Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv CSS kev txhawb nqa.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis lov tes taw Compatibility Hom hauv Internet Explorer).Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav ua rau lub xaib tsis muaj qauv thiab JavaScript.
Liquid biopsy (LB) yog ib lub tswv yim uas tau txais kev muaj koob meej sai hauv biomedical field.Lub tswv yim feem ntau yog ua raws li kev tshawb pom ntawm cov khoom tawg ntawm cov kab mob DNA (ccfDNA), uas feem ntau tso tawm raws li cov khoom me me tom qab cell tuag hauv ntau cov ntaub so ntswg.Ib feem me me ntawm cov khoom tawg no yog los ntawm cov ntaub so ntswg txawv teb chaws los yog cov kab mob.Hauv kev ua haujlwm tam sim no, peb tau siv lub tswv yim no rau cov mussels, ib hom kab mob uas paub txog lawv cov dej hiav txwv pom muaj peev xwm.Peb siv lub peev xwm ntawm mussels los ua cov ntxaij lim dej ntuj los ntes cov DNA ib puag ncig los ntawm ntau qhov chaw los muab cov ntaub ntawv hais txog biodiversity ntawm marine ntug dej hiav txwv ecosystems.Peb cov txiaj ntsig tau pom tias mussel hemolymph muaj cov kab DNA uas sib txawv loj heev, los ntawm 1 txog 5 kb.Shotgun sequencing qhia tau hais tias muaj coob tus DNA fragments yog txawv teb chaws microbial keeb kwm.Ntawm lawv, peb pom DNA tawg ntawm cov kab mob, archaea, thiab cov kab mob, suav nrog cov kab mob uas paub tias kis tau rau ntau tus tswv tsev feem ntau pom nyob rau hauv ntug dej hiav txwv ecosystems.Hauv kev xaus, peb txoj kev tshawb fawb pom tau tias lub tswv yim ntawm LB siv rau cov mussels sawv cev rau cov nplua nuj tab sis tseem tsis tau tshawb nrhiav qhov kev paub txog microbial ntau haiv neeg hauv cov dej hiav txwv ntug dej hiav txwv.
Qhov cuam tshuam ntawm kev hloov pauv huab cua (CC) ntawm biodiversity ntawm marine ecosystems yog ib qho chaw loj hlob sai ntawm kev tshawb fawb.Kev ua kom sov hauv ntiaj teb tsis tsuas yog ua rau muaj kev ntxhov siab ntawm lub cev, tab sis kuj thawb cov kev txwv ntawm kev hloov pauv ntawm thermal stability ntawm cov kab mob hauv hiav txwv, cuam tshuam rau thaj chaw ntawm ntau hom, ua rau lawv tshawb nrhiav ntau yam zoo [1, 2].Ntxiv rau qhov cuam tshuam rau biodiversity ntawm metazoans, CC cuam tshuam qhov sib npaug ntawm tus tswv-microbial kev sib cuam tshuam.Cov kab mob microbial dysbacteriosis no ua rau muaj kev hem thawj loj rau marine ecosystems vim nws ua rau cov kab mob hauv hiav txwv muaj kev cuam tshuam rau cov kab mob sib kis [3, 4].Nws ntseeg tau tias SS ua lub luag haujlwm tseem ceeb hauv kev tuag coob, uas yog qhov teeb meem loj rau kev tswj hwm thoob ntiaj teb marine ecosystems [5, 6].Qhov no yog ib qho teeb meem tseem ceeb uas muab kev lag luam, ecological thiab khoom noj khoom haus cuam tshuam ntawm ntau hom marine.Qhov no yog tshwj xeeb tshaj yog muaj tseeb rau bivalves nyob rau hauv lub polar cheeb tsam, qhov twg cov teebmeem ntawm CK yog ntau tam sim ntawd thiab hnyav [6, 7].Qhov tseeb, bivalves xws li Mytilus spp.tau dav siv los saib xyuas qhov cuam tshuam ntawm CC ntawm marine ecosystems.Tsis yog qhov xav tsis thoob, ntau tus lej ntawm biomarkers tau tsim los saib xyuas lawv txoj kev noj qab haus huv, feem ntau siv ob-tier txoj hauv kev uas muaj kev ua haujlwm biomarkers raws li kev ua haujlwm enzymatic lossis cellular zog xws li cell viability thiab phagocytic kev ua haujlwm [8].Cov txheej txheem no kuj suav nrog kev ntsuas qhov concentration ntawm cov ntsuas ntsuas tshwj xeeb uas sib sau hauv cov ntaub so ntswg tom qab nqus dej hiav txwv ntau.Txawm li cas los xij, lub peev xwm pom zoo thiab ib nrab qhib cov hlab ntsha ntawm bivalves muab lub sijhawm los tsim cov hemolymph biomarkers tshiab siv lub tswv yim ntawm kua biopsy (LB), ib qho yooj yim thiab tsis tshua muaj kev cuam tshuam rau kev tswj tus neeg mob.ntshav kuaj [9, 10].Txawm hais tias muaj ntau hom circulating molecules tuaj yeem pom hauv tib neeg LB, lub tswv yim no feem ntau yog raws li DNA sequencing tsom xam ntawm circulating extracellular DNA (ccfDNA) fragments nyob rau hauv plasma.Qhov tseeb, lub xub ntiag ntawm kev nthuav dav DNA hauv tib neeg cov ntshav tau paub txij li nruab nrab xyoo pua 20th [11], tab sis nws tsuas yog nyob rau xyoo tas los no uas qhov tshwm sim ntawm cov txheej txheem sib luag tau ua rau kev kuaj mob raws li ccfDNA.Lub xub ntiag ntawm cov circulating DNA fragments yog vim ib feem ntawm passive tso tawm ntawm genomic DNA (nuclear thiab mitochondrial) tom qab cell tuag. Hauv cov tib neeg noj qab haus huv, qhov concentration ntawm ccfDNA feem ntau qis (<10 ng / mL) tab sis tuaj yeem nce 5-10 zaug hauv cov neeg mob uas muaj ntau yam pathologies lossis raug kev ntxhov siab, ua rau cov ntaub so ntswg puas. Hauv cov tib neeg noj qab haus huv, qhov concentration ntawm ccfDNA feem ntau qis (<10 ng / mL) tab sis tuaj yeem nce 5-10 zaug hauv cov neeg mob uas muaj ntau yam pathologies lossis raug kev ntxhov siab, ua rau cov ntaub so ntswg puas. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 разонон ральрет гией или подвергающихся стрессу, приводящему к повреждению тканей. Hauv cov neeg noj qab haus huv, qhov concentration ntawm cccDNA feem ntau qis (<10 ng / mL), tab sis nws tuaj yeem nce 5-10 zaug hauv cov neeg mob uas muaj ntau yam kab mob lossis muaj kev ntxhov siab uas ua rau cov ntaub so ntswg puas.在健康个体中，ccfDNA 的浓度通常较低（<10 ng/mL），但在患有各种病理或承受压力的患倅 5导致组织损伤.在健康个体中，ccfdna 的浓度较低（（<10 ng/ml）但在各种病理或承受压倅 0倍 ， 从而 组织。。。 损伤 损伤 损伤 损伤 损伤 损伤Концентрации ccfDNA обычно низкие (<10 нг/мл) у здоровых людей, но могут быть увеличены в 5-10 ратразу ологиями или стрессом, что приводит к повреждению тканей. ccfDNA cov ntsiab lus feem ntau yog qis (<10 ng / ml) hauv cov tib neeg noj qab haus huv, tab sis tuaj yeem nce 5-10-fold hauv cov neeg mob uas muaj ntau yam kab mob lossis kev ntxhov siab, ua rau cov ntaub so ntswg puas.Qhov loj ntawm ccfDNA fragments sib txawv, tab sis feem ntau yog li ntawm 150 txog 200 bp.[12].Kev soj ntsuam ntawm tus kheej-derived ccfDNA, piv txwv li, ccfDNA los ntawm ib txwm lossis hloov pauv cov tswv tsev, tuaj yeem siv los txheeb xyuas cov noob caj noob ces thiab epigenetic hloov pauv tam sim no nyob rau hauv cov genome thiab / lossis mitochondrial genome, yog li pab cov kws kho mob xaiv cov kev kho mob tshwj xeeb rau cov tshuaj molecular [13] .Txawm li cas los xij, ccfDNA tuaj yeem tau txais los ntawm cov chaw txawv teb chaws xws li ccfDNA los ntawm cov menyuam hauv plab thaum cev xeeb tub lossis los ntawm cov kabmob hloov pauv [14,15,16,17].ccfDNA kuj tseem yog ib qho tseem ceeb ntawm cov ntaub ntawv txhawm rau txhawm rau kuaj pom muaj cov nucleic acids ntawm tus neeg sawv cev kis kab mob (hloov txawv teb chaws), uas tso cai rau kev tshawb nrhiav tsis muaj kab mob kis thoob plaws uas tsis tau txheeb xyuas los ntawm cov kab lis kev cai ntshav, tsis txhob cuam tshuam cov kab mob ntawm cov ntaub so ntswg [18].Cov kev tshawb fawb tsis ntev los no tau pom tseeb tias tib neeg cov ntshav muaj cov ntaub ntawv nplua nuj uas tuaj yeem siv los txheeb xyuas cov kab mob thiab kab mob, thiab hais tias kwv yees li 1% ntawm ccfDNA pom hauv tib neeg cov ntshav yog los ntawm txawv teb chaws [19].Cov kev tshawb fawb no qhia tau hais tias cov biodiversity ntawm ib tug kab mob lub circulating microbiome yuav raug soj ntsuam siv ccfDNA tsom xam.Txawm li cas los xij, txog thaum tsis ntev los no, lub tswv yim no tau siv tshwj xeeb rau tib neeg thiab, rau qhov tsawg dua, hauv lwm cov vertebrates [20, 21].
Nyob rau hauv daim ntawv tam sim no, peb siv lub peev xwm LB los soj ntsuam ccfDNA ntawm Aulacomya atra, ib hom kab yav qab teb uas pom nyob rau hauv subantarctic Kerguelen Islands, ib pawg ntawm cov Islands tuaj saum toj siab loj uas tsim 35 lab xyoo dhau los.volcanic eruption.Siv qhov kev sim hauv vitro, peb pom tias DNA tawg hauv dej hiav txwv tau sai sai los ntawm mussels thiab nkag mus rau hauv hemolymph compartment.Shotgun sequencing tau qhia tias mussel hemolymph ccfDNA muaj DNA tawg ntawm nws tus kheej thiab tsis yog nws tus kheej keeb kwm, suav nrog cov kab mob symbiotic thiab DNA tawg los ntawm biomes ib yam ntawm cov dej txias dej hiav txwv ntug dej hiav txwv ecosystems.Hemolymph ccfDNA kuj muaj cov kab mob sib kis tau los ntawm cov kab mob nrog ntau tus tswv tsev sib txawv.Peb kuj pom DNA tawg ntawm cov tsiaj txhu ntau yam xws li cov ntses pob txha, hiav txwv anemones, algae thiab kab.Hauv kev xaus, peb txoj kev tshawb fawb pom tau tias LB lub tswv yim tuaj yeem ua tiav rau cov tsiaj txhu hauv hiav txwv kom tsim tau ib qho kev nplua nuj genomic repertoire hauv marine ecosystems.
Cov neeg laus (55-70 mm ntev) Mytilus platensis (M. platensis) thiab Aulacomya atra (A. atra) tau sau los ntawm intertidal rocky shores ntawm Port-au-Fabkis (049°21.235 S, 070°13.490 E.).Kerguelen Islands nyob rau lub Kaum Ob Hlis 2018. Lwm cov neeg laus xiav mussels (Mytilus spp.) tau txais los ntawm cov chaw muag khoom lag luam (PEI Mussel King Inc., Prince Edward Island, Canada) thiab muab tso rau hauv qhov ntsuas kub (4 ° C) aerated tank uas muaj 10-20 L ntawm 32‰ dag brine.(Artificial hiav txwv ntsev Reef Crystal, Instant Ocean, Virginia, USA).Rau txhua qhov kev sim, qhov ntev thiab qhov hnyav ntawm tus kheej lub plhaub tau ntsuas.
Txoj cai qhib pub dawb rau txoj haujlwm no muaj nyob online (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).Luv luv, LB hemolymph tau sau los ntawm cov leeg abductor raws li tau piav qhia [22].Lub hemolymph tau qhia meej los ntawm centrifugation ntawm 1200 × g rau 3 feeb, lub supernatant tau khov (-20 ° C) kom txog thaum siv.Rau kev sib cais thiab kev ua kom huv ntawm cfDNA, cov qauv (1.5-2.0 ml) tau thawed thiab ua tiav siv cov khoom siv NucleoSnap cfDNA (Macherey-Nagel, Bethlehen, PA) raws li cov chaw tsim khoom cov lus qhia.ccfDNA tau khaws cia ntawm -80 ° C kom txog thaum kev tshuaj xyuas ntxiv.Hauv qee qhov kev sim, ccfDNA raug cais tawm thiab ua kom huv si siv QIAamp DNA Kev Tshawb Fawb Cov Khoom Siv (QIAGEN, Toronto, Ontario, Canada).Purified DNA tau txheeb xyuas siv tus qauv PicoGreen kuaj.Kev faib tawm ntawm cov ccfDNA cais tau raug tshuaj xyuas los ntawm capillary electrophoresis siv Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) siv cov khoom siv DNA siab.Qhov kev ntsuam xyuas tau ua tiav siv 1 µl ntawm cov qauv ccfDNA raws li cov chaw tsim khoom cov lus qhia.
Rau sequencing ntawm hemolymph ccfDNA fragments, Génome Québec (Montreal, Quebec, Canada) tau npaj cov tsev qiv ntawv tua phom siv Illumina DNA Mix cov khoom siv ntawm Illumina MiSeq PE75 cov khoom siv.Tus qauv adapter (BioO) tau siv.Cov ntaub ntawv nyoos muaj nyob hauv NCBI Sequence Read Archive (SRR8924808 thiab SRR8924809).Kev nyeem ntawv yooj yim tau raug soj ntsuam siv FastQC [23].Trimmomatic [24] tau siv rau clipping adapters thiab nyeem tsis zoo.Shotgun nyeem nrog kev sib tw kawg tau FLASH muab tso rau hauv ib qho kev nyeem ntev dua nrog qhov sib tshooj tsawg kawg ntawm 20 bp kom tsis txhob muaj qhov tsis sib xws [25]. Cov ntawv nyeem sib koom ua ke tau sau tseg nrog BLASTN siv bivalve NCBI Taxonomy database (e tus nqi < 1e−3 thiab 90% homology), thiab npog cov txheej txheem tsis sib xws tau ua los ntawm DUST [26]. Cov ntawv nyeem sib koom ua ke tau sau tseg nrog BLASTN siv bivalve NCBI Taxonomy database (e tus nqi < 1e−3 thiab 90% homology), thiab npog cov txheej txheem tsis sib xws tau ua los ntawm DUST [26]. Объединенные чтения были анннотированы с помощью BLASTN с использованием базы данных таксономии двуслтворазы e <1e-3 thiab 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с исполь 2. Pooled nyeem tau sau tseg nrog BLASTN siv NCBI bivalve taxonomy database (e tus nqi <1e-3 thiab 90% homology), thiab tsis tshua muaj qhov sib txawv ntawm qhov npog ntsej muag tau ua los ntawm DUST [26].使用双壳类 NCBI 分类数据库 (e 值 < 1e-3 和90% 同源性) 用BLASTN 注释合并的读潰甍 并迎 T杂度序列的掩蔽.使用双壳类 ncbi 分类（（（<1e-3和 90%同源））用用用注释合并读数，幨忽6甶忽甶用忽甶用忽。序列的。。。掩蔽掩蔽掩蔽掩蔽掩蔽Объединенные чтения были анннотированы с помощью BLASTN с использованием таксономической базы данныхв с вус чение e <1e-3 и 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с ис. Cov ntawv nyeem sib sau tau sau tseg nrog BLASTN siv NCBI bivalve taxonomic database (e tus nqi <1e-3 thiab 90% homology), thiab tsis tshua muaj qhov sib txawv ntawm qhov npog ntsej muag tau ua los ntawm DUST [26].Kev nyeem tau muab faib ua ob pawg: ntsig txog bivalve sequences (no hu ua self-reads) thiab unrelated (non-self-reads).Ob pawg tau sib sau ua ke siv MEGAHIT los tsim contigs [27].Lub caij no, kev faib se tawm ntawm cov neeg txawv teb chaws microbiome nyeem tau raug cais siv Kraken2 [28] thiab cov duab sawv cev los ntawm Krona ncuav qab zib ntawm Galaxy [29, 30].Qhov zoo tshaj plaws kmers tau txiav txim siab yog kmers-59 los ntawm peb qhov kev sim ua ntej. Tus kheej contigs tau raug txheeb xyuas los ntawm kev sib haum xeeb nrog BLASTN (bivalve NCBI database, e tus nqi <1e−10 thiab 60% homology) rau qhov kawg annotation. Tus kheej contigs tau raug txheeb xyuas los ntawm kev sib haum xeeb nrog BLASTN (bivalve NCBI database, e tus nqi <1e−10 thiab 60% homology) rau qhov kawg annotation. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двусытвор чата двусытвор чата) <1e-10 thiab гомология 60%) для окончательной аннотации. Tus kheej-contigs raug txheeb xyuas los ntawm kev sib piv nrog BLASTN (NCBI bivalve database, e tus nqi <1e-10 thiab 60% homology) rau qhov kawg annotation.然后通过与 BLASTN (双壳贝类NCBI 数据库, e 值< 1e-10 和60% 同源性)注释.然后通过与BLASTN (双壳贝类NCBI 数据库, e 值 < 1e-10 和60% Затем были идентифицированы собственные контиги для окончательной анннотации путем сопоста вления NBI с устворчатых моллюсков, значение e <1e-10 thiab гомология 60%). Tus kheej-contigs raug txheeb xyuas rau qhov kawg annotation los ntawm kev sib piv nrog BLASTN (NCBI bivalve database, e tus nqi <1e-10 thiab 60% homology). Nyob rau hauv parallel, nonself pawg contigs tau annotated nrog BLASTN (nt NCBI database, e tus nqi <1e−10 thiab 60% homology). Nyob rau hauv parallel, nonself pawg contigs tau annotated nrog BLASTN (nt NCBI database, e tus nqi <1e−10 thiab 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, зна <чиони.-1гога дананых nt NCBI, зна <чиоeни.-1гога) Nyob rau tib lub sijhawm, cov pab pawg txawv teb chaws tau sau tseg nrog BLASTN (NT NCBI database, e tus nqi <1e-10 thiab 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 (<базачни NT 1 зенны) thiab 60%). Nyob rau tib lub sijhawm, pawg tsis yog tus kheej tau sau tseg nrog BLASTN (nt NCBI database, e tus nqi <1e-10 thiab 60% homology). BLASTX kuj tau ua los ntawm non-self contigs siv cov nr thiab RefSeq protein NCBI databases (e tus nqi <1e−10 thiab 60% homology). BLASTX kuj tau ua los ntawm non-self contigs siv cov nr thiab RefSeq protein NCBI databases (e tus nqi <1e−10 thiab 60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значо 1% гего значо 1% . BLASTX kuj tau ua rau cov tsis-tus kheej contigs siv cov nr thiab RefSeq NCBI protein databases (e tus nqi <1e-10 thiab 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% 倧源).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% 倧源). BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значиение % 0 года <1 года). BLASTX kuj tau ua los ntawm cov tsis-tus kheej contigs siv cov nr thiab RefSeq NCBI protein databases (e tus nqi <1e-10 thiab 60% homology).Lub BLASTN thiab BLASTX pas dej ntawm cov tsis-tus kheej-contigs sawv cev rau qhov kawg contigs (saib cov ntaub ntawv ntxiv).
Cov primers siv rau PCR yog teev nyob rau hauv Table S1.Taq DNA polymerase (Bio Basic Canada, Markham, ON) tau siv los ua kom nthuav dav ccfDNA hom phiaj noob.Cov tshuaj tiv thaiv hauv qab no tau siv: denaturation ntawm 95 ° C rau 3 feeb, 95 ° C rau 1 feeb, teem annealing kub rau 1 feeb, elongation ntawm 72 ° C rau 1 feeb, 35 cycles, thiab thaum kawg 72 ° C nyob rau hauv 10 feeb..PCR cov khoom raug cais los ntawm electrophoresis hauv agarose gels (1.5%) uas muaj SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Canada) ntawm 95 V.
Mussels (Mytilus spp.) tau acclimatized hauv 500 ml oxygenated seawater (32 PSU) rau 24 teev ntawm 4 ° C.Plasmid DNA uas muaj qhov ntxig encoding tib neeg galectin-7 cDNA ib ntus (NCBI tus lej L07769) tau ntxiv rau lub vial ntawm qhov kawg ntawm 190 μg / μl.Mussels incubated nyob rau hauv tib yam mob yam tsis muaj DNA ntxiv yog tus tswj.Qhov thib peb tswj tank muaj DNA tsis muaj mussels.Txhawm rau saib xyuas qhov zoo ntawm DNA hauv dej hiav txwv, cov qauv dej hiav txwv (20 μl; peb rov ua dua) raug coj los ntawm txhua lub tank ntawm lub sijhawm qhia.Rau plasmid DNA traceability, LB mussels tau sau thaum lub sij hawm qhia thiab soj ntsuam los ntawm qPCR thiab ddPCR.Vim muaj cov ntsiab lus ntsev ntawm dej hiav txwv, aliquots tau diluted hauv PCR dej zoo (1:10) ua ntej txhua qhov kev ntsuam xyuas PCR.
Digital droplet PCR (ddPCR) tau ua tiav siv BioRad QX200 raws tu qauv (Mississauga, Ontario, Canada).Siv qhov kub thiab txias profile los txiav txim siab qhov kub thiab txias (Table S1).Poob tau tsim siv QX200 lub tshuab hluav taws xob poob (BioRad).ddPCR tau ua raws li hauv qab no: 95 ° C rau 5 min, 50 cycles ntawm 95 ° C rau 30 s thiab muab annealing kub rau 1 min thiab 72 ° C rau 30 s, 4 ° C rau 5 min thiab 90 ° C hauv 5 feeb.Tus naj npawb ntawm cov tee thiab cov tshuaj tiv thaiv zoo (ntau cov ntawv luam / µl) tau ntsuas los ntawm QX200 tus nyeem ntawv nco (BioRad).Cov qauv uas muaj tsawg dua 10,000 tee dej tau raug tso tseg.Tus qauv tswj tsis tau ua txhua zaus ddPCR khiav.
qPCR tau ua tiav siv Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) thiab LGALS7 tshwj xeeb primers.Tag nrho cov PCRs tau ua tiav hauv 20 µl siv QuantFast SYBR Green PCR Kit (QIAGEN).qPCR tau pib nrog 15 min incubation ntawm 95 ° C tom qab 40 lub voj voog ntawm 95 ° C rau 10 vib nas this thiab ntawm 60 ° C rau 60 vib nas this nrog ib qho kev sau cov ntaub ntawv.Melting curves tau tsim los siv kev ntsuas ua tiav ntawm 95 ° C rau 5 s, 65 ° C rau 60 s, thiab 97 ° C thaum kawg ntawm qPCR.Txhua qPCR tau ua nyob rau hauv triplicate, tshwj tsis yog rau cov qauv tswj.
Txij li thaum mussels paub txog lawv qhov kev pom zoo, peb tau tshawb xyuas thawj zaug seb lawv puas tuaj yeem lim thiab khaws cov DNA seem nyob hauv dej hiav txwv.Peb kuj tau xav txog seb cov khoom tawg no puas nyob hauv lawv lub cev ib nrab qhib lymphatic.Peb tau daws qhov teeb meem no sim los ntawm kev taug qab txoj hmoo ntawm cov DNA soluble tawg ntxiv rau xiav mussel tso tsheb hlau luam.Txhawm rau pab txhawb kev taug qab ntawm DNA tawg, peb siv txawv teb chaws (tsis yog tus kheej) plasmid DNA uas muaj tib neeg galectin-7 noob.ddPCR taug qab plasmid DNA fragments nyob rau hauv dej hiav txwv thiab mussels.Peb cov txiaj ntsig tau pom tias yog tias tus nqi ntawm DNA tawg hauv dej hiav txwv tseem nyob ib puag ncig lub sijhawm (txog 7 hnub) thaum tsis muaj mussels, tom qab ntawd nyob rau hauv lub xub ntiag ntawm mussels qib no yuav luag tag tsis pub dhau 8 teev (Fig. 1a,b).Fragments ntawm exogenous DNA tau yooj yim pom nyob rau hauv 15 min nyob rau hauv intravalvular kua thiab hemolymph (Fig. 1c).Cov tawg no tseem tuaj yeem tshawb pom txog li 4 teev tom qab kis tau.Qhov kev lim dej no nrog rau kev hwm ntawm DNA tawg yog piv rau kev lim dej ntawm cov kab mob thiab cov algae [31].Cov txiaj ntsig no qhia tias mussels tuaj yeem lim thiab khaws cov DNA txawv teb chaws hauv lawv cov dej ntim.
Kev txheeb ze ntawm plasmid DNA nyob rau hauv dej hiav txwv nyob rau hauv lub xub ntiag (A) los yog tsis muaj (B) ntawm mussels, ntsuas los ntawm ddPCR.Hauv A, cov txiaj ntsig tau nthuav tawm raws li feem pua, nrog ciam teb ntawm cov thawv sawv cev rau 75th thiab 25 feem pua.Qhov haum logarithmic nkhaus yog pom nyob rau hauv liab, thiab qhov chaw ntxoov ntxoo grey sawv cev rau 95% kev ntseeg siab ib ntus.Hauv B, kab liab sawv cev rau qhov txhais tau tias thiab kab xiav sawv cev rau 95% kev ntseeg siab lub sijhawm rau qhov concentration.C Kev sib sau ntawm plasmid DNA hauv hemolymph thiab valvular kua ntawm mussels nyob rau lub sijhawm sib txawv tom qab sib ntxiv ntawm plasmid DNA.Cov txiaj ntsig tau nthuav tawm raws li cov ntawv luam pom tseeb / mL (± SE).
Tom ntej no, peb tshawb xyuas lub hauv paus chiv keeb ntawm ccfDNA nyob rau hauv mussels sau los ntawm mussel txaj nyob rau hauv Kerguelen Islands, ib tug tej thaj chaw deb pab pawg neeg ntawm Islands tuaj nrog txwv anthropogenic cawv.Rau lub hom phiaj no, cccDNA los ntawm mussel hemolymphs raug cais tawm thiab ua kom huv los ntawm txoj kev uas feem ntau siv los ua kom huv tib neeg cccDNA [32, 33].Peb pom tias qhov nruab nrab hemolymph ccfDNA concentrations hauv mussels yog nyob rau hauv qis micrograms ib ml hemolymph ntau yam (saib Table S2, Cov Lus Qhia Ntxiv).Qhov ntau ntawm cov ntsiab lus no ntau dua li cov neeg noj qab haus huv (tsawg nanograms ib milliliter), tab sis tsawg zaus, hauv cov neeg mob qog noj ntshav, qib ccfDNA tuaj yeem ncav cuag ntau micrograms ib milliliter [34, 35].Kev soj ntsuam ntawm qhov loj me ntawm hemolymph ccfDNA tau pom tias cov khoom tawg no sib txawv loj heev, xws li ntawm 1000 bp txog 1000 bp.mus txog 5000 bp (Fig. 2).Cov txiaj ntsig zoo sib xws tau txais los ntawm cov khoom siv silica-based QIAamp Investigator Kit, ib txoj hauv kev uas feem ntau siv hauv kev tshawb fawb forensic kom cais tawm sai sai thiab ntxuav cov genomic DNA los ntawm cov qauv DNA tsawg, suav nrog ccfDNA [36].
Tus sawv cev ccfDNA electrophoregram ntawm mussel hemolymph.Extracted nrog NucleoSnap Plasma Kit (sab saum toj) thiab QIAamp DNA Investigator Kit.B Violin zaj duab xis qhia txog kev faib tawm ntawm hemolymph ccfDNA concentrations (± SE) hauv mussels.Cov kab dub thiab liab sawv cev rau nruab nrab thiab thawj thiab thib peb quartiles, raws li.
Kwv yees li 1% ntawm ccfDNA nyob rau hauv tib neeg thiab primates muaj qhov chaw txawv teb chaws [21, 37].Muab cov kab ke ib nrab qhib ntawm bivalves, microbial-nplua nuj seawater, thiab qhov loj me ntawm mussel ccfDNA, peb xav tias mussel hemolymph ccfDNA tej zaum yuav muaj ib tug nplua nuj thiab ntau haiv neeg pas dej ua ke ntawm microbial DNA.Txhawm rau ntsuas qhov kev xav no, peb tau ua raws cov hemolymph ccfDNA los ntawm Aulacomya atra cov qauv sau los ntawm Kerguelen Islands, yielding tshaj 10 lab nyeem, 97.6% uas dhau los tswj kev ua tau zoo.Cov kev nyeem ntawv tau muab cais raws li tus kheej thiab tsis yog tus kheej qhov chaw siv BLASTN thiab NCBI bivalve databases (Fig. S1, Cov Ntaub Ntawv Ntxiv).
Hauv tib neeg, ob qho tib si nuclear thiab mitochondrial DNA tuaj yeem tso rau hauv cov hlab ntsha [38].Txawm li cas los xij, hauv txoj kev tshawb fawb tam sim no, nws tsis tuaj yeem piav qhia meej txog cov DNA genomic ntawm cov mussels, vim tias A. atra genome tsis tau ua raws lossis piav qhia.Txawm li cas los xij, peb muaj peev xwm txheeb xyuas ntau qhov ccfDNA tawg ntawm peb tus kheej keeb kwm siv lub tsev qiv ntawv bivalve (Fig. S2, Cov Ntaub Ntawv Ntxiv).Peb kuj tau lees paub tias muaj DNA tawg ntawm peb tus kheej keeb kwm los ntawm kev qhia PCR amplification ntawm cov A. atra genes uas tau sequenced (Fig. 3).Ib yam li ntawd, muab hais tias mitochondrial genome ntawm A. atra muaj nyob rau hauv cov ntaub ntawv pej xeem, ib tus tuaj yeem nrhiav pov thawj rau qhov muaj cov mitochondrial ccfDNA fragments hauv hemolymph ntawm A. atra.Lub xub ntiag ntawm mitochondrial DNA fragments tau lees paub los ntawm PCR amplification (Fig. 3).
Ntau yam mitochondrial genes muaj nyob rau hauv hemolymph ntawm A. atra (liab dots - Tshuag tooj: SRX5705969) thiab M. platensis (xiav dots - Tshuag tooj: SRX5705968) amplified los ntawm PCR.Daim duab yoog los ntawm Breton li al., 2011 B Amplification ntawm hemolymph supernatant los ntawm A. atra Khaws rau ntawm daim ntawv FTA.Siv 3 hli punch los ntxiv ncaj qha rau PCR raj uas muaj cov PCR sib xyaw.
Muab cov ntsiab lus microbial ntau nyob rau hauv dej hiav txwv, peb pib tsom mus rau tus yam ntxwv ntawm microbial DNA sequences hauv hemolymph.Txhawm rau ua qhov no, peb siv ob lub tswv yim sib txawv.Thawj lub tswv yim siv Kraken2, ib qho kev qhia ua ntu zus raws li kev faib tawm uas tuaj yeem txheeb xyuas cov kab mob microbial nrog qhov tseeb piv rau BLAST thiab lwm yam cuab yeej [28].Ntau tshaj 6719 nyeem tau txiav txim siab tias yog kab mob keeb kwm, thaum 124 thiab 64 yog los ntawm archaea thiab kab mob, raws li (Fig. 4).Cov kab mob DNA feem ntau yog Firmicutes (46%), Proteobacteria (27%), thiab Bacteroidetes (17%) (Fig. 4a).Qhov kev faib tawm no zoo ib yam nrog cov kev tshawb fawb yav dhau los ntawm marine blue mussel microbiome [39, 40].Gammaproteobacteria yog cov chav kawm tseem ceeb ntawm Proteobacteria (44%), suav nrog ntau Vibrionales (Fig. 4b).Txoj kev ddPCR tau lees paub tias muaj Vibrio DNA tawg nyob rau hauv ccfDNA ntawm A. atra hemolymph (Fig. 4c) [41].Txhawm rau kom tau txais cov ntaub ntawv ntau ntxiv txog cov kab mob hauv paus chiv keeb ntawm ccfDNA, ib qho ntxiv mus kom ze (Daim duab S2, Cov Lus Qhia Ntxiv). Nyob rau hauv cov ntaub ntawv no, nyeem tias overlapped tau sib sau ua khub-kawg nyeem thiab raug cais raws li tus kheej (bivalves) los yog tsis yog tus kheej keeb kwm siv BLASTN thiab tus nqi ntawm 1e−3 thiab txiav tawm nrog> 90% homology. Nyob rau hauv cov ntaub ntawv no, nyeem tias overlapped tau sib sau ua khub-kawg nyeem thiab raug cais raws li tus kheej (bivalves) los yog tsis yog tus kheej keeb kwm siv BLASTN thiab tus nqi ntawm 1e−3 thiab txiav tawm nrog> 90% homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были клоессифыцирова рчатые моллюски) или чужие по происхождению с использованием BLASTN и значения e 1e-3 и отсечения с ги0йол> Nyob rau hauv rooj plaub no, sib tshooj nyeem tau sau ua khub-kawg nyeem thiab raug cais raws li haiv neeg (bivalve) lossis tsis yog thawj siv BLASTN thiab e tus nqi ntawm 1e-3 thiab txiav tawm nrog> 90% homology.在这种情况下，重叠的读数组装为配对末端读数，并使用BLASTN 和1e-3 的e 值和> 90% 姱戼同自身（双壳类）或非自身来源.在这种情况下，重叠读数组装 为配末端读数，使用的使用 使用 blastn 和 1 %源性的分类自身（双壳类）非自身。。。。。。。。。。。。。 В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицироввсны каноссифицироввста е моллюски) или несобственные по происхождению с использованием значений e BLASTN и 1e-3 и порога гим>ололом 9%. Nyob rau hauv cov ntaub ntawv no, overlapping nyeem tau sau ua khub-kawg nyeem thiab cais raws li tus kheej (bivalves) lossis tsis yog thawj siv e BLASTN thiab 1e-3 qhov tseem ceeb thiab homology pib> 90%.Txij li thaum A. atra genome tseem tsis tau ua tiav, peb tau siv lub tswv yim de novo los ua ke ntawm MEGAHIT Next Generation Sequencing (NGS) assembler.Tag nrho ntawm 147,188 contigs tau raug txheeb xyuas tias yog nyob ntawm (bivalves) ntawm keeb kwm.Cov contigs no tau tawg nrog e-tus nqi ntawm 1e-10 siv BLASTN thiab BLASTX.Lub tswv yim no tau tso cai rau peb txheeb xyuas 482 cov khoom tsis yog bivalve tam sim no hauv A. atra ccfDNA.Ntau tshaj li ib nrab (57%) ntawm cov DNA tawg no tau txais los ntawm cov kab mob, feem ntau yog los ntawm cov kab mob gill, suav nrog sulfotrophic symbionts, thiab los ntawm gill symbionts Solemya velum (Fig. 5).
kwv tij abundance ntawm hom theem.B Microbial ntau haiv neeg ntawm ob lub ntsiab phyla (Firmicutes thiab Proteobacteria).Tus neeg sawv cev amplification ntawm ddPCR C Vibrio spp.A. Fragments ntawm 16S rRNA noob (xiav) nyob rau hauv peb atra hemolymphs.
Tag nrho ntawm 482 sau contigs tau txheeb xyuas.General profile ntawm taxonomic tis ntawm metagenomic contig annotations (prokaryotes thiab eukaryotes).B Cov ncauj lus kom ntxaws ntawm cov kab mob DNA tawg tau txheeb xyuas los ntawm BLASTN thiab BLASTX.
Kraken2 tsom xam kuj tau pom tias mussel ccfDNA muaj archaeal DNA fragments, nrog rau DNA fragments ntawm Euryarchaeota (65%), Crenarchaeota (24%), thiab Thaurmarcheota (11%) (Fig. 6a).Lub xub ntiag ntawm DNA tawg tau muab los ntawm Euryarchaeota thiab Crenarchaeota, yav tas los pom nyob rau hauv lub zej zog microbial ntawm Californian mussels, yuav tsum tsis txhob tuaj raws li ib tug surprise [42].Txawm hais tias Euryarchaeota feem ntau cuam tshuam nrog cov xwm txheej hnyav, tam sim no tau lees paub tias ob qho tib si Euryarchaeota thiab Crenarcheota yog cov feem ntau prokaryotes nyob rau hauv marine cryogenic ib puag ncig [43, 44].Lub xub ntiag ntawm methanogenic microorganisms nyob rau hauv mussels tsis yog ib qho xav tsis thoob, muab cov ntaub ntawv tsis ntev los no ntawm cov methane ntws tawm hauv qab los ntawm Kerguelen Plateau [45] thiab ua tau microbial methane ntau lawm tau pom tawm ntawm ntug dej hiav txwv ntawm Kerguelen Islands [46].
Peb qhov kev xav tom qab ntawd hloov mus rau kev nyeem ntawv los ntawm cov kab mob DNA.Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, qhov no yog thawj qhov kev tshawb fawb tawm ntawm tus kab mob cov ntsiab lus ntawm mussels.Raws li qhov xav tau, peb pom DNA tawg ntawm bacteriophages (Caudovirales) (Fig. 6b).Txawm li cas los xij, cov kab mob DNA feem ntau yog los ntawm lub cev ntawm nucleocytoviruses, tseem hu ua nuclear cytoplasmic loj DNA virus (NCLDV), uas muaj cov genome loj tshaj plaws ntawm txhua tus kab mob.Nyob rau hauv cov kab mob no, feem ntau DNA sequences yog cov tsev neeg Mimimidoviridae (58%) thiab Poxviridae (21%), uas nws cov tswv ntuj muaj xws li vertebrates thiab arthropods, thaum ib feem me me ntawm cov DNA sequences yog paub virological algae.Kab mob marine eukaryotic algae.Cov kab ke kuj tau txais los ntawm tus kab mob Pandora, tus kab mob loj heev uas muaj cov genome loj tshaj plaws ntawm txhua yam kab mob uas paub txog.Interestingly, qhov ntau ntawm cov tswv uas paub tias muaj tus kab mob, raws li tau txiav txim los ntawm hemolymph ccfDNA sequencing, kuj loj (Daim duab S3, Cov ntaub ntawv ntxiv).Nws suav nrog cov kab mob uas kis kab mob xws li Baculoviridae thiab Iridoviridae, nrog rau cov kab mob uas kis tau amoeba, algae thiab vertebrates.Peb kuj pom cov kab ke sib txuam nrog Pithovirus sibericum genome.Pitoviruses (tseem hu ua "zombie viruses") yog thawj zaug cais tawm ntawm 30,000 xyoo permafrost hauv Siberia [47].Yog li, peb cov txiaj ntsig tau ua raws li cov ntaub ntawv dhau los uas qhia tias tsis yog txhua hom kab mob niaj hnub no tau ploj mus [48] thiab tias cov kab mob no tuaj yeem muaj nyob rau hauv thaj chaw deb subarctic marine ecosystems.
Thaum kawg, peb tau sim saib seb peb puas tuaj yeem pom DNA tawg ntawm lwm cov tsiaj txhu ntau.Tag nrho ntawm 482 txawv teb chaws contigs raug txheeb xyuas los ntawm BLASTN thiab BLASTX nrog nt, nr thiab RefSeq cov tsev qiv ntawv (genomic thiab protein).Peb cov txiaj ntsig tau pom tias ntawm cov khoom txawv teb chaws ntawm ccfDNA ntawm ntau cov tsiaj txhu DNA ntawm cov pob txha pob txha predominates (Fig. 5).DNA tawg ntawm kab thiab lwm hom kuj tau pom.Ib feem loj ntawm DNA tawg tsis tau txheeb xyuas, tej zaum yog vim muaj qhov tsis txaus ntseeg ntawm ntau tus tsiaj hauv hiav txwv hauv cov ntaub ntawv genomic piv rau cov tsiaj hauv av [49].
Nyob rau hauv daim ntawv tam sim no, peb siv lub tswv yim LB rau mussels, sib cav hais tias hemolymph ccfDNA txhaj koob tshuaj tivthaiv tuaj yeem muab kev nkag siab rau hauv cov dej hiav txwv ntug dej hiav txwv ecosystems.Tshwj xeeb, peb pom tias 1) mussel hemolymph muaj cov ntsiab lus siab (microgram theem) ntawm qhov loj (~ 1-5 kb) circulating DNA fragments;2) cov DNA tawg no yog ob leeg ywj pheej thiab tsis ywj pheej 3) Ntawm cov chaw txawv teb chaws ntawm cov DNA tawg no, peb pom cov kab mob, cov kab mob thiab kab mob DNA, nrog rau DNA ntawm lwm cov tsiaj txhu ntau ntau;4) Kev sib sau ntawm cov ccfDNA txawv teb chaws nyob rau hauv hemolymph tshwm sim sai heev thiab pab txhawb rau kev lim dej sab hauv ntawm mussels.Hauv kev xaus, peb txoj kev tshawb fawb pom tau tias lub tswv yim ntawm LB, uas tam sim no tau siv feem ntau hauv kev kho mob biomedicine, encodes ib qho kev nplua nuj tab sis tsis tau tshawb nrhiav qhov kev paub uas tuaj yeem siv los nkag siab zoo txog kev sib cuam tshuam ntawm cov kab xaum thiab lawv ib puag ncig.
Ntxiv nrog rau primates, ccfDNA kev cais tawm tau tshaj tawm hauv cov tsiaj txhu, suav nrog nas, dev, miv, thiab nees [50, 51, 52].Txawm li cas los xij, rau peb txoj kev paub, peb txoj kev tshawb fawb yog thawj zaug qhia txog kev tshawb nrhiav thiab kev sib txuas ntawm ccfDNA hauv cov tsiaj hiav txwv nrog rau kev qhib cov kab ke.Qhov no anatomical feature thiab filtering peev xwm ntawm mussels yuav, tsawg kawg yog ib feem, piav qhia txog qhov sib txawv me me yam ntxwv ntawm circulating DNA fragments piv rau lwm hom.Nyob rau hauv tib neeg, feem ntau DNA tawg nyob rau hauv cov ntshav yog ib tug me me fragments xws li qhov loj ntawm 150 mus rau 200 bp.nrog qhov siab tshaj plaws ntawm 167 bp [34, 53].Ib qho me me tab sis tseem ceeb ntawm DNA tawg yog nyob nruab nrab ntawm 300 thiab 500 bp hauv qhov loj, thiab txog 5% yog ntev dua 900 bp.[54].Yog vim li cas rau qhov loj me no yog qhov tseem ceeb ntawm ccfDNA hauv plasma tshwm sim los ntawm kev tuag ntawm tes, vim yog kev tuag ntawm tes los yog vim necrosis ntawm cov kab mob hematopoietic hauv cov tib neeg noj qab haus huv los yog vim apoptosis ntawm cov qog hlwb hauv cov neeg mob qog noj ntshav (lub npe hu ua circulating qog DNA)., ctDNA).Qhov loj me me ntawm hemolymph ccfDNA uas peb pom nyob rau hauv mussels muaj li ntawm 1000 mus rau 5000 bp, qhia tias mussel ccfDNA muaj qhov sib txawv.Qhov no yog qhov kev xav tsis zoo, txij li cov mussels muaj ib lub vascular semi-qhib thiab nyob hauv cov dej hauv dej hiav txwv uas muaj cov ntsiab lus siab ntawm microbial genomic DNA.Qhov tseeb, peb cov kev sim hauv kev sim siv exogenous DNA tau pom tias mussels khaws cov DNA tawg hauv dej hiav txwv, tsawg kawg tom qab ob peb teev lawv raug degraded tom qab cellular uptake thiab / los yog tso tawm thiab / los yog khaws cia nyob rau hauv ntau lub koom haum.Muab qhov tsis tshua muaj ntawm cov hlwb (ob leeg prokaryotic thiab eukaryotic), kev siv cov intravalvular compartments yuav txo tus nqi ntawm ccfDNA los ntawm tus kheej qhov chaw thiab los ntawm txawv teb chaws.Xav txog qhov tseem ceeb ntawm bivalve innate kev tiv thaiv thiab ntau ntawm cov phagocytes circulating, peb ntxiv hypothesized tias txawm txawv teb chaws ccfDNA yog enriched nyob rau hauv circulating phagocytes uas accumulates txawv teb chaws DNA thaum noj cov kab mob thiab / los yog cellular khib nyiab.Ua ke, peb cov txiaj ntsig tau pom tias bivalve hemolymph ccfDNA yog qhov chaw cia khoom tshwj xeeb ntawm cov ntaub ntawv molecular thiab txhawb nqa lawv cov xwm txheej raws li hom kab xaum.
Peb cov ntaub ntawv qhia tau hais tias kev sib luag thiab kev tshuaj xyuas ntawm cov kab mob uas tau los ntawm hemolymph ccfDNA cov khoom tuaj yeem muab cov ntaub ntawv tseem ceeb txog tus tswv tsev kab mob thiab cov kab mob uas muaj nyob rau hauv ib puag ncig marine ecosystem.Cov txheej txheem txhaj tshuaj tua kab mob tau nthuav tawm cov kab mob commensal A. atra gill uas yuav tau plam yog tias cov txheej txheem kev txheeb xyuas cov qauv 16S rRNA tau raug siv, vim yog ib feem ntawm lub tsev qiv ntawv kev tsis ncaj ncees.Qhov tseeb, peb siv LB cov ntaub ntawv sau los ntawm M. platensis nyob rau tib lub mussel txheej ntawm Kerguelen tau pom tias muaj pes tsawg leeg ntawm cov kab mob gill-associated bacterial symbionts yog tib yam rau ob hom mussel (Fig. S4, Cov Lus Qhia Ntxiv).Qhov zoo sib xws ntawm ob lub noob caj noob ces sib txawv tuaj yeem cuam tshuam txog cov kab mob hauv zej zog hauv qhov txias, sulfurous, thiab volcanic deposits ntawm Kerguelen [55, 56, 57, 58].Qib siab ntawm cov kab mob sulfur-txo cov kab mob tau piav qhia zoo thaum sau cov qwj nplais los ntawm thaj chaw bioturbated ntug hiav txwv [59], xws li ntug dej hiav txwv ntawm Port-au-Fabkis.Lwm qhov ua tau yog tias cov kab mob commensal mussel flora tuaj yeem cuam tshuam los ntawm kab rov tav kis [60, 61].Kev tshawb fawb ntxiv yog xav tau los txiav txim qhov kev sib raug zoo ntawm ib puag ncig marine, seafloor nto, thiab muaj pes tsawg leeg ntawm cov kab mob symbiotic hauv mussels.Cov kev tshawb fawb no tseem niaj hnub no.
Qhov ntev thiab qhov siab ntawm hemolymph ccfDNA, nws yooj yim ntawm kev ntxuav, thiab kev ua haujlwm siab kom tso cai rau kev txhaj tshuaj sai sai yog qee qhov zoo ntawm kev siv mussel ccfDNA los ntsuas biodiversity hauv marine ntug hiav txwv ecosystems.Txoj hauv kev no muaj txiaj ntsig tshwj xeeb rau kev ua tus yam ntxwv ntawm cov zej zog kis kab mob (viromes) hauv ib qho chaw muab khoom [62, 63].Tsis zoo li cov kab mob, archaea, thiab eukaryotes, kab mob genomes tsis muaj phylogenetically conserved noob xws li 16S sequences.Peb cov txiaj ntsig tau qhia tias cov kua dej biopsies los ntawm cov cim qhia hom xws li mussels tuaj yeem siv los txheeb xyuas tus lej loj ntawm ccfDNA tus kab mob cov kab mob uas paub tias kis tus tswv tsev uas feem ntau nyob hauv ntug dej hiav txwv ecosystems.Qhov no suav nrog cov kab mob hu ua protozoa, arthropods, kab, nroj tsuag, thiab kab mob kab mob (xws li, bacteriophages).Ib qho kev faib tawm zoo sib xws tau pom thaum peb kuaj xyuas hemolymph ccfDNA virome ntawm xiav mussels (M. platensis) sau nyob rau hauv tib lub mussel txheej ntawm Kerguelen (Table S2, Cov Lus Qhia Ntxiv).Shotgun sequencing of ccfDNA yog qhov tseeb txoj hauv kev tshiab uas tau txais lub zog hauv txoj kev kawm ntawm virome ntawm tib neeg lossis lwm hom [21, 37, 64].Txoj hauv kev no yog qhov tshwj xeeb tshaj yog rau kev kawm cov kab mob DNA ob leeg, txij li tsis muaj ib lub noob tau khaws cia ntawm txhua tus kab mob DNA ob leeg, sawv cev rau cov kab mob sib txawv thiab dav hauv Baltimore [65].Txawm hais tias feem ntau ntawm cov kab mob no tseem tsis tau cais tawm thiab tuaj yeem suav nrog cov kab mob los ntawm qhov tsis paub meej ntawm lub ntiaj teb kab mob [66], peb pom tias cov kab mob viromes thiab host ranges ntawm mussels A. atra thiab M. platensis poob ntawm ob hom.zoo sib xws (saib daim duab S3, cov ntaub ntawv ntxiv).Qhov zoo sib xws no tsis yog qhov xav tsis thoob, vim nws tuaj yeem cuam tshuam qhov tsis muaj kev xaiv hauv kev txhawb nqa DNA tam sim no hauv ib puag ncig.Cov kev tshawb fawb yav tom ntej siv purified RNA yog tam sim no xav tau los ua tus cwj pwm RNA virome.
Hauv peb txoj kev tshawb fawb, peb tau siv cov raj xa dej nruj heev uas tau hloov kho los ntawm kev ua haujlwm ntawm Kowarski thiab cov npoj yaig [37], uas tau siv ob-kauj ruam tshem tawm ntawm cov ntawv nyeem thiab contigs ua ntej thiab tom qab sib sau ua ke ntawm haiv neeg ccfDNA, uas ua rau muaj feem ntau ntawm cov ntawv nyeem tsis tau piav qhia.Yog li ntawd, peb tsis tuaj yeem txiav txim siab tias qee qhov ntawm cov ntawv nyeem tsis tau piav qhia yuav tseem muaj lawv tus kheej keeb kwm, feem ntau vim tias peb tsis muaj cov ntaub ntawv siv genome rau hom mussel no.Peb kuj tau siv cov raj xa dej no vim peb txhawj xeeb txog cov chimeras ntawm tus kheej thiab tsis yog tus kheej nyeem thiab cov ntawv nyeem ntev uas tsim los ntawm Illumina MiSeq PE75.Lwm qhov laj thawj rau feem ntau ntawm cov ntawv nyeem tsis tau piav qhia yog tias ntau ntawm cov kab mob marine, tshwj xeeb tshaj yog nyob rau thaj chaw deb xws li Kerguelen, tsis tau sau tseg.Peb siv Illumina MiSeq PE75, piv txwv li ccfDNA fragment lengths zoo ib yam li tib neeg ccfDNA.Rau cov kev tshawb fawb yav tom ntej, muab peb cov txiaj ntsig qhia tias hemolymph ccfDNA tau nyeem ntev dua li tib neeg thiab / lossis tsiaj txhu, peb pom zoo kom siv lub platform sib txuas kom haum rau ntev ccfDNA tawg.Qhov kev coj ua no yuav ua rau nws yooj yim dua los txheeb xyuas cov lus qhia ntxiv rau kev tsom xam tob.Tau txais qhov tsis muaj tam sim no ua tiav A. atra nuclear genome ib ntus kuj tseem yuav pab txhawb kev ntxub ntxaug ntawm ccfDNA los ntawm tus kheej thiab tsis yog tus kheej.Muab hais tias peb cov kev tshawb fawb tau tsom mus rau qhov ua tau ntawm kev siv lub tswv yim ntawm cov kua biopsy mussels, peb cia siab tias raws li lub tswv yim no tau siv rau hauv kev tshawb fawb yav tom ntej, cov cuab yeej tshiab thiab cov kav dej yuav raug tsim los ua kom muaj peev xwm ntawm txoj kev no los kawm txog microbial ntau haiv neeg ntawm mussels.marine ecosystem.
Raws li qhov tsis yog-invasive soj ntsuam biomarker, nce tib neeg plasma qib ntawm ccfDNA cuam tshuam nrog ntau yam kab mob, cov ntaub so ntswg puas, thiab kev ntxhov siab [67,68,69].Qhov kev nce no yog txuam nrog kev tso tawm ntawm DNA tawg ntawm nws tus kheej keeb kwm tom qab cov ntaub so ntswg puas.Peb tau hais txog qhov teeb meem no uas siv cov kev ntxhov siab kub ntxhov, uas cov mussels raug luv luv rau qhov kub ntawm 30 ° C.Peb tau ua qhov kev tshuaj ntsuam no ntawm peb hom mussels hauv peb qhov kev sim ywj pheej.Txawm li cas los xij, peb tsis pom muaj kev hloov pauv hauv ccfDNA qib tom qab kev ntxhov siab kub ntxhov (saib daim duab S5, cov ntaub ntawv ntxiv).Qhov kev tshawb pom no tuaj yeem piav qhia, yam tsawg kawg ntawm ib feem, qhov tseeb tias cov mussels muaj ib lub voj voog ib nrab qhib thiab sib sau ntau cov DNA txawv teb chaws vim lawv cov dej lim dej siab.Ntawm qhov tod tes, mussels, zoo li ntau tus neeg tsis muaj zog, yuav tiv taus ntau dua rau kev ntxhov siab vim cov ntaub so ntswg puas tsuaj, yog li txwv kev tso tawm ccfDNA hauv lawv cov hemolymph [70, 71].
Txog niaj hnub no, DNA tsom xam ntawm biodiversity hauv dej ecosystems tau tsom mus rau ib puag ncig DNA (eDNA) metabarcoding.Txawm li cas los xij, txoj kev no feem ntau txwv rau kev tshuaj ntsuam biodiversity thaum siv primers.Kev siv cov phom phom sij hla dhau qhov kev txwv ntawm PCR thiab kev xaiv tsis ncaj ncees ntawm cov txheej txheem primer.Yog li, hauv kev txiav txim siab, peb txoj kev yog ze rau qhov tsis ntev los no tau siv high-throughput eDNA Shotgun sequencing method, uas muaj peev xwm ncaj qha ua ntu zus DNA thiab txheeb xyuas yuav luag tag nrho cov kab mob [72, 73].Txawm li cas los xij, muaj ntau qhov teeb meem tseem ceeb uas paub qhov txawv LB los ntawm cov txheej txheem eDNA.Tau kawg, qhov sib txawv tseem ceeb ntawm eDNA thiab LB yog kev siv lub tshuab lim dej.Kev siv hom marine xws li daim txhuam cev thiab bivalves (Dresseina spp.) ua cov lim dej rau kev kawm eDNA tau tshaj tawm [74, 75].Txawm li cas los xij, Dreissena txoj kev tshawb fawb siv cov ntaub so ntswg biopsies los ntawm DNA tau muab rho tawm.Kev tshuaj xyuas ccfDNA los ntawm LB tsis tas yuav tsum muaj cov ntaub so ntswg biopsy, tshwj xeeb thiab qee zaum cov cuab yeej kim thiab kev thauj mus los cuam tshuam nrog eDNA lossis cov ntaub so ntswg biopsy.Qhov tseeb, peb tsis ntev los no tau tshaj tawm tias ccfDNA los ntawm LB tuaj yeem khaws thiab txheeb xyuas nrog FTA kev txhawb nqa yam tsis muaj kev tswj xyuas cov hlua txias, uas yog qhov nyuaj rau kev tshawb fawb hauv thaj chaw deb [76].Kev rho tawm ntawm ccfDNA los ntawm cov kua biopsies kuj yog qhov yooj yim thiab muab cov DNA zoo rau kev txhaj tshuaj tiv thaiv kab mob thiab kev tshuaj xyuas PCR.Qhov no yog qhov txiaj ntsig zoo uas tau muab qee qhov kev txwv kev cuam tshuam nrog eDNA tsom xam [77].Qhov yooj yim thiab tus nqi qis ntawm cov qauv qauv kuj yog tshwj xeeb tshaj yog haum rau cov kev saib xyuas mus sij hawm ntev.Ntxiv nrog rau lawv lub peev xwm lim dej siab, lwm qhov kev paub zoo ntawm bivalves yog cov tshuaj mucopolysaccharide muaj pes tsawg leeg ntawm lawv cov hnoos qeev, uas txhawb nqa kev nqus ntawm cov kab mob [78, 79].Qhov no ua rau bivalves lub lim dej zoo tshaj plaws rau kev qhia txog biodiversity thiab kev cuam tshuam ntawm kev hloov pauv huab cua hauv cov dej hauv dej.Txawm hais tias muaj tus tswv tsev-derived DNA fragments tuaj yeem pom tau tias yog qhov txwv ntawm txoj kev piv rau eDNA, tus nqi cuam tshuam nrog kev muaj xws li ib txwm muaj ccfDNA piv rau eDNA ib txhij nkag siab rau ntau cov ntaub ntawv muaj rau kev tshawb fawb txog kev noj qab haus huv.offset host.Qhov no suav nrog qhov muaj cov kab mob sib kis ua ke rau hauv genome ntawm tus tswv tsev.Qhov no yog qhov tseem ceeb tshwj xeeb tshaj yog rau cov mussels, muab qhov muaj kab rov tav kis kab mob leukemic retroviruses hauv bivalves [80, 81].Lwm qhov zoo dua ntawm LB tshaj eDNA yog tias nws siv phagocytic kev ua haujlwm ntawm cov qe ntshav ncig hauv hemolymph, uas cuam tshuam cov kab mob (thiab lawv cov genomes).Phagocytosis yog lub luag haujlwm tseem ceeb ntawm cov qe ntshav hauv bivalves [82].Thaum kawg, txoj kev siv qhov zoo ntawm qhov muaj peev xwm lim dej ntawm cov mussels (nruab nrab 1.5 l / h ntawm cov dej hiav txwv) thiab ob hnub ncig, uas ua rau muaj kev sib xyaw ntawm cov dej hiav txwv sib txawv, tso cai rau kev ntes ntawm heterologous eDNA.[83, 84] ib.Yog li, mussel ccfDNA tsom xam yog ib qho kev nthuav qhia kev noj haus, kev lag luam, thiab ib puag ncig cuam tshuam ntawm mussels.Zoo ib yam li kev soj ntsuam ntawm LB sau los ntawm tib neeg, txoj kev no tseem qhib qhov muaj peev xwm ntsuas cov noob caj noob ces thiab epigenetic hloov pauv hauv tus tswv DNA hauv cov lus teb rau cov tshuaj exogenous.Piv txwv li, cov thev naus laus zis tiam thib peb tuaj yeem pom los ua genome-wide methylation tsom hauv haiv neeg ccfDNA siv nanopore sequencing.Cov txheej txheem no yuav tsum tau ua kom yooj yim los ntawm qhov tseeb tias qhov ntev ntawm cov mussel ccfDNA fragments yog qhov zoo tshaj plaws nrog ntev-nyeem sequencing platforms uas tso cai rau genome-wide DNA methylation tsom los ntawm ib qho kev sib txuas ua ke yam tsis tas yuav muaj tshuaj hloov pauv.85,86] Qhov no yog ib qho nthuav tau, raws li nws tau pom tias DNA methylation ib puag ncig muaj kev cuam tshuam ntau yam.Yog li ntawd, nws tuaj yeem muab kev pom zoo rau hauv cov txheej txheem tswj xyuas cov lus teb tom qab raug rau kev hloov pauv huab cua lossis cov pa phem [87].Txawm li cas los xij, kev siv LB tsis muaj kev txwv.Needless hais, qhov no yuav tsum tau muaj cov cim qhia hom nyob rau hauv lub ecosystem.Raws li tau hais los saum toj no, siv LB los soj ntsuam cov biodiversity ntawm ib qho kev muab ecosystem kuj yuav tsum tau muaj ib tug nruj bioinformatics pipeline uas yuav siv sij hawm mus rau hauv tus account lub xub ntiag ntawm DNA fragments los ntawm lub hauv paus.Lwm qhov teeb meem loj yog qhov muaj cov genomes siv rau marine hom.Nws cia siab tias cov kev pib xws li Marine Mammal Genomes Project thiab qhov uas nyuam qhuav tsim Fish10k project [88] yuav pab txhawb kev tsom xam yav tom ntej.Daim ntawv thov ntawm LB lub tswv yim rau marine filter-feeding kab mob kuj tau sib xws nrog cov kev nce qib tshiab tshaj plaws hauv kev sib txuas cov thev naus laus zis, ua kom nws zoo rau kev tsim cov multi-ohm biomarkers los muab cov ntaub ntawv tseem ceeb txog kev noj qab haus huv ntawm cov chaw nyob hauv hiav txwv teb rau ib puag ncig kev ntxhov siab.
Genome sequencing cov ntaub ntawv tau muab tso rau hauv NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 nyob rau hauv Bioprojects SRR8924808.
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