-shotgun lubonise ukuba i-mussel hemolymph ccfDNA inamaqhekeza e-DNA eyakhe nengeyiyo imvelaphi, kubandakanywa iibhaktheriya ze-symbiotic kunye namaqhekeza e-DNA asuka kwi-biomes eqhelekileyo ye-ecosystem ebandayo yaselwandle yentaba-mlilo.IHemolymph ccfDNA ikwaqulathe ulandelelwano lwentsholongwane ephuma kwiintsholongwane ezinoluhlu olwahlukileyo lomamkeli.Siphinde safumana amaqhekeza e-DNA kwizilwanyana ezininzi ezifana neentlanzi zamathambo, i-anemones yolwandle, i-algae kunye nezinambuzane.Ukuqukumbela, uphononongo lwethu lubonisa ukuba ingcamango ye-LB ingasetyenziswa ngempumelelo kwii-invertebrates zaselwandle ukuvelisa i-genomic repertoire ecebileyo kwizinto eziphilayo zaselwandle.
Abantu abadala (55-70 mm ubude) iMytilus platensis (M. platensis) kunye neAulacomya atra (A. atra) zaqokelelwa kunxweme olunamatye oluphakathi kwePort-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., Prince Edward Island, Canada) kwaye zibekwe kwiqondo lokushisa elilawulwayo (4 ° C) itanki ene-aerated equkethe i-10-20 L ye-32 ‰ i-brine eyenziweyo.(ityuwa yolwandle eyenziweyo iReef Crystal, i-Instant Ocean, eVirginia, eU.SA).Kuvavanyo ngalunye, ubude kunye nobunzima beqokobhe ngalinye balinganiswa.
Iprothokholi yasimahla yokufikelela evulekileyo yale nkqubo iyafumaneka kwi-intanethi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Ngokufutshane, i-LB hemolymph yaqokelelwa kwimisipha ye-abductor njengoko kuchaziwe [22].I-hemolymph yacaciswa nge-centrifugation kwi-1200 × g imizuzu ye-3, i-supernatant yayikhenkce (-20 ° C) de isetyenziswe.Ukwahlukaniswa kunye nokucocwa kwe-cfDNA, iisampulu (1.5-2.0 ml) zanyibilikiswa kwaye zalungiswa kusetyenziswa i-NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) ngokwemiyalelo yomenzi.I-ccfDNA yagcinwa kwi -80 ° C de kube uhlalutyo olulongezelelweyo.Kwezinye iimvavanyo, i-ccfDNA yahlukaniswa kwaye yahlanjululwa kusetyenziswa i-QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada).I-DNA ehlanjululweyo yalinganiswa ngokusebenzisa i-PicoGreen assay.Ukuhanjiswa kweqhekeza le-ccfDNA eyedwa yahlalutywa yi-capillary electrophoresis usebenzisa i-Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) usebenzisa i-High Sensitivity DNA Kit.Uvavanyo lwenziwe kusetyenziswa i-1 µl yesampulu ye-ccfDNA ngokwemiyalelo yomenzi.
Ukulandelelanisa amaqhekeza e-hemolymph ccfDNA, i-Génome Québec (eMontreal, eQuebec, eKhanada) yalungisa amathala eencwadi asebenzisa i-Illumina DNA Mix yekhithi ye-Illumina MiSeq PE75.I-adapter eqhelekileyo (i-BioO) isetyenzisiwe.Iifayile zedatha eluhlaza zifumaneka kwi-NCBI Sequence Read Archive (SRR8924808 kunye ne-SRR8924809).Umgangatho wokufunda osisiseko wavavanywa kusetyenziswa i-FastQC [23].I-Trimmomatic [24] isetyenziselwe ukunqunqa iiadaptha kunye nokufundwa okukumgangatho ophantsi.I-Shotgun ifundeka ngeziphelo ezidityanisiweyo ibiye IFLASH idityaniswe kufundo olunye olude kunye nokudityaniswa okuncinci kwe-20 bp ukunqanda ukungahambelani [25]. Ukufundwa okudityanisiweyo kwachazwa kunye ne-BLASTN kusetyenziswa i-bivalve NCBI Taxonomy database (ixabiso le-<1e-3 kunye ne-90% ye-homology), kunye nokufihla ukulandelelana kobunzima obuphantsi kwenziwa kusetyenziswa i-DUST [26]. Ukufundwa okudityanisiweyo kwachazwa kunye ne-BLASTN kusetyenziswa i-bivalve NCBI Taxonomy database (ixabiso le-<1e-3 kunye ne-90% ye-homology), kunye nokufihla ukulandelelana kobunzima obuphantsi kwenziwa kusetyenziswa i-DUST [26]. Usetyenziso olungakumbi nge- BLASTN базы данных таксономии двустворчатых моллюсков% NCBI (Z10) маскирование последовательностей низкой сложности было выполнено с использованием Uthuli [26]. Ukufundwa okuhlanganisiweyo kwachazwa nge-BLASTN kusetyenziswa i-NCBI bivalve taxonomy database (ixabiso le-<1e-3 kunye ne-90% ye-homology), kunye ne-masking ephantsi yolandelelwano oluntsonkothileyo lwenziwa kusetyenziswa i-DUST [26].使用双壳类NCBI 分类数据库.掩蔽.使用 双 壳类 ncbi 分类 (((<1e-3 和 90% 同源) 用 用 注释 合并读数 , 并 使用公物 [26] .掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽Usetyenziso olungakumbi nge- BLASTN , usetyenziso lwe-intanethi ye-intanethi а маскирование последовательностей низкой сложности было выполнено с использованием Uthuli [26]. Ukufundwa okuhlanganisiweyo kwachazwa nge-BLASTN kusetyenziswa i-NCBI bivalve taxonomic database (ixabiso le-<1e-3 kunye ne-90% ye-homology), kunye ne-masking ephantsi yolandelelwano oluntsonkothileyo lwenziwa kusetyenziswa i-DUST [26].Ufundo lohlulahlulwe lwaba ngamaqela amabini: anxulumene nolandelelwano lwe-bivalve (apha lubizwa ngokuba kukufunda ngokwakho) kunye nokunganxulumananga (okungazifundiyo).Amaqela amabini ahlanganiswe ngokwahlukeneyo esebenzisa i-MEGAHIT ukuvelisa i-contigs [27].Okwangoku, ukuhanjiswa kwetaxonomic yokufundwa kwe-microbiome yangaphandle kwahlelwa kusetyenziswa i-Kraken2 [28] kwaye imelwe ngokubonakalayo yitshathi yephayi ye-Krona kwiGalaxy [29, 30].Ezona khilomitha zifanelekileyo zamiselwa ukuba zibe zii-kmers-59 ukusuka kumfuniselo wethu wokuqala. I-Self contigs yaye yachongwa ngolungelelwaniso ne-BLASTN (idathabheyisi ye-bivalve NCBI, ixabiso le-<1e−10 kunye ne-60% ye-homology) kwinkcazo yokugqibela. I-Self contigs yaye yachongwa ngolungelelwaniso ne-BLASTN (idathabheyisi ye-bivalve NCBI, ixabiso le-<1e−10 kunye ne-60% ye-homology) kwinkcazo yokugqibela. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюсков NCBI, значени10 e) нчательной аннотации. I-self-contigs yachongwa ngokuhambelana ne-BLASTN (i-NCBI yedatha ye-bivalve, ixabiso le-<1e-10 kunye ne-60% ye-homology) ye-annotation 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 , е e <1e-10 kwaye гомология 60%). I-Self-contigs yaye yachongwa kwi-annotation yokugqibela ngokuhambelana ne-BLASTN (i-NCBI bivalve database, ixabiso le-<1e-10 kunye ne-60% ye-homology). Ngokunxuseneyo, i-nonself group contigs yachazwa ngeBLASTN (nt NCBI database, e value <1e−10 kunye ne-60% homology). Ngokunxuseneyo, i-nonself group contigs yachazwa ngeBLASTN (nt NCBI database, e value <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%). Ngokuhambelanayo, i-contigs yeqela langaphandle ichazwe nge-BLASTN (idatha ye-NT NCBI, ixabiso le-<1e-10 kunye ne-60% ye-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-%). Ngokuhambelanayo, ii-contigs zeqela ezingezizo ezakhe zachazwa nge-BLASTN (nt NCBI database, ixabiso le-<1e-10 kunye ne-60% ye-homology). I-BLASTX nayo yaqhutywa kwii-nonself contigs kusetyenziswa i-nr kunye ne-RefSeq protein ye-NCBI yogcino-lwazi (e value <1e−10 kunye ne-60% homology). I-BLASTX nayo yaqhutywa kwii-nonself contigs kusetyenziswa i-nr kunye ne-RefSeq protein ye-NCBI yogcino-lwazi (e value <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 kwakhona kwi-non-self contigs usebenzisa i-nr kunye ne-RefSeq NCBI yogcino lweprotheyini (ixabiso le-<1e-10 kunye ne-60% ye-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 kwakhona kwi-non-self contigs usebenzisa i-nr kunye ne-RefSeq NCBI yogcino lweprotheyini (ixabiso le-<1e-10 kunye ne-60% ye-homology).I-BLASTN kunye ne-BLASTX iiphuli ze-non-self-contigs zimele i-contigs yokugqibela (jonga ifayile eyoNgezelelweyo).
Iiprimers ezisetyenziselwa i-PCR zidweliswe kwiTheyibhile S1.I-Taq DNA polymerase (i-Bio Basic Canada, i-Markham, i-ON) yayisetyenziselwa ukukhulisa i-ccfDNA ekujoliswe kuyo.Ezi meko zilandelayo zokusabela zisetyenzisiwe: i-denaturation kwi-95 ° C kwimizuzu ye-3, i-95 ° C ngomzuzu we-1, i-annealing yemizuzu ye-1, i-elongation kwi-72 ° C ye-1 ngomzuzu, i-35 imijikelezo, kwaye ekugqibeleni i-72 ° C phakathi kwemizuzu eyi-10..Iimveliso ze-PCR zahlulwe nge-electrophoresis kwi-agarose gels (1.5%) equkethe i-SYBRTM Ekhuselekileyo ye-DNA Gel Stain (Invitrogen, Burlington, ON, Canada) kwi-95 V.
Iimbaza (Mytilus spp.) zaye zaqhelaniswa ne-500 ml yamanzi olwandle aneoksijini (32 PSU) iiyure ezingama-24 kwi-4°C.I-Plasmid DNA equlethe i-insert encoding i-galectin-7 ye-cDNA yokulandelelana (inombolo ye-NCBI yokufikelela i-L07769) yongezwa kwi-vial kwi-concentration yokugqibela ye-190 μg / μl.Iimbaza ezifakwe phantsi kweemeko ezifanayo ngaphandle kokudibanisa i-DNA yayilulawulo.Itanki yesithathu yokulawula iqulethe i-DNA ngaphandle kweembaza.Ukujonga umgangatho we-DNA emanzini olwandle, iisampuli zamanzi olwandle (20 μl; ukuphindaphinda okuthathu) zathathwa kwitanki nganye ngexesha elibonisiweyo.Ukulandelela i-plasmid DNA, i-LB mussels yavunwa ngamaxesha abonakalisiweyo kwaye yahlalutywa yi-qPCR kunye ne-ddPCR.Ngenxa yetyuwa eninzi yamanzi olwandle, i-aliquots yaxutywa kumanzi asemgangathweni we-PCR (1:10) ngaphambi kwazo zonke iimvavanyo ze-PCR.
I-Digital droplet PCR (ddPCR) yenziwe ngokusebenzisa i-BioRad QX200 protocol (Mississauga, Ontario, Canada).Sebenzisa iprofayile yeqondo lobushushu ukumisela elona qondo lobushushu liphezulu (Itheyibhile S1).Iidrophu zenziwe kusetyenziswa i-QX200 drop generator (BioRad).I-ddPCR yenziwa ngolu hlobo lulandelayo: i-95 ° C kwi-5 min, i-50 cycles ye-95 ° C kwi-30 s kunye ne-annealing ye-annealing ye-1 min kunye ne-72 ° C ye-30 s, i-4 ° C ye-5 min kunye ne-90 ° C phakathi kwemizuzu emi-5.Inani lamathontsi kunye neempendulo ezilungileyo (inani leekopi/µl) zalinganiswa kusetyenziswa i-QX200 drop reader (BioRad).Iisampulu ezinamathontsi angaphantsi kwe-10,000 zaliwe.Ulawulo lwepateni alwenziwanga ngalo lonke ixesha i-ddPCR iqhutywa.
I-qPCR yenziwa kusetyenziswa i-Rotor-Gene® 3000 (uPhando lweCorbett, eSydney, e-Australia) kunye ne-LGALS7 iiprimers ezikhethekileyo.Zonke ii-PCR zobungakanani zenziwe kwi-20 µl kusetyenziswa i-QuantiFast SYBR Green PCR Kit (QIAGEN).I-qPCR yaqalwa nge-15 min incubation kwi-95 ° C ilandelwa yimijikelo ye-40 kwi-95 ° C imizuzwana ye-10 kunye ne-60 ° C imizuzwana engama-60 kunye nokuqokelela idatha enye.Iingqungquthela ezinyibilikayo zenziwe ngokusebenzisa imilinganiselo elandelelanayo kwi-95 ° C kwi-5 s, i-65 ° C ye-60 s, kunye ne-97 ° C ekupheleni kwe-qPCR.I-qPCR nganye yenziwe ngokuphindwe kathathu, ngaphandle kweesampuli zolawulo.
Kuba iimbaza zisaziwa ngomlinganiselo ophezulu wokuhluza, siqale saphanda ukuba zingahluza kwaye zigcine amaqhekeza eDNA ekhoyo emanzini olwandle.Sikwanomdla wokuba la maqhekeza aqokelelana kwinkqubo yawo ye-semi-open lymphatic.Siwusombulule lo mba ngovavanyo ngokulanda ikamva lamaqhekeza e-DNA anyibilikayo afakwe kwiitanki zeembaza eziluhlaza.Ukuququzelela ukulandelela amaqhekeza e-DNA, sasebenzisa i-plasmid ye-plasmid yangaphandle (hayi i-self) equlethe i-galectin-7 gene.I-ddPCR ilanda amaqhekeza e-plasmid DNA emanzini olwandle nakwiimbaza.Iziphumo zethu zibonisa ukuba ukuba isixa DNA amaqhekeza kumanzi olwandle wahlala ngokwentelekiso ngokwentelekiso ngokuhamba kwexesha (ukuya kwiintsuku ezisi-7) ngokungabikho imbaza, ngoko phambi iimbaza eli nqanaba phantse wanyamalala ngokupheleleyo kwiiyure ezisi-8 (Fig. 1a,b).Iziqwenga ze-DNA exogenous zifunyenwe lula ngaphakathi kwe-15 min kwi-intravalvular fluid kunye ne-hemolymph (umzobo 1c).La maqhekeza asenokubonwa ukuya kutsho kwiiyure ezi-4 emva kokuba sesichengeni.Lo msebenzi wokucoca ngokubhekiselele kumaqhekeza e-DNA unokuthelekiswa nomsebenzi wokucoca iibhaktheriya kunye ne-algae [31].Ezi ziphumo zibonisa ukuba i-mussels inokucoca kwaye iqokelele i-DNA yangaphandle kwiindawo zabo zolwelo.
Imilinganiselo ezalanayo ye-plasmid DNA emanzini olwandle kwindawo (A) okanye ukungabikho (B) kwembaza, ilinganiswa nge-ddPCR.Kwi-A, iziphumo zibonakaliswa njengeepesenti, kunye nemida yeebhokisi ezimele i-75 kunye ne-25 yepesenti.Ijika lelogarithmic elifakiweyo liboniswe ngombala obomvu, kwaye indawo enombala ongwevu imele i-95% yexesha lokuzithemba.Kwi-B, umgca obomvu ubonisa intsingiselo kunye nomgca ohlaza okwesibhakabhaka ubonisa i-95% yexesha lokuzithemba kwingqwalasela.C Ukuqokelela kwe-plasmid DNA kwi-hemolymph kunye ne-valvular fluid ye-mussels ngamaxesha ahlukeneyo emva kokongezwa kwe-plasmid DNA.Iziphumo zinikezelwa njengeekopi ezipheleleyo ezichongiweyo/mL (±SE).
Okulandelayo, siphande ngemvelaphi ye-ccfDNA kwiimbaza eziqokelelwe kwiibhedi zeembaza kwiZiqithi zeKerguelen, iqela elikude leziqithi ezinefuthe elilinganiselweyo le-anthropogenic.Ngenxa yale njongo, i-cccDNA esuka kwi-mussel hemolymphs yahlukaniswa kwaye yahlanjululwa ngeendlela eziqhelekileyo ezisetyenziselwa ukuhlambulula i-cccDNA yabantu [32, 33].Siye safumanisa ukuba i-avareji ye-hemolymph ccfDNA concentrations kwi-mussels ikwi-micrograms ezisezantsi ngoluhlu lwe-hemolymph (bona iTheyibhuli S2, iNkcukacha eyoNgezelelweyo).Olu luhlu logxininiso lukhulu kakhulu kunabantu abanempilo (i-nanograms ephantsi nge-milliliter), kodwa kwiimeko ezinqabileyo, kwizigulane zomhlaza, inqanaba le-ccfDNA linokufikelela kwii-micrograms ezininzi nge-milliliter [34, 35].Uhlalutyo lobungakanani bokusabalalisa i-hemolymph ccfDNA lubonise ukuba la maqhekeza ahluka kakhulu ngobukhulu, ukusuka kwi-1000 bp ukuya kwi-1000 bp.ukuya kuthi ga kwi-5000 bp (umzobo 2).Iziphumo ezifanayo zifunyenwe kusetyenziswa i-silica-based QIAamp Investigator Kit, indlela esetyenziswa ngokuqhelekileyo kwisayensi ye-forensic ukuba ihlukanise ngokukhawuleza kwaye ihlambulule i-DNA ye-genomic kwiisampuli eziphantsi ze-DNA, kuquka i-ccfDNA [36].
Ummeli we-ccfDNA electrophoregram ye-mussel hemolymph.Itsalwe nge-NucleoSnap Plasma Kit (phezulu) kunye neQIAamp DNA Investigator Kit.B Iploti yeViolin ebonisa ukuhanjiswa kwe-hemolymph ccfDNA concentrations (±SE) kwiimbaza.Imigca emnyama nebomvu imele i-median kunye ne-quartiles yokuqala neyesithathu, ngokulandelanayo.
Ngokumalunga ne-1% ye-ccfDNA ebantwini kunye neeprimates inomthombo wangaphandle [21, 37].Ukunika isemi-open circulatory system ye-bivalves, amanzi olwandle atyebileyo ngemicrobial, kunye nobungakanani bosasazo lwembaza ccfDNA, siye saqikelela ukuba imbaza ihemolymph ccfDNA inokuqulatha ichibi elityebileyo nelohlukeneyo lemicrobial DNA.Ukuvavanya le ngcamango, silandelelanisa i-hemolymph ccfDNA kwiisampuli ze-Aulacomya atra eziqokelelwe kwii-Kerguelen Islands, zivelise ngaphezu kwe-10 yezigidi ezifundwayo, i-97.6% ephumelele ulawulo lomgangatho.Ufundo lwaye lwahlelwa ngokwemithombo yobuqu kunye nengeyobuqu kusetyenziswa i-BLASTN kunye ne-NCBI yolwazi lwe-bivalve (umzobo we-S1, uLwazi oloNgezelelweyo).
Ebantwini, zombini i-DNA yenyukliya kunye ne-mitochondrial inokukhutshwa egazini [38].Nangona kunjalo, kwisifundo esikhoyo, kwakungenakwenzeka ukuchaza ngokweenkcukacha i-DNA ye-nyukliya ye-mussels, ngenxa yokuba i-A. atra genome ayizange ilandelelaniswe okanye ichazwe.Nangona kunjalo, sakwazi ukuchonga inani lamaqhekeza e-ccfDNA emvelaphi yethu sisebenzisa ithala leencwadi le-bivalve (Umfanekiso we-S2, uLwazi oloNgezelelweyo).Siphinde saqinisekisa ubukho bamaqhekeza e-DNA yemvelaphi yethu ngokukhuliswa kwe-PCR echanekileyo kwezo zifuza ze-A. atra ezilandelelanisiweyo (Umfanekiso 3).Ngokufanayo, xa kunikwa ukuba i-mitochondrial genome ye-A. atra iyafumaneka kwi-database yoluntu, umntu unokufumana ubungqina bobukho be-mitochondrial ccfDNA fragments kwi-hemolymph ye-A. atra.Ubukho be-mitochondrial DNA fragments kwaqinisekiswa yi-PCR amplification (Umfanekiso 3).
Iintlobo ezahlukeneyo zofuzo ze-mitochondrial zazikho kwi-hemolymph ye-A. atra (amachaphaza abomvu - inombolo yesitokhwe: SRX5705969) kunye ne-M. platensis (amachaphaza aluhlaza - inombolo yesitokhwe: SRX5705968) inyuswe yi-PCR.Umzobo uthatyathwe kwiBreton et al., 2011 B Ukwandiswa kwe-hemolymph supernatant ukusuka kwi-A. atra Egcinwe kwiphepha le-FTA.Sebenzisa i-punch ye-3 mm yokongeza ngokuthe ngqo kwi-tube ye-PCR equkethe umxube we-PCR.
Ukunikezelwa komxholo we-microbial obuninzi kumanzi olwandle, ekuqaleni sagxila ekubonakalisweni kwe-microbial DNA ukulandelelana kwi-hemolymph.Ukwenza oku, sisebenzisa iindlela ezimbini ezahlukeneyo.Isicwangciso sokuqala esisetyenzisiweyo i-Kraken2, i-algorithm-based based classification program ekwazi ukuchonga ukulandelelana kwe-microbial ngokuchaneka okuthelekiswa ne-BLAST kunye nezinye izixhobo [28].Ngaphezulu kokufundwa kwe-6719 kunqunywe ukuba yimvelaphi yebhaktheriya, ngelixa i-124 kunye ne-64 yayivela kwi-archaea kunye neentsholongwane, ngokulandelanayo (umzobo 4).Iziqwenga ze-DNA zebhaktheriya ezininzi zaziyi-Firmicutes (46%), i-Proteobacteria (27%), kunye ne-Bacteroidetes (17%) (umzobo 4a).Olu lwabiwo luyahambelana nezifundo zangaphambili ze-marine blue mussel microbiome [39, 40].I-Gammaproteobacteria yayiyeyona klasi iphambili yeProteobacteria (44%), kuquka iVibrionales ezininzi (umzobo 4b).Indlela ye-ddPCR iqinisekisile ubukho be-Vibrio DNA fragments kwi-ccfDNA ye-A. atra hemolymph (Umfanekiso 4c) [41].Ukufumana ulwazi oluthe kratya malunga nemvelaphi yebhaktheriya ye-ccfDNA, indlela eyongezelelweyo yathathwa (Umfanekiso we-S2, Ulwazi olongezelelweyo). Kulo mzekelo, ukufunda okudityanisiweyo kuye kwadityaniswa njengofundo oludityanisiweyo kwaye lwahlelwa njengalo lwesiqu (i-bivalves) okanye imvelaphi engeyoyathu kusetyenziswa i-BLASTN kunye nexabiso le-e elingu-1e−3 kunye nokusikwa nge > 90% yehomology. Kulo mzekelo, ukufunda okudityanisiweyo kuye kwadityaniswa njengofundo oludityanisiweyo kwaye lwahlelwa njengalo lwesiqu (i-bivalves) okanye imvelaphi engeyoyathu kusetyenziswa i-BLASTN kunye nexabiso le-e elingu-1e−3 kunye nokusikwa nge > 90% yehomology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как собственные (двый) происхождению с использованием BLASTN kunye ne-значения e 1e-3 kunye ne-отсечения с гомологией> 90%. Kule meko, ukufundwa okugqithisiweyo kwaqokelelwa njengokufundwa okudibeneyo kwaye kwahlelwa njengemveli (i-bivalve) okanye engeyiyo yokuqala usebenzisa i-BLASTN kunye nexabiso le-e le-1e-3 kunye ne-cutoff nge> 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 kunye ne-1e-3 iimali kunye>90% ngokuthengisa imveliso yemoto.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 ne 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 собственбраные происхождению с использованием значений e BLASTN kunye ne-1e-3 kunye ne-90%. Kule meko, ukufundeka okungaphezulu kwaqokelelwa njengofundo olugqityiweyo kwaye lwahlelwa njengaye (i-bivalves) okanye engeyiyo eyemvelaphi kusetyenziswa i-e BLASTN kunye ne-1e-3 yamaxabiso kunye nomda we-homology> 90%.Ekubeni i-A. atra genome ingekalandeleli, sisebenzise isicwangciso sendibano ye-de novo ye-MEGAHIT Next Generation Sequencing (NGS) assembler.Iyonke i-147,188 contigs ichongiwe njengabaxhomekeke (bivalves) kwimvelaphi.Ezi contigs emva koko zaqhushumba ngamaxabiso e-e-1e-10 kusetyenziswa iBLASTN kunye neBLASTX.Eli qhinga lisivumele ukuba sichonge amaqhekeza angama-482 angekho kwi-bivalve akhoyo kwi-A. atra ccfDNA.Ngaphezu kwesiqingatha (57%) yala maqhekeza e-DNA afunyenwe kwiibhaktheriya, ngakumbi kwii-symbionts zegill, kubandakanywa ne-sulfotrophic symbionts, kunye ne-gill symbionts i-Solemya velum (Fig. 5).
Ubuninzi obunxulumeneyo kwinqanaba lohlobo.B Ukwahlukana kweMicrobial yeephyla ezimbini eziphambili (iFirmicutes kunye neProteobacteria).Ummeli wokukhulisa i-ddPCR C Vibrio spp.A. Iziqwenga ze-16S rRNA gene (blue) kwii-atra hemolymphs ezintathu.
Iyonke i-482 eqokelelweyo ye-contigs iye yahlalutywa.Iprofayili ngokubanzi yokusabalalisa i-taxonomic ye-metagenomic contig annotations (iprokaryotes kunye ne-eukaryotes).B Usasazo oluneenkcukacha lwe-bacterial DNA fragments ezichongwe yi-BLASTN kunye ne-BLASTX.
Uhlalutyo lwe-Kraken2 lubonise kwakhona ukuba i-mussel ccfDNA iqulethe i-archaeal DNA fragments, kuquka i-DNA fragments ye-Euryarchaeota (65%), i-Crenarchaeota (24%), kunye ne-Thaurmarcheota (11%) (Fig. 6a).Ubukho bamaqhekeza e-DNA athathwe kwi-Euryarchaeota kunye ne-Crenarchaeota, eyayifudula ifunyenwe kwindawo ye-microbial ye-mussels yase-California, akufuneki simangalise [42].Nangona i-Euryarchaeota ihlala idityaniswa neemeko ezigqithisileyo, ngoku iyaqondwa ukuba zombini i-Euryarchaeota kunye neCrenarcheota ziphakathi kwezona prokaryotes zixhaphakileyo kwindawo ye-cryogenic yolwandle [43, 44].Ubukho bemethane microorganisms zemethane kwimbaza akumangalisi, xa kunikwa iingxelo zakutsha nje zokuvuza okukhulu kwemethane ukusuka kwiindawo ezivuzayo eKerguelen Plateau [45] kunye nokuveliswa kwemethane encinci enokwenzeka kunxweme lweZiqithi zaseKerguelen [46].
Ingqwalasela yethu emva koko yatshintshela ekufundeni kwiintsholongwane zeDNA.Ngokolwazi lwethu, esi sisifundo sokuqala ebesingasijongisisi sona somxholo wentsholongwane kwiimbaza.Njengoko kulindelwe, sifumene iziqwenga ze-DNA ze-bacteriophages (Caudovirales) (umzobo 6b).Nangona kunjalo, i-DNA yentsholongwane eqhelekileyo ivela kwi-phylum ye-nucleocytoviruses, eyaziwa ngokuba yi-nuclear cytoplasmic big DNA virus (NCLDV), ene-genome enkulu kuyo nayiphi na intsholongwane.Ngaphakathi kule phylum, uninzi lolandelelwano lwe-DNA luzezosapho lwe-Miimidoviridae (58%) kunye ne-Poxviridae (21%), ezinemikhosi yendalo equka ii-vertebrates kunye ne-arthropods, ngelixa inxalenye encinci yolu landelelwano lwe-DNA iyingxenye ye-virological algae eyaziwayo.Yosulela i-algae ye-eukaryotic yolwandle.Ulandelelwano lwafunyanwa kwakhona kwintsholongwane yePandora, intsholongwane enkulu enobungakanani obukhulu bejenome kuyo nayiphi na intsholongwane eyaziwayo.Okubangel 'umdla kukuba, uluhlu lwemikhosi eyaziwayo ukuba yosulelwe yintsholongwane, njengoko igqitywe yi-hemolymph ccfDNA sequencing, yayinkulu kakhulu (Umfanekiso S3, Ulwazi oloNgezelelweyo).Iquka iintsholongwane ezosulela izinambuzane ezifana ne-Baculoviridae kunye ne-Iridoviridae, kunye neentsholongwane ezosulela i-amoeba, i-algae kunye ne-vertebrates.Siphinde safumana ulandelelwano oluhambelana nePithovirus sibericum genome.I-pitoviruses (ekwaziwa nangokuthi "iintsholongwane ze-zombie") zaqala ukubekwa zodwa kwi-permafrost eneminyaka engama-30,000 ubudala eSiberia [47].Ngaloo ndlela, iziphumo zethu zihambelana neengxelo zangaphambili ezibonisa ukuba ayizizo zonke iintlobo zanamhlanje zezi ntsholongwane eziphelileyo [48] kwaye ezi ntsholongwane zinokubakho kwiindawo ezikude zaselwandle.
Ekugqibeleni, siye savavanya ukuze sibone ukuba singakwazi ukufumana amaqhekeza eDNA kwezinye izilwanyana ezininzi.Iyonke i-482 ye-contigs yangaphandle ichongiwe yi-BLASTN kunye ne-BLASTX ngamathala eencwadi e-nt, nr kunye ne-RefSeq (i-genomic kunye neprotheni).Iziphumo zethu zibonisa ukuba phakathi kwamaqhekeza angaphandle kwe-ccfDNA yezilwanyana ezininzi ze-DNA zamathambo amathambo zihamba phambili (Umfanekiso 5).Kwakhona kuye kwafunyanwa amaqhekeza eDNA ezinambuzane nezinye iintlobo.Inxalenye enkulu ye-DNA fragments ayichongwa, mhlawumbi ngenxa yokungamelani kwenani elikhulu leentlobo zaselwandle kwii-database ze-genomic xa kuthelekiswa neentlobo ze-terrestrial [49].
Kweli phepha langoku, sisebenzisa ingqikelelo ye-LB kwiimbaza, sixoxa ukuba i-hemolymph ccfDNA shot sequencing inokubonelela ngengqiqo ekubunjweni kwezinto eziphilayo zaselwandle.Ngokukodwa, sifumene ukuba i-1) i-mussel hemolymph iqulethe i-concentrations ephezulu (amanqanaba e-microgram) enkulu kakhulu (~ 1-5 kb) ejikelezayo amaqhekeza e-DNA;2) ezi ziqwenga ze-DNA zombini ezizimeleyo kwaye azizimele 3) Phakathi kwemithombo yangaphandle yale maqhekeza e-DNA, sifumene ibhaktheriya, i-archaeal kunye ne-viral DNA, kunye ne-DNA yezinye izilwanyana ezininzi;I-4) Ukuqokelela kwezi ziqwenga ze-ccfDNA zangaphandle kwi-hemolymph kwenzeka ngokukhawuleza kwaye kufake isandla kumsebenzi wokucoca wangaphakathi we-mussels.Ukuqukumbela, uphando lwethu lubonisa ukuba ingcamango ye-LB, esele isetyenziswe ngokuyinhloko kwintsimi ye-biomedicine, ifaka ikhowudi yomthombo ocebileyo kodwa ongahlolisiyo ongasetyenziselwa ukuqonda kangcono ukusebenzisana phakathi kweentlobo ze-sentinel kunye nokusingqongileyo.
Ukongeza kwiiprimates, ukwahlukaniswa kwe-ccfDNA kuye kwaxelwa kwizilwanyana ezanyisayo, kubandakanya iimpuku, izinja, iikati, namahashe [50, 51, 52].Nangona kunjalo, kulwazi lwethu, uphononongo lwethu lolokuqala ukunika ingxelo yokufunyanwa kunye nolandelelwano lwe-ccfDNA kwiintlobo zaselwandle kunye nenkqubo evulekileyo yokujikeleza.Olu phawu lwe-anatomical kunye nekhono lokucoca lembaza, linokuthi, ubuncinci ngokuyinxenye, lichaze iimpawu zobukhulu ezahlukeneyo zokujikeleza kwamaqhekeza e-DNA xa kuthelekiswa nezinye iintlobo.Ebantwini, uninzi lwamaqhekeza e-DNA ajikeleza egazini ngamaqhekeza amancinci asuka kwi-150 ukuya kwi-200 bp.kunye nencopho ephezulu ye-167 bp [34, 53].Inxalenye encinci kodwa ebalulekileyo ye-DNA fragments iphakathi kwe-300 kunye ne-500 bp ngobukhulu, kwaye malunga ne-5% ubude kune-900 bp.[54].Isizathu solu lwabiwo lobungakanani kukuba owona mthombo we-ccfDNA kwiplasma uvela ngenxa yokufa kweeseli, mhlawumbi ngenxa yokufa kweeseli okanye ngenxa ye-necrosis yeeseli ezijikelezayo ze-hematopoietic kubantu abaphilileyo okanye ngenxa ye-apoptosis yeeseli zethumba kwizigulana zomhlaza (eyaziwa ngokuba yi-DNA ejikelezayo)., ctDNA).Ubungakanani bokuhanjiswa kwe-hemolymph ccfDNA esiyifumene kwimbaza ukusuka kwi-1000 ukuya kwi-5000 bp, ebonisa ukuba i-ccfDNA yembaza inemvelaphi eyahlukileyo.Le yingcinga enengqondo, kuba iimbaza zinenkqubo yemithambo evulekileyo evulekileyo kwaye zihlala kwindawo ezihlala emanzini eziqulathe ukugxilwa okuphezulu kwe-microbial genomic DNA.Ngapha koko, iimvavanyo zethu zelabhoratri zisebenzisa i-DNA yangaphandle zibonise ukuba iimbaza ziqokelela amaqhekeza e-DNA kumanzi olwandle, ubuncinci emva kweeyure ezimbalwa zonakaliswa emva kokuthathwa kweeseli kunye / okanye ukukhutshwa kunye / okanye ukugcinwa kwimibutho eyahlukeneyo.Ukunika ukunqaba kweeseli (zombini iprokaryotic kunye ne-eukaryotic), ukusetyenziswa kwe-intravalvular compartments kuya kunciphisa isixa se-ccfDNA kwimithombo yobuqu kunye nakwimithombo yangaphandle.Ukuqwalasela ukubaluleka kwe-bivalve innate immunity kunye nenani elikhulu le-phagocytes ejikelezayo, siqhubela phambili sicinga ukuba i-ccfDNA yangaphandle ityetyiswe ekujikelezeni i-phagocytes eqokelela i-DNA yangaphandle xa kufakwe ii-microorganisms kunye / okanye i-debris yeselula.Xa zidibene, iziphumo zethu zibonisa ukuba i-bivalve hemolymph ccfDNA yindawo eyodwa yolwazi lwemolekyuli kwaye yomeleza isimo sabo njengeentlobo ze-sentinel.
Idatha yethu ibonisa ukuba ulandelelwano kunye nohlalutyo lwamaqhekeza e-hemolymph ccfDNA ephuma kwibhaktheriya inokubonelela ngolwazi oluphambili malunga neentyantyambo zebhaktheriya ezibambayo kunye neebhaktheriya ezikhoyo kwindawo engqongileyo yaselwandle.Ubuchule bokulandelanisa ukudubula buveze ulandelelwano lwebhaktiriya e-commensal i-A. atra gill ebeya kuphoswa ukuba iindlela eziqhelekileyo zokuchonga i-16S rRNA bezisetyenzisiwe, ngenxa yenxalenye yokuthambekela kwethala leencwadi lereferensi.Enyanisweni, ukusetyenziswa kwethu kwedatha ye-LB eqokelelwe kwi-M. platensis kwi-mussel layer e-Kerguelen ibonise ukuba ukubunjwa kwee-symbionts zebhaktheriya ezinxulumene ne-gill kwakufana kuzo zombini iintlobo zeembaza (Umfanekiso we-S4, Ulwazi olongezelelweyo).Oku kufana kweembaza ezimbini ezahlukeneyo zofuzo zingabonakalisa ukubunjwa koluntu lwebhaktheriya kwindawo ebandayo, i-sulfurous, kunye ne-volcanic ye-Kerguelen [55, 56, 57, 58].Amanqanaba aphezulu eemicroorganisms zokunciphisa isulfure achazwe kakuhle xa kuvunwa iimbaza kwiindawo eziselunxwemeni ezinebhayoturbated [59], ezifana nonxweme lwasePort-au-France.Enye into enokwenzeka kukuba i-commensal mussel flora inokuchaphazeleka ngokusasazwa okuthe tye [60, 61].Uphando oluthe kratya luyafuneka ukumisela unxulumano phakathi kwemekobume yaselwandle, umphezulu wolwandle, kunye nokwakheka kweebhaktheriya ze-symbiotic kwiimbaza.Ezi zifundo ziyaqhubeka ngoku.
Ubude kunye noxinzelelo lwe-hemolymph ccfDNA, ukucocwa kwayo ngokulula, kunye nomgangatho ophezulu ukuvumela ukulandelelana kompu okhawulezayo zezinye zezibonelelo ezininzi zokusebenzisa i-ccfDNA yembaza ukuvavanya i-biodiversity kwi-ikhosistim yonxweme yolwandle.Le ndlela isebenzayo ngokukodwa ekubonakaliseni uluntu lwentsholongwane (i-viromes) kwi-ecosystem enikeziweyo [62, 63].Ngokungafani neebhaktheriya, i-archaea, kunye ne-eukaryotes, i-genomes yentsholongwane ayinayo i-phylogenetically conserved genes efana nokulandelelana kwe-16S.Iziphumo zethu zibonisa ukuba i-biopsy yolwelo evela kwiintlobo zezalathisi ezifana nembaza zinokusetyenziselwa ukuchonga inani elikhulu lamaqhekeza entsholongwane ye-ccfDNA eyaziwa ngokosulela iindwendwe ezihlala kwindalo yaselwandle engaselunxwemeni.Oku kubandakanya iintsholongwane ezaziwayo ukuba zosulela iprotozoa, i-arthropods, izinambuzane, izityalo, kunye neentsholongwane zebhaktiriya (umzekelo, i-bacteriophages).Ukusabalalisa okufanayo kwafunyanwa xa sihlola i-hemolymph ccfDNA virome ye-blue mussels (M. platensis) eqokelelwe kwi-mussel layer e-Kerguelen (Itheyibhile S2, Ulwazi olongezelelweyo).Ukulandelelana kompu we-ccfDNA ngenene yindlela entsha efumana amandla kuphononongo lwe-virome yabantu okanye ezinye iintlobo zezilwanyana [21, 37, 64].Le ndlela iluncedo ngakumbi ekufundeni iintsholongwane ze-DNA ezinemisonto ephindwe kabini, kuba akukho jini linye lilondolozwe kuzo zonke iintsholongwane ze-DNA ezinemisonto ephindwe kabini, emele olona didi lwahlukeneyo nolubanzi lweentsholongwane eBaltimore [65].Nangona uninzi lwezi ntsholongwane zihlala zingahlelwanga kwaye zinokubandakanya iintsholongwane ezisuka kwindawo engaziwayo ngokupheleleyo yentsholongwane yehlabathi [66], sifumanise ukuba iiviromes kunye noluhlu lweembaza A. atra kunye neM. platensis ziwela phakathi kwezi ntlobo zimbini.ngokufanayo (jonga umfanekiso S3, ulwazi olongezelelweyo).Oku kufana akumangalisi, njengoko kunokubonakalisa ukungabikho kokukhetha ekuthathweni kwe-DNA ekhoyo kwindawo.Izifundo zexesha elizayo zisebenzisa i-RNA ecocekileyo ziyafuneka ngoku ukubonisa i-RNA virome.
Kuphononongo lwethu, sisebenzise umbhobho ongqongqo kakhulu othathwe kumsebenzi ka-Kowarski kunye noogxa [37], abasebenzisa ukucinywa kwamanyathelo amabini okufundwa kunye ne-contigs ngaphambi nasemva kokudityaniswa kwe-ccfDNA yomthonyama, okubangele umlinganiselo ophezulu wokufundwa okungabhalwanga.Ke ngoko, asinakukhuphela ngaphandle ukuba ezinye zezi ncwadi zifundwayo zingachazwanga zisenokuba nemvelaphi yazo, ikakhulu ngenxa yokuba asinayo i-genome yolu didi lweembaza.Siphinde sasebenzisa lo mbhobho kuba sasinenkxalabo malunga ne-chimeras phakathi kokuzifundela kunye nokungazifundanga kunye nobude obufundwayo obuveliswe yi-Illumina MiSeq PE75.Esinye isizathu sokufunda okuninzi okungabhalwanga kukuba uninzi lweentsholongwane zaselwandle, ngakumbi kwiindawo ezikude njengeKerguelen, azikachazwa.Sisebenzise i-Illumina MiSeq PE75, sicinga ukuba ubude beqhekeza le-ccfDNA bufana ne-ccfDNA yabantu.Kwizifundo zexesha elizayo, kunikwe iziphumo zethu ezibonisa ukuba i-hemolymph ccfDNA ifundeka ixesha elide kunabantu kunye/okanye izilwanyana ezanyisayo, sicebisa ukuba kusetyenziswe iqonga lolandelelwano olulungele ngakumbi amaqhekeza amade e-ccfDNA.Lo mkhuba uya kwenza kube lula kakhulu ukuchonga izibonakaliso ezininzi zohlalutyo olunzulu.Ukufumana ulandelelwano lwegenome yenyukliya yeAtra engafumanekiyo kukwalungiselela kakhulu ucalucalulo lwe-ccfDNA kwimithombo yobuqu kunye nengeyiyo.Njengoko uphando lwethu lugxininise kumathuba okusebenzisa ingqikelelo ye-liquid biopsy kwiimbaza, sinethemba lokuba njengoko le ngcamango isetyenziswa kuphando lwexesha elizayo, izixhobo ezitsha kunye nemibhobho iya kuphuhliswa ukwandisa amandla ale ndlela yokufunda iyantlukwano yemicrobial yembaza.i-ecosystem yolwandle.
Njenge-biomarker yeklinikhi engabonakaliyo, amanqanaba aphezulu e-plasma ye-ccfDNA adibene nezifo ezahlukahlukeneyo, umonakalo wezicubu, kunye neemeko zoxinzelelo [67,68,69].Olu lwando lunxulunyaniswa nokukhululwa kwamaqhekeza e-DNA emvelaphi yawo emva komonakalo wezicubu.Siwusombulule lo mba sisebenzisa uxinzelelo oluqatha lobushushu, apho iimbaza ziye zavezwa kancinci kubushushu obungama-30 °C.Senze olu hlalutyo kwiindidi ezintathu ezahlukeneyo zeembaza kwimifuniselo emithathu ezimeleyo.Nangona kunjalo, asizange sifumane naluphi na utshintsho kumanqanaba e-ccfDNA emva koxinzelelo oluphezulu lobushushu (jonga umfanekiso we-S5, ulwazi olongezelelweyo).Oku kufunyaniswayo kunokuchaza, ubuncinci, into yokuba i-mussels ine-semi-open circulatory system kwaye iqokelele isixa esikhulu se-DNA yangaphandle ngenxa yokucoca okuphezulu.Kwelinye icala, iimbaza, njengezilwanyana ezininzi ezingenamqolo, zinokunganyangeki ngakumbi kumonakalo wezicubu ezibangelwa luxinzelelo, ngaloo ndlela zinciphisa ukukhutshwa kwe-ccfDNA kwi-hemolymph [70, 71].
Ukuza kuthi ga ngoku, uhlalutyo lwe-DNA lweentlobo ngeentlobo zezinto eziphilayo kwi-ecosystems yasemanzini lujolise ikakhulu kwi-metabarcoding ye-DNA (eDNA) yokusingqongileyo.Nangona kunjalo, le ndlela ihlala ilinganiselwe kuhlalutyo lwezinto eziphilayo xa kusetyenziswa iiprimers.Ukusetyenziswa kokulandelelana kompu kuthintela imida ye-PCR kunye nokhetho olucalucalulo lweeseti zeprimer.Ngaloo ndlela, ngandlela ithile, indlela yethu isondele kwindlela esandul 'ukusetyenziswa kwe-eDNA Shotgun yokulandelelana ngokuthe ngqo, ekwazi ukulandelelanisa ngokuthe ngqo i-DNA eqhekezayo kwaye ihlalutye phantse zonke izinto eziphilayo [72, 73].Nangona kunjalo, kukho imiba emininzi ebalulekileyo eyahlula i-LB kwiindlela eziqhelekileyo ze-eDNA.Ngokuqinisekileyo, umahluko omkhulu phakathi kwe-eDNA kunye ne-LB kukusetyenziswa kwemikhosi yokucoca kwendalo.Ukusetyenziswa kweentlobo zaselwandle ezifana ne-sponges kunye ne-bivalves (i-Dresseina spp.) Njengesihluzo sendalo sokufunda i-eDNA kuye kwabikwa [74, 75].Nangona kunjalo, uphando lukaDreissena lwasebenzisa i-biopsy yezicubu apho i-DNA yakhutshwa khona.Uhlalutyo lwe-ccfDNA evela kwi-LB ayifuni i-biopsy yezicubu, izixhobo ezikhethekileyo kwaye ngamanye amaxesha ezibiza kakhulu kunye nezinto ezinxulumene ne-eDNA okanye i-tissue biopsy.Enyanisweni, kutshanje siye sabika ukuba i-ccfDNA evela kwi-LB inokugcinwa kwaye ihlalutywe ngenkxaso ye-FTA ngaphandle kokugcina ikhonkco ebandayo, eyona mngeni omkhulu wophando kwiindawo ezikude [76].Ukutsalwa kwe-ccfDNA kwi-biopsies yolwelo nayo ilula kwaye ibonelela ngomgangatho ophezulu we-DNA yokulandelelana kompu kunye nohlalutyo lwe-PCR.Le yinzuzo enkulu enikwe ezinye zeemida zobugcisa ezinxulumene nohlalutyo lwe-eDNA [77].Ubulula kunye neendleko eziphantsi zendlela yesampulu nazo zifanelekile ngokukodwa kwiinkqubo zokubeka iliso ixesha elide.Ukongeza kubuchule babo bokucoca okuphezulu, enye into eyaziwayo ye-bivalves yikhemikhali ye-mucopolysaccharide ye-mucus yabo, ekhuthaza ukufunxwa kweentsholongwane [78, 79].Oku kwenza ukuba i-bivalves ibe sisihluzo sendalo esifanelekileyo sokuphawula izinto ezahlukeneyo zebhayoloji kunye neempembelelo zokutshintsha kwemozulu kwindawo ephilayo yasemanzini.Nangona ubukho bamaqhekeza e-DNA athathwe kumkhosi bungabonwa njengomda wendlela xa kuthelekiswa ne-eDNA, iindleko ezinxulumene nokuba ne-ccfDNA yemveli xa kuthelekiswa ne-eDNA iyaqondakala ngaxeshanye kuninzi lolwazi olukhoyo kwizifundo zempilo.i-offset host.Oku kubandakanya ubukho bokulandelelana kwentsholongwane edibeneyo kwi-genome yomninimzi.Oku kubaluleke kakhulu kwiimbaza, ngenxa yobukho be-retroviruses ye-leukemic edluliselwe ngokuthe tye kwi-bivalves [80, 81].Enye inzuzo ye-LB ngaphezu kwe-eDNA kukuba ixhaphaza umsebenzi we-phagocytic wokujikeleza iiseli zegazi kwi-hemolymph, eyenza i-microorganisms (kunye ne-genomes yazo).I-Phagocytosis ngowona msebenzi uphambili weeseli zegazi kwi-bivalves [82].Ekugqibeleni, indlela ithatha ithuba lokuhluza okuphezulu kwe-mussels (i-avareji ye-1.5 l / h yamanzi olwandle) kunye nokujikeleza kweentsuku ezimbini, okwandisa ukuxutywa kweendlela ezahlukeneyo zamanzi olwandle, okuvumela ukuba kubanjwe i-eDNA ye-heterologous.[83,84].Ke ngoko, uhlalutyo lwembaza ccfDNA yindlela enomdla enikwe isondlo, uqoqosho, kunye neempembelelo zokusingqongileyo kwimbaza.Ngokufana nohlalutyo lwe-LB eqokelelwe ebantwini, le ndlela iphinda ivule ithuba lokulinganisa utshintsho lwezofuzo kunye ne-epigenetic kwi-DNA yomkhosi ekuphenduleni izinto ezingaphandle.Umzekelo, itekhnoloji yolandelelwano lwesizukulwana sesithathu inokucingelwa ukwenza uhlalutyo lwe-genome-wide methylation kwi-ccfDNA yasekhaya kusetyenziswa ukulandelelana kwe-nanopore.Le nkqubo kufuneka iququzelelwe yinto yokuba ubude beengqungquthela ze-ccfDNA ze-mussel zihambelana ngokufanelekileyo kunye neeplatifomu zokulandelelanisa ixesha elide ezivumela uhlalutyo lwe-DNA methylation ye-genome-wide ukusuka kulandelelwano olulodwa oluqhutywe ngaphandle kwesidingo sokuguqulwa kweekhemikhali.85,86] Le yinto enomdla, njengoko kubonisiwe ukuba impendulo ye-DNA methylistation imilinganiselo yoxinzelelo lwendalo kunye neepatheni ezininzi ze-methylistation zendalo zibonisa.Ngoko ke, inokubonelela ngengqiqo ebalulekileyo kwiindlela eziphantsi ezilawula impendulo emva kokuvezwa kokutshintsha kwemozulu okanye ukungcola [87].Nangona kunjalo, ukusetyenziswa kweLB akukho mida.Ngokungafunekiyo ukuthetha, oku kufuna ubukho beentlobo zesalathisi kwi-ecosystem.Njengoko kukhankanyiwe ngasentla, ukusebenzisa i-LB ukuvavanya i-biodiversity ye-ecosystem enikiweyo ikwafuna umbhobho ongqongqo we-bioinformatics othathela ingqalelo ubukho bamaqhekeza e-DNA asuka kumthombo.Enye ingxaki enkulu kukufumaneka kwee-genomes ezibhekiselele kwiintlobo zaselwandle.Kuyathenjwa ukuba amalinge afana neProjekthi yeMarine Mammal Genomes kunye neprojekthi esandul' ukusekwa yeFish10k [88] iya kuququzelela uhlalutyo olunjalo kwixesha elizayo.Ukusetyenziswa kwengqikelelo ye-LB kwizinto eziphilayo zaselwandle ezitya izihluzo nazo ziyahambelana nenkqubela phambili yamva nje yokulandelelanisa ubuchwepheshe, okwenza ukuba ifaneleke kakuhle ukuphuhliswa kwe-multi-ohm biomarkers ukunika ulwazi olubalulekileyo malunga nempilo yeendawo zokuhlala zaselwandle ekuphenduleni uxinzelelo lokusingqongileyo.
Idatha yolandelelwano lwe-genome ifakwe kwi-NCBI yokuSeka i-Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 phantsi kwe-Bioprojects SRR8924808.
Brierley AS, Kingsford MJ Impembelelo yokutshintsha kwemozulu kubomi baselwandle kunye ne-ecosystem.Cole Biology.2009;19: P602–P614.
Gissi E, Manea E, Mazaris AD, Fraschetti S, Almpanidou V, Bevilacqua S, et al.Qwalasela iimpembelelo ezidityanisiweyo zokutshintsha kwemozulu kunye nezinye izinto ezicinezelayo kwimo engqongileyo yaselwandle.indawo jikelele yenzululwazi.2021;755:142564.
Carella F, Antuofermo E, Farina S, Salati F, Mandas D, Prado P, et al.).Inzululwazi yokuqala kaMatshi.2020;7:48.
I-Seront L, i-Nicastro CR, i-Zardi GI, i-Goberville E. Ukunciphisa ukunyamezela ukushisa phantsi kweemeko eziphindaphindiweyo zokuxinzelela ukushisa kuchaza ukufa okuphezulu kwehlobo le-blue mussels.Ingxelo yezeNzululwazi 2019;9:17498.
Fey SB, Siepielski AM, Nussle S, Cervantes-Yoshida K, Hwan JL, Huber ER, et al.Utshintsho lwakutsha nje kumaxesha, oonobangela kunye nobungakanani bokusweleka 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.
Bradley M, Coutts SJ, Jenkins E, O'Hara TM.Impembelelo enokubakho yokutshintsha kwemozulu kwizifo ze-Arctic zoonotic.Int J Circumpolar impilo.2005;64:468–77 .
Beyer J., Greene NW, Brooks S., Allan IJ, Ruus A., Gomez T. et al.Iimbaza eziluhlaza (Mytilus edulis spp.) njengezidalwa ezibonisa umqondiso kuhlolo longcoliseko lonxweme: uphononongo.Mar Environ Res 2017;130:338-65 .
USiravegna G, uMarsoni S, uSiena S, uBardelli A. Ukudityaniswa kwe-biopsy yolwelo kunyango lomhlaza.Nat Rev Clean Oncol.2017;14:531–48 .
Wan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, et al.Ukuvuthwa kwe-biopsy yolwelo: Ivumela i-tumor DNA ukuba ijikeleze.Nat Rev Cancer.2017;17:223–38.
Mandel P., Metais P. Nucleic acids kwiplasma yabantu.Imizuzu yeentlanganiso zeSoc Biol encedisayo.1948;142:241-3 .
Bronkhorst AJ, Ungerer W, Holdenrieder S. Indima entsha ye-cell-free DNA njengophawu lwemolekyuli yonyango lomhlaza.Ubungakanani bohlalutyo lwe-biomolar.2019;17:100087.
Ignatiadis M., Sledge GW, Jeffrey SS Liquid biopsy ingena kwiklinikhi - imiba yokuphunyezwa kunye nemingeni yexesha elizayo.Nat Rev Clin Oncol.2021;18:297–312 .
Lo YM, Corbetta N., Chamberlain PF, Rai W., Sargent IL, Redman CW kunye nabanye.I-DNA ye-Fetal ikhona kwi-plasma yomama kunye ne-serum.I-Lancet.1997;350:485-7.
I-Mufarray MN, i-Wong RJ, uShaw GM, uStevenson DK, i-Quake SR Uphononongo lwekhosi yokukhulelwa kunye neengxaki zayo usebenzisa i-RNA ejikelezayo ye-extracellular egazini labasetyhini ngexesha lokukhulelwa.Unyango lwabantwana.2020;8:605219.
Ollerich M, Sherwood K, Keown P, Schütz E, Beck J, Stegbauer J, et al.I-Liquid biopsy: i-donor cell-free DNA isetyenziselwa ukufumanisa izilonda ze-allogeneic kwi-graft yezintso.Nat uMfundisi uNephroli.2021;17:591–603 .
UJuan FC, uLo YM Innovations kuxilongo lwaphambi kokubeleka: ulandelelwano lwe-plasma genome yoomama.UAna MD.2016;67:419-32.
Gu W, Deng X, Lee M, Sucu YD, Arevalo S, Stryke D, et al.Ukufunyaniswa kwe-pathogen ngokukhawuleza kunye nesizukulwana esilandelayo solandelelwano lwe-metagenomic yolwelo olusulelekileyo lomzimba.Nat Medicine.2021;27:115-24.
Ixesha lokuposa: Aug-14-2022