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ʻO ka biopsy wai (LB) kahi manaʻo e kaulana koke nei i ke kahua biomedical. Hoʻokumu nui ʻia ke kumumanaʻo ma ka ʻike ʻana i nā ʻāpana o ka DNA extracellular e kaapuni ana (ccfDNA), kahi i hoʻokuʻu nui ʻia ma ke ʻano he mau ʻāpana liʻiliʻi ma hope o ka make ʻana o nā cell i nā ʻiʻo like ʻole. Loaʻa kahi hapa liʻiliʻi o kēia mau ʻāpana mai nā ʻiʻo haole (haole) a i ʻole nā ​​​​ola. Ma ka hana o kēia manawa, ua hoʻopili mākou i kēia manaʻo i nā mussels, kahi ʻano kiaʻi i ʻike ʻia no ko lākou hiki ke kānana wai kai kiʻekiʻe. Hoʻohana mākou i ka hiki o nā mussels e hana ma ke ʻano he kānana kūlohelohe e hopu i nā ʻāpana DNA kaiapuni mai nā kumu like ʻole e hāʻawi i ka ʻike e pili ana i ka biodiversity o nā kaiaola kai kai. Hōʻike kā mākou mau hopena aia nā ʻāpana DNA o ka mussel hemolymph i ʻokoʻa loa ka nui, mai 1 a 5 kb. Ua hōʻike ka hoʻonohonoho ʻana o Shotgun he nui nā ʻāpana DNA mai nā microbial haole. I waena o lākou, ua loaʻa iā mākou nā ʻāpana DNA mai nā bacteria, archaea, a me nā virus, me nā virus i ʻike ʻia e hoʻopili i nā ʻano host like ʻole i loaʻa pinepine i nā kaiaola kai kai. I ka hopena, hōʻike kā mākou noiʻi ʻana ʻo ke kumumanaʻo o LB i hoʻopili ʻia i nā ʻūpā he kumu waiwai akā ʻaʻole i ʻimi ʻia o ka ʻike e pili ana i ka ʻokoʻa o nā microbial i nā kaiaolaola kahakai kai.
ʻO ka hopena o ka loli o ke aniau (CC) ma ke ʻano like ʻole o nā kaiaolaola kai he wahi noiʻi e ulu wikiwiki ana. ʻAʻole wale ka hoʻomehana honua e hoʻoulu i nā pilikia physiological koʻikoʻi, akā hoʻokuke pū i nā palena evolutionary o ke kūpaʻa wela o nā meaola kai, e hoʻopilikia ana i ka noho ʻana o kekahi mau ʻano, e koi ana iā lākou e ʻimi i nā kūlana maikaʻi aʻe [1, 2]. Ma waho aʻe o ka hoʻopilikia ʻana i ka biodiversity o nā metazoans, hoʻopilikia ʻo CC i ke kaulike palupalu o nā pilina host-microbial. ʻO kēia microbial dysbacteriosis kahi hoʻoweliweli koʻikoʻi i nā kaiaolaola kai no ka mea e hoʻolilo i nā meaola kai i mea maʻalahi i nā pathogens infectious [3, 4]. Ua manaʻoʻiʻo ʻia he kuleana koʻikoʻi ʻo SS i nā make nui, he pilikia koʻikoʻi ia no ka hoʻokele ʻana i nā kaiaolaola kai honua [5, 6]. He pilikia koʻikoʻi kēia i hāʻawi ʻia i nā hopena waiwai, kaiaolaola a me ka meaʻai o nā ʻano kai he nui. He ʻoiaʻiʻo loa kēia no nā bivalves e noho ana ma nā wahi polar, kahi i ʻoi aku ka koke a me ka koʻikoʻi o nā hopena o CK [6, 7]. ʻO kaʻoiaʻiʻo, hoʻohana nui ʻia nā bivalves e like me Mytilus spp. e nānā i nā hopena o CC ma nā kaiaolaola kai. ʻAʻole ia he mea kupanaha, ua hoʻomohala ʻia kahi helu nui o nā biomarkers e nānā i ko lākou olakino, me ka hoʻohana pinepine ʻana i kahi ala ʻelua-papa e pili ana i nā biomarkers hana e pili ana i ka hana enzymatic a i ʻole nā ​​​​hana cellular e like me ke ola o ke kelepona a me ka hana phagocytic [8]. Hoʻokomo pū kēia mau ʻano hana i ke ana ʻana o ka nui o nā hōʻailona kaomi kikoʻī e hōʻiliʻili ana i nā ʻiʻo palupalu ma hope o ka omo ʻana o ka nui o ka wai kai. Eia nō naʻe, ʻo ka mana kānana kiʻekiʻe a me ka ʻōnaehana circulatory semi-open o nā bivalves e hāʻawi i kahi manawa kūpono e hoʻomohala i nā biomarkers hemolymph hou me ka hoʻohana ʻana i ke kumumanaʻo o ka biopsy wai (LB), kahi ala maʻalahi a me ka minimally invasive i ka hoʻokele maʻi. nā laʻana koko [9, 10]. ʻOiai hiki ke loaʻa kekahi mau ʻano o nā molekala e kahe ana i loko o ke kanaka LB, ua hoʻokumu nui ʻia kēia manaʻo ma ka nānā ʻana o ka DNA sequencing o nā ʻāpana DNA extracellular circulating (ccfDNA) i loko o ka plasma. ʻO ka ʻoiaʻiʻo, ua ʻike ʻia ke alo o ka DNA e kahe ana i loko o ka plasma kanaka mai ka waena o ke kenekulia 20 [11], akā i nā makahiki i hala iho nei wale nō i alakaʻi ai ka hiki ʻana mai o nā ʻano sequencing kiʻekiʻe i ka hōʻailona lapaʻau e pili ana i ka ccfDNA. ʻO ke alo o kēia mau ʻāpana DNA e kaapuni ana ma muli o ka hoʻokuʻu ʻole ʻia o ka DNA genomic (nuclear a me mitochondrial) ma hope o ka make ʻana o nā cell. I nā kānaka olakino, haʻahaʻa ka nui o ka ccfDNA (<10 ng/mL) akā hiki ke hoʻonui ʻia e 5-10 mau manawa i nā poʻe maʻi e ʻeha nei i nā ʻano maʻi like ʻole a i ʻole i kau ʻia i ke kaumaha, e hopena ana i ka hōʻino ʻia o nā ʻiʻo. I nā kānaka olakino, haʻahaʻa ka nui o ka ccfDNA (<10 ng/mL) akā hiki ke hoʻonui ʻia e 5-10 mau manawa i nā poʻe maʻi e ʻeha nei i nā ʻano maʻi like ʻole a i ʻole i kau ʻia i ke kaumaha, e hopena ana i ka hōʻino ʻia o nā ʻiʻo. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 раз ухльчо патологией или подвергающихся стрессу, приводящему к повреждению тканей. I nā kānaka olakino, haʻahaʻa ka nui o ka cccDNA (<10 ng/mL), akā hiki ke hoʻonui ʻia ma 5-10 mau manawa i nā poʻe maʻi me nā ʻano maʻi like ʻole a i ʻole ma lalo o ke kaumaha e alakaʻi ai i ka hōʻino ʻana o nā ʻiʻo.在健康个体中，ccfDNA 的浓度通常较低（<10 ng/mL），但在患有各种病理或承受压力的患者中可增加5-10 倍，从而导致组织损他。在 健康 个体 中 ， ccfdna 的 浓度 较 低 （（<10 ng/ml） 但 在 各 种 病理 或 承受 或 承受可 增加 5-10 倍 ， 从而 组织。。Концентрации ccfDNA обычно низкие (<10 нг/мл) у здоровых людей, но могут быть увеличены в 5-10 раз у плица патологиями или стрессом, что приводит к повреждению тканей. ʻO ka maʻamau, haʻahaʻa ka nui o ka ccfDNA (<10 ng/ml) i nā poʻe olakino, akā hiki ke hoʻonui ʻia he 5-10-fold i nā maʻi me nā ʻano maʻi like ʻole a i ʻole ke kaumaha, e hopena ana i ka hōʻino ʻia o nā ʻiʻo.ʻOkoʻa loa ka nui o nā ʻāpana ccfDNA, akā maʻamau mai 150 a 200 bp. [12]. Hiki ke hoʻohana ʻia ka loiloi o ka ccfDNA i loaʻa iā ia iho, ʻo ia hoʻi, ccfDNA mai nā cell host maʻamau a i ʻole i hoʻololi ʻia, e ʻike i nā loli genetic a me epigenetic i loaʻa i loko o ka genome nuclear a/a i ʻole mitochondrial, no laila e kōkua ana i nā kauka e koho i nā lāʻau lapaʻau i kuhikuhi ʻia i ka molekala [13]. Eia nō naʻe, hiki ke loaʻa ka ccfDNA mai nā kumu haole e like me ccfDNA mai nā cell fetal i ka wā hāpai a i ʻole mai nā ʻōpū i hoʻoili ʻia [14,15,16,17]. He kumu ʻike koʻikoʻi nō hoʻi ka ccfDNA no ka ʻike ʻana i ke alo o nā waikawa nucleic o kahi mea maʻi (haole), e ʻae ana i ka ʻike ʻole ʻana o nā maʻi ākea i ʻike ʻole ʻia e nā moʻomeheu koko, e pale ana i ka biopsy invasive o nā ʻiʻo i loaʻa i ka maʻi [18]. Ua hōʻike maoli nā haʻawina hou he nui ka ʻike i loko o ke koko kanaka e hiki ke hoʻohana ʻia e ʻike i nā pathogens viral a me bacterial, a ma kahi o 1% o ka ccfDNA i loaʻa i ka plasma kanaka mai ka ʻāina ʻē [19]. Hōʻike kēia mau haʻawina e hiki ke loiloi ʻia ke ʻano olaola o ka microbiome e kaapuni ana o kahi meaola me ka hoʻohana ʻana i ka nānā ʻana o ccfDNA. Eia nō naʻe, a hiki i kēia manawa, ua hoʻohana wale ʻia kēia manaʻo i loko o nā kānaka a, i kahi liʻiliʻi, i nā vertebrates ʻē aʻe [20, 21].
Ma ka pepa o kēia manawa, hoʻohana mākou i ka hiki LB e kālailai i ka ccfDNA o Aulacomya atra, kahi ʻano hema i loaʻa pinepine ʻia ma nā mokupuni ʻo Kerguelen subantarctic, kahi hui o nā mokupuni ma luna o kahi pāpū nui i hoʻokumu ʻia he 35 miliona mau makahiki i hala. pele pele. Me ka hoʻohana ʻana i kahi ʻōnaehana hoʻokolohua in vitro, ua ʻike mākou ua lawe koke ʻia nā ʻāpana DNA i loko o ka wai kai e nā mussels a komo i loko o ka ʻāpana hemolymph. Ua hōʻike ka hoʻonohonoho ʻana o Shotgun aia nā ʻāpana DNA o ka mussel hemolymph ccfDNA o kona kumu ponoʻī a me ke kumu ʻole, me nā bacteria symbiotic a me nā ʻāpana DNA mai nā biomes maʻamau o nā kaiaolaola kai pele anu. Loaʻa pū ka Hemolymph ccfDNA i nā moʻo viral i loaʻa mai nā virus me nā pae host like ʻole. Ua ʻike pū mākou i nā ʻāpana DNA mai nā holoholona multicellular e like me ka iʻa bony, nā anemones kai, nā algae a me nā pepeke. I ka hopena, hōʻike kā mākou noiʻi hiki ke hoʻopili pono ʻia ka manaʻo LB i nā invertebrates kai e hana i kahi repertoire genomic waiwai i nā kaiaolaola kai.
Ua hōʻiliʻili ʻia nā ʻiʻo makua (55-70 mm ka lōʻihi) ʻo Mytilus platensis (M. platensis) a me Aulacomya atra (A. atra) mai nā kahakai pōhaku intertidal o Port-au-France (049°21.235 S, 070°13.490 E .). Nā mokupuni ʻo Kerguelen i Dekemaba 2018. Ua loaʻa nā ʻiʻo polū makua ʻē aʻe (Mytilus spp.) mai kahi mea hoʻolako kalepa (PEI Mussel King Inc., Prince Edward Island, Kanada) a ua waiho ʻia i loko o kahi pahu earated i kāohi ʻia ka mahana (4°C) me 10-20 L o ka brine artificial 32‰. (paʻakai kai artificial Reef Crystal, Instant Ocean, Virginia, USA). No kēlā me kēia hoʻokolohua, ua ana ʻia ka lōʻihi a me ke kaumaha o nā pūpū pākahi.
Loaʻa kahi protocol komo ākea manuahi no kēia polokalamu ma ka pūnaewele (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). I ka pōkole, ua hōʻiliʻili ʻia ka hemolymph LB mai nā ʻiʻo abductor e like me ka mea i wehewehe ʻia [22]. Ua hoʻomālamalama ʻia ka hemolymph ma o ka centrifugation ma 1200 × g no 3 mau minuke, ua hoʻopaʻahau ʻia ka supernatant (-20 ° C) a hiki i ka hoʻohana ʻana. No ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe ʻana o cfDNA, ua hoʻoheheʻe ʻia nā laʻana (1.5-2.0 ml) a ua hana ʻia me ka hoʻohana ʻana i ka pahu NucleoSnap cfDNA (Macherey-Nagel, Bethlehen, PA) e like me nā kuhikuhi a ka mea hana. Ua mālama ʻia ka ccfDNA ma -80 ° C a hiki i ka nānā hou ʻana. I kekahi mau hoʻokolohua, ua hoʻokaʻawale ʻia a hoʻomaʻemaʻe ʻia ka ccfDNA me ka hoʻohana ʻana i ka QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Kanada). Ua helu ʻia ka DNA i hoʻomaʻemaʻe ʻia me ka hoʻohana ʻana i kahi assay PicoGreen maʻamau. Ua kālailai ʻia ka hoʻolaha ʻāpana o ka ccfDNA i hoʻokaʻawale ʻia e ka electrophoresis capillary me ka hoʻohana ʻana i kahi Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) me ka hoʻohana ʻana i kahi High Sensitivity DNA Kit. Ua hana ʻia ka hoʻāʻo me ka hoʻohana ʻana i 1 µl o ka hāpana ccfDNA e like me nā kuhikuhi a ka mea hana.
No ke kaʻina hana ʻana o nā ʻāpana hemolymph ccfDNA, ua hoʻomākaukau ʻo Génome Québec (Montreal, Quebec, Kanada) i nā waihona puke shotgun me ka hoʻohana ʻana i ka Illumina DNA Mix kit o ka Illumina MiSeq PE75 kit. Ua hoʻohana ʻia kahi adapter maʻamau (BioO). Loaʻa nā faila ʻikepili maka mai ka NCBI Sequence Read Archive (SRR8924808 a me SRR8924809). Ua loiloi ʻia ke ʻano heluhelu kumu me ka hoʻohana ʻana iā FastQC [23]. Ua hoʻohana ʻia ʻo Trimmomatic [24] no ka ʻoki ʻana i nā adapters a me nā heluhelu maikaʻi ʻole. Ua hoʻohui ʻia nā heluhelu shotgun me nā wēlau i hui pū ʻia me FLASH i nā heluhelu hoʻokahi lōʻihi me ka liʻiliʻi o 20 bp e pale aku i nā mismatches [25]. Ua hoʻopaʻa inoa ʻia nā heluhelu i hui pū ʻia me BLASTN me ka hoʻohana ʻana i kahi waihona ʻikepili NCBI Taxonomy bivalve (waiwai e < 1e−3 a me 90% homology), a ua hana ʻia ka uhi ʻana o nā moʻo paʻakikī haʻahaʻa me ka hoʻohana ʻana iā DUST [26]. Ua hoʻopaʻa inoa ʻia nā heluhelu i hui pū ʻia me BLASTN me ka hoʻohana ʻana i kahi waihona ʻikepili NCBI Taxonomy bivalve (waiwai e < 1e−3 a me 90% homology), a ua hana ʻia ka uhi ʻana o nā moʻo paʻakikī haʻahaʻa me ka hoʻohana ʻana iā DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN с использованием базы данных таксономии двустворчозолючнич < 1e-3 и 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST []. Ua hoʻopaʻa inoa ʻia nā heluhelu i hui pū ʻia me BLASTN me ka hoʻohana ʻana i ka waihona ʻikepili taxonomy bivalve NCBI (waiwai e < 1e-3 a me 90% homology), a ua hana ʻia ka uhi ʻana o ke kaʻina paʻakikī haʻahaʻa me ka hoʻohana ʻana iā DUST [26].使用双壳类NCBI 分类数据库（e 值< 1e-3 和90% 同源性）用 BLASTN 注释合并的读敽，用数，用并进行低复杂度序列的掩蔽。使用 双 壳类 ncbi 分类 （（（<1e-3 和 90% 同源） 用 用 用 注释 合并 读数 ， 6用 ， 6用进行 复杂度 序列 的。。。 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽 掩蔽掩蔽 掩蔽 掩蔽Объединенные чтения были аннотированы с помощью BLASTN с использованием таксономической базы данных двуствослюмча (значение e <1e-3 и 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с исполнено с использозо. Ua hoʻopaʻa inoa ʻia nā heluhelu i hui pū ʻia me BLASTN me ka hoʻohana ʻana i ka ʻikepili taxonomic bivalve NCBI (waiwai e <1e-3 a me 90% homology), a ua hana ʻia ka masking sequence haʻahaʻa me ka hoʻohana ʻana iā DUST [26].Ua māhele ʻia nā heluhelu i ʻelua mau pūʻulu: pili i nā moʻo bivalve (i kapa ʻia ma aneʻi he heluhelu ponoʻī) a pili ʻole (nā heluhelu ponoʻī ʻole). Ua hōʻuluʻulu ʻia ʻelua mau pūʻulu me ka hoʻohana ʻana iā MEGAHIT e hana i nā contigs [27]. I kēia manawa, ua hoʻokaʻawale ʻia ka hoʻokaʻawale taxonomic o nā heluhelu microbiome malihini me ka hoʻohana ʻana iā Kraken2 [28] a hōʻike ʻia ma ke ʻano kiʻi e kahi pakuhi pai Krona ma Galaxy [29, 30]. Ua hoʻoholo ʻia nā kmers kūpono he kmers-59 mai kā mākou mau hoʻokolohua mua. A laila ua ʻike ʻia nā contigs ponoʻī ma o ka hoʻohālikelike ʻana me BLASTN (bivalve NCBI database, e value < 1e−10 a me 60% homology) no kahi annotation hope loa. A laila ua ʻike ʻia nā contigs ponoʻī ma o ka hoʻohālikelike ʻana me BLASTN (bivalve NCBI database, e value < 1e−10 a me 60% homology) no kahi annotation hope loa. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюбиск, 1 моллючения и гомология 60%) для окончательной аннотации. A laila ua ʻike ʻia nā mea hoʻohui ponoʻī ma o ka hoʻohālikelike ʻana me BLASTN (NCBI bivalve database, e value <1e-10 a me 60% homology) no ka annotation hope loa.然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60%同源性）对齐来识别自身重叠群以进行最终注释。然后通过与BLASTN（双壳贝类NCBI 数据库，e 值< 1e-10 和60% Затем были идентифицированы собственные контиги для окончательной аннотации путем сопоставления с BLASTN (базал да NCBIн двустворчатых моллюсков, значение e <1e-10 и гомология 60%). A laila ua ʻike ʻia nā contigs ponoʻī no ka hōʻike hope loa ma o ka hoʻohālikelike ʻana me BLASTN (NCBI bivalve database, e value <1e-10 a me 60% homology). Ma ka ʻaoʻao like, ua hoʻopaʻa inoa ʻia nā contigs hui ponoʻī me BLASTN (ʻikepili nt NCBI, waiwai e < 1e−10 a me 60% homology). Ma ka ʻaoʻao like, ua hoʻopaʻa inoa ʻia nā contigs hui ponoʻī me BLASTN (ʻikepili nt NCBI, waiwai e < 1e−10 a me 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN (база данных nt NCBI, значение e <1e-10 %) Ma ka ʻaoʻao like, ua hoʻopaʻa inoa ʻia nā contigs hui haole me BLASTN (NT NCBI database, e value <1e-10 a me 60% homology).平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。 Параллельно контиги, не относящиеся к собственной группе, были аннотированы с помощью BLASTN (база данных nt nt 1 гомология 60%). Ma ka ʻaoʻao like, ua hoʻopaʻa inoa ʻia nā contigs hui ʻole me BLASTN (ʻikepili nt NCBI, waiwai e <1e-10 a me 60% homology). Ua hana ʻia ʻo BLASTX ma nā contigs nonself me ka hoʻohana ʻana i nā waihona ʻikepili nr a me RefSeq protein NCBI (e value < 1e−10 a me 60% homology). Ua hana ʻia ʻo BLASTX ma nā contigs nonself me ka hoʻohana ʻana i nā waihona ʻikepili nr a me RefSeq protein NCBI (e value < 1e−10 a me 60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значогие лио <1e%)-10 e <1e. Ua hana ʻia ʻo BLASTX ma nā contigs non-self me ka hoʻohana ʻana i nā waihona protein nr a me RefSeq NCBI (e value < 1e-10 a me 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） 怌 。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） 怌 。 BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значение e <1e-10 %) Ua hana ʻia ʻo BLASTX ma nā contigs non-self me ka hoʻohana ʻana i nā waihona protein nr a me RefSeq NCBI (waiwai e <1e-10 a me 60% homology).ʻO nā loko BLASTN a me BLASTX o nā non-self-contigs e hōʻike ana i nā contigs hope loa (e ʻike i ka faila Supplementary).
Ua helu ʻia nā primers i hoʻohana ʻia no ka PCR ma ka Papa S1. Ua hoʻohana ʻia ʻo Taq DNA polymerase (Bio Basic Canada, Markham, ON) e hoʻonui i nā genes target ccfDNA. Ua hoʻohana ʻia nā kūlana hopena penei: denaturation ma 95°C no 3 mau minuke, 95°C no 1 minuke, hoʻonohonoho i ka mahana annealing no 1 minuke, elongation ma 72°C no 1 minuke, 35 mau pōʻaiapuni, a ma hope loa 72°C i loko o 10 mau minuke. . Ua hoʻokaʻawale ʻia nā huahana PCR e ka electrophoresis i loko o nā gels agarose (1.5%) e loaʻa ana iā SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Kanada) ma 95 V.
Ua hoʻomaʻamaʻa ʻia nā ʻūhini (Mytilus spp.) i loko o 500 ml wai kai oxygenated (32 PSU) no 24 mau hola ma 4°C. Ua hoʻohui ʻia ka DNA plasmid e loaʻa ana kahi mea hoʻokomo e hoʻopili ana i ka moʻo cDNA galectin-7 kanaka (NCBI accession number L07769) i loko o ka vial ma kahi hoʻohui hope loa o 190 μg/μl. ʻO nā ʻūhini i hoʻoulu ʻia ma lalo o nā kūlana like me ka ʻole o ka hoʻohui ʻana o ka DNA ka mea hoʻomalu. ʻO ke kolu o ka pahu hoʻomalu i loaʻa ka DNA me ka ʻole o nā ʻūhini. No ka nānā ʻana i ka maikaʻi o ka DNA i loko o ka wai kai, ua lawe ʻia nā laʻana wai kai (20 μl; ʻekolu mau hana hou) mai kēlā me kēia pahu i ka manawa i hōʻike ʻia. No ka hiki ke hahai ʻia ka DNA plasmid, ua ʻohi ʻia nā ʻūhini LB i nā manawa i hōʻike ʻia a ua kālailai ʻia e qPCR a me ddPCR. Ma muli o ke kiʻekiʻe o ka paʻakai o ka wai kai, ua hoʻoheheʻe ʻia nā aliquots i loko o ka wai maikaʻi PCR (1:10) ma mua o nā hoʻāʻo PCR āpau.
Ua hana ʻia ka PCR kulu kikohoʻe (ddPCR) me ka hoʻohana ʻana i ka protocol BioRad QX200 (Mississauga, Ontario, Kanada). E hoʻohana i ka ʻikepili mahana e hoʻoholo ai i ka mahana kūpono (Papa S1). Ua hana ʻia nā kulu me ka hoʻohana ʻana i kahi mea hana kulu QX200 (BioRad). Ua hana ʻia ka ddPCR penei: 95°C no 5 min, 50 mau pōʻaiapuni o 95°C no 30 s a me kahi mahana annealing i hāʻawi ʻia no 1 min a me 72°C no 30 s, 4°C no 5 min a me 90°C i loko o 5 mau minuke. Ua ana ʻia ka helu o nā kulu a me nā hopena maikaʻi (helu o nā kope/µl) me ka hoʻohana ʻana i kahi mea heluhelu kulu QX200 (BioRad). Ua hōʻole ʻia nā laʻana me ka emi ma mua o 10,000 mau kulu. ʻAʻole i hana ʻia ka kaohi ʻano i kēlā me kēia manawa i holo ʻia ai ka ddPCR.
Ua hana ʻia ʻo qPCR me ka hoʻohana ʻana iā Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) a me nā primers kikoʻī LGALS7. Ua hana ʻia nā PCR quantitative āpau i loko o 20 µl me ka hoʻohana ʻana i ka QuantiFast SYBR Green PCR Kit (QIAGEN). Ua hoʻomaka ʻia ʻo qPCR me kahi incubation 15 min ma 95°C i ukali ʻia e 40 mau pōʻaiapuni ma 95°C no 10 kekona a ma 60°C no 60 kekona me hoʻokahi hōʻiliʻili ʻikepili. Ua hoʻokumu ʻia nā piʻo hoʻoheheʻe me ka hoʻohana ʻana i nā ana like ʻole ma 95°C no 5 s, 65°C no 60 s, a me 97°C ma ka hopena o ka qPCR. Ua hana ʻia kēlā me kēia qPCR ma ke ʻano ʻekolu, koe wale nō nā laʻana kaohi.
ʻOiai ua ʻike ʻia nā mussels no ko lākou helu kānana kiʻekiʻe, ua noiʻi mua mākou inā hiki iā lākou ke kānana a mālama i nā ʻāpana DNA i loko o ka wai kai. Ua hoihoi pū mākou inā hōʻiliʻili kēia mau ʻāpana i loko o kā lākou ʻōnaehana lymphatic hapa-hāmama. Ua hoʻoponopono mākou i kēia pilikia ma ke ʻano hoʻokolohua ma ka huli ʻana i ka hopena o nā ʻāpana DNA hiki ke hoʻoheheʻe ʻia i hoʻohui ʻia i nā pahu mussel polū. No ka hoʻomaʻalahi ʻana i ka hahai ʻana i nā ʻāpana DNA, ua hoʻohana mākou i ka DNA plasmid haole (ʻaʻole ponoʻī) i loaʻa ka gene galectin-7 kanaka. Hoʻopaʻa ʻo ddPCR i nā ʻāpana DNA plasmid i loko o ka wai kai a me nā mussels. Hōʻike kā mākou mau hopena inā ua mau ka nui o nā ʻāpana DNA i loko o ka wai kai i ka hala ʻana o ka manawa (a hiki i 7 mau lā) i ka loaʻa ʻole o nā mussels, a laila i ke alo o nā mussels ua aneane nalowale loa kēia pae i loko o 8 mau hola (Kiʻi 1a, b). Ua ʻike maʻalahi ʻia nā ʻāpana o ka DNA exogenous i loko o 15 min i loko o ka wai intravalvular a me ka hemolymph (Kiʻi 1c). Hiki ke ʻike ʻia kēia mau ʻāpana a hiki i 4 mau hola ma hope o ka hōʻike ʻana. ʻO kēia hana kānana e pili ana i nā ʻāpana DNA e like me ka hana kānana o ka bacteria a me ka algae [31]. Hōʻike kēia mau hopena hiki i nā ʻūpā ke kānana a hōʻiliʻili i ka DNA haole i loko o kā lākou mau keʻena wai.
ʻO nā ʻano pili o ka DNA plasmid i loko o ka wai kai i ke alo (A) a i ʻole ka ʻole (B) o nā mussels, i ana ʻia e ddPCR. Ma A, ua hōʻike ʻia nā hopena ma ke ʻano he pakeneka, me nā palena o nā pahu e hōʻike ana i ka 75th a me 25th percentiles. Hōʻike ʻia ka piʻo logarithmic i hoʻopili ʻia i ka ʻulaʻula, a ʻo ka wahi i hoʻoluʻu ʻia i ka hina hina e hōʻike ana i ka manawa hilinaʻi 95%. Ma B, hōʻike ka laina ʻulaʻula i ka awelika a hōʻike ka laina polū i ka manawa hilinaʻi 95% no ka ʻano. C Hōʻiliʻili ʻana o ka DNA plasmid i loko o ka hemolymph a me ka wai valvular o nā mussels i nā manawa like ʻole ma hope o ka hoʻohui ʻana o ka DNA plasmid. Hōʻike ʻia nā hopena ma ke ʻano he kope paʻa i ʻike ʻia/mL (±SE).
ʻO ka mea aʻe, ua noiʻi mākou i ke kumu o ka ccfDNA i loko o nā mussels i hōʻiliʻili ʻia mai nā moena mussel ma nā mokupuni ʻo Kerguelen, kahi hui mamao o nā mokupuni me ka mana anthropogenic palena. No kēia kumu, ua hoʻokaʻawale ʻia a hoʻomaʻemaʻe ʻia ka cccDNA mai nā hemolymph mussel e nā ʻano hana i hoʻohana pinepine ʻia e hoʻomaʻemaʻe i ka cccDNA kanaka [32, 33]. Ua ʻike mākou ʻo ka awelika o ka nui o ka hemolymph ccfDNA i loko o nā mussels aia i loko o ka pae haʻahaʻa micrograms no ka ml hemolymph (e nānā i ka Papa S2, ʻIke Hoʻohui). ʻOi aku ka nui o kēia pae o nā nui ma mua o nā poʻe olakino (nā nanograms haʻahaʻa no ka milliliter), akā i nā hihia kakaikahi, i nā maʻi maʻi kanesa, hiki i ka pae o ka ccfDNA ke hiki i kekahi mau micrograms no ka milliliter [34, 35]. Ua hōʻike ʻia kahi loiloi o ka hoʻolaha nui o ka hemolymph ccfDNA he ʻokoʻa loa ka nui o kēia mau ʻāpana, mai 1000 bp a i 1000 bp. a hiki i 5000 bp (Kiʻi 2). Ua loaʻa nā hopena like me ka hoʻohana ʻana i ka silica-based QIAamp Investigator Kit, kahi ʻano hana i hoʻohana pinepine ʻia i ka ʻepekema forensic e hoʻokaʻawale koke a hoʻomaʻemaʻe i ka genomic DNA mai nā laʻana DNA haʻahaʻa haʻahaʻa, me ccfDNA [36].
ʻO ka electrophoregram ccfDNA hōʻike o ka hemolymph mussel. Hoʻopuka ʻia me ka NucleoSnap Plasma Kit (luna) a me QIAamp DNA Investigator Kit. B Violin chart e hōʻike ana i ka hoʻolaha ʻana o nā ʻano hemolymph ccfDNA (±SE) i loko o nā mussels. Hōʻike nā laina ʻeleʻele a me ka ʻulaʻula i ka median a me nā quartiles mua a me ke kolu.
Ma kahi o 1% o ka ccfDNA i loko o ke kanaka a me nā primates he kumu haole [21, 37]. Ma muli o ka ʻōnaehana kahe koko hapa-hāmama o nā bivalves, ka wai kai momona i ka microbial, a me ka nui o ka hoʻolaha ʻana o ka mussel ccfDNA, ua kuhiakau mākou he loaʻa paha i ka mussel hemolymph ccfDNA kahi waihona waiwai a ʻano like ʻole o ka microbial DNA. No ka hoʻāʻo ʻana i kēia kuhiakau, ua hoʻonohonoho mākou i ka hemolymph ccfDNA mai nā laʻana Aulacomya atra i hōʻiliʻili ʻia mai nā mokupuni ʻo Kerguelen, e loaʻa ana ma mua o 10 miliona mau heluhelu, 97.6% o ia mau mea i hala i ka mana maikaʻi. A laila ua hoʻokaʻawale ʻia nā heluhelu e like me nā kumu ponoʻī a me nā kumu ʻole ponoʻī me ka hoʻohana ʻana i nā waihona ʻikepili bivalve BLASTN a me NCBI (Fig. S1, Supplementary Information).
I loko o ke kanaka, hiki ke hoʻokuʻu ʻia ka DNA nuklea a me ka mitochondrial i loko o ke koko [38]. Eia naʻe, i loko o ke aʻo ʻana o kēia manawa, ʻaʻole hiki ke wehewehe kikoʻī i ka DNA genomic nuklea o nā mussels, ʻoiai ʻaʻole i hoʻonohonoho ʻia a wehewehe ʻia paha ka genome A. atra. Eia naʻe, ua hiki iā mākou ke ʻike i kahi helu o nā ʻāpana ccfDNA o ko mākou kumu ponoʻī me ka hoʻohana ʻana i ka waihona puke bivalve (Fig. S2, Supplementary Information). Ua hōʻoia pū mākou i ke alo o nā ʻāpana DNA o ko mākou kumu ponoʻī ma o ka hoʻonui ʻana o PCR i kuhikuhi ʻia o kēlā mau genes A. atra i hoʻonohonoho ʻia (Fig. 3). Pēlā nō, ʻoiai ua loaʻa ka genome mitochondrial o A. atra i nā waihona ʻikepili lehulehu, hiki i kekahi ke loaʻa nā hōʻike no ke alo o nā ʻāpana ccfDNA mitochondrial i loko o ka hemolymph o A. atra. Ua hōʻoia ʻia ke alo o nā ʻāpana DNA mitochondrial e ka hoʻonui ʻana o PCR (Fig. 3).
Aia nā ʻano genes mitochondrial like ʻole i loko o ka hemolymph o A. atra (nā kiko ʻulaʻula - helu waihona: SRX5705969) a me M. platensis (nā kiko polū - helu waihona: SRX5705968) i hoʻonui ʻia e PCR. Hoʻololi ʻia ke kiʻi mai Breton et al., 2011 B Hoʻonui ʻia o ka supernatant hemolymph mai A. atra Mālama ʻia ma ka pepa FTA. E hoʻohana i kahi punch 3 mm e hoʻohui pololei i ka ʻōmole PCR e loaʻa ana ka hui PCR.
Ma muli o ka nui o ka microbial i loko o ka wai kai, ua kālele mua mākou i ke ʻano o nā moʻo DNA microbial i loko o ka hemolymph. No ka hana ʻana i kēia, hoʻohana mākou i ʻelua mau hoʻolālā like ʻole. ʻO ke hoʻolālā mua i hoʻohana iā Kraken2, kahi papahana hoʻokaʻawale moʻo i hoʻokumu ʻia i ka algorithm e hiki ke ʻike i nā moʻo microbial me ka pololei e like me BLAST a me nā mea hana ʻē aʻe [28]. Ua hoʻoholo ʻia ma mua o 6719 mau heluhelu he kumu bacteria, ʻoiai ʻo 124 a me 64 mai archaea a me nā virus, kēlā me kēia (Kiʻi 4). ʻO nā ʻāpana DNA bacteria nui loa ʻo Firmicutes (46%), Proteobacteria (27%), a me Bacteroidetes (17%) (Kiʻi 4a). Kūlike kēia hoʻolaha me nā haʻawina mua o ka microbiome mussel polū kai [39, 40]. ʻO Gammaproteobacteria ka papa nui o Proteobacteria (44%), me nā Vibrionales he nui (Kiʻi 4b). Ua hōʻoia ke ʻano ddPCR i ke alo o nā ʻāpana DNA Vibrio i loko o ka ccfDNA o A. atra hemolymph (Kiʻi 4c) [41]. No ka loaʻa ʻana o ka ʻike hou aku e pili ana i ke kumu bacteria o ccfDNA, ua lawe ʻia kahi ala hou aʻe (Kiʻi S2, ʻIke Hoʻohui). Ma kēia hihia, ua hōʻuluʻulu ʻia nā heluhelu i hoʻopili ʻia ma ke ʻano he mau heluhelu i hui pū ʻia a ua hoʻokaʻawale ʻia e like me ke kumu ponoʻī (bivalves) a i ʻole ke kumu ponoʻī ʻole me ka hoʻohana ʻana iā BLASTN a me kahi waiwai e o 1e−3 a me kahi ʻoki me ka >90% homology. Ma kēia hihia, ua hōʻuluʻulu ʻia nā heluhelu i hoʻopili ʻia ma ke ʻano he mau heluhelu i hui pū ʻia a ua hoʻokaʻawale ʻia e like me ke kumu ponoʻī (bivalves) a i ʻole ke kumu ponoʻī ʻole me ka hoʻohana ʻana iā BLASTN a me kahi waiwai e o 1e−3 a me kahi ʻoki me ka >90% homology. В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и были классифицированы как стонби моллюски) или чужие по происхождению с использованием BLASTN и значения e 1e-3 и отсечения с гомологией> 90%. Ma kēia hihia, ua hōʻiliʻili ʻia nā heluhelu i hoʻopili ʻia ma ke ʻano he mau heluhelu i hoʻopau ʻia a ua hoʻokaʻawale ʻia he ʻōiwi (bivalve) a i ʻole ʻaʻole kumu me ka hoʻohana ʻana iā BLASTN a me ka waiwai e o 1e-3 a me ka ʻoki me ka >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 会和>90%同源性的截止值分类为自身（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 使用 使用 的3> blastn 的和和90% 同源性 的 分类 自身 （双 壳类） 非 自身。。。。。。。。 В этом случае перекрывающиеся чтения были собраны как чтения с парными концами и классифицированы как собственный моллюски) a i ole несобственные по происхождению с использованием значений e BLASTN и 1e-3 и порога гомологии> 90%. Ma kēia hihia, ua hōʻiliʻili ʻia nā heluhelu i hoʻopili ʻia ma ke ʻano he mau heluhelu i hoʻopau ʻia a ua hoʻokaʻawale ʻia he ponoʻī (bivalves) a i ʻole ʻaʻole kumu me ka hoʻohana ʻana i nā waiwai e BLASTN a me 1e-3 a me kahi paepae homology >90%.ʻOiai ʻaʻole i hoʻonohonoho ʻia ka genome A. atra, ua hoʻohana mākou i ke ʻano hoʻākoakoa de novo o ka mea hoʻākoakoa MEGAHIT Next Generation Sequencing (NGS). Ua ʻike ʻia he 147,188 mau contigs he mau mea hilinaʻi (bivalves) o ke kumu. A laila ua pahū ʻia kēia mau contigs me nā e-values ​​​​o 1e-10 me ka hoʻohana ʻana iā BLASTN a me BLASTX. Ua ʻae kēia hoʻolālā iā mākou e ʻike i 482 mau ʻāpana non-bivalve i loaʻa i loko o A. atra ccfDNA. Ua loaʻa ma mua o ka hapalua (57%) o kēia mau ʻāpana DNA mai nā bacteria, ʻo ka hapa nui mai nā symbionts gill, me nā symbionts sulfotrophic, a mai nā symbionts gill Solemya velum (Kiʻi 5).
Ka nui o ka nui ma ka pae ʻano. B ʻOkoʻa o nā microbial o ʻelua phyla nui (Firmicutes a me Proteobacteria). Hoʻonui ʻia ʻana o ddPCR C Vibrio spp. A. Nā ʻāpana o ka gene 16S rRNA (uliuli) i ʻekolu atra hemolymphs.
Ua kālailai ʻia he 482 mau contigs i hōʻiliʻili ʻia. ʻIke laulā o ka hoʻolaha taxonomic o nā annotations contig metagenomic (prokaryotes a me eukaryotes). B Hoʻolaha kikoʻī o nā ʻāpana DNA bacteria i ʻike ʻia e BLASTN a me BLASTX.
Ua hōʻike pū ka loiloi ʻo Kraken2 ua loaʻa i ka mussel ccfDNA nā ʻāpana DNA archaeal, me nā ʻāpana DNA o Euryarchaeota (65%), Crenarchaeota (24%), a me Thaurmarcheota (11%) (Kiʻi 6a). ʻO ke alo o nā ʻāpana DNA i loaʻa mai Euryarchaeota lāua ʻo Crenarchaeota, i loaʻa mua ma ke kaiāulu microbial o nā mussels Kaleponi, ʻaʻole ia he mea kupanaha [42]. ʻOiai ua pili pinepine ʻo Euryarchaeota me nā kūlana koʻikoʻi, ua ʻike ʻia i kēia manawa ʻo Euryarchaeota lāua ʻo Crenarcheota kekahi o nā prokaryotes maʻamau i ke kaiapuni cryogenic kai [43, 44]. ʻAʻole he mea kupanaha ke alo o nā microorganisms methanogenic i loko o nā mussels, hāʻawi ʻia i nā hōʻike hou o nā leaka methane nui mai nā leaka lalo ma ka Kerguelen Plateau [45] a me ka hana microbial methane hiki ke ʻike ʻia ma waho o ke kahakai o nā mokupuni ʻo Kerguelen [46].
A laila ua neʻe ko mākou manaʻo i nā heluhelu ʻana mai nā virus DNA. I ko mākou ʻike maikaʻi loa, ʻo kēia ka haʻawina mua ma waho o ka pahuhopu o ka ʻike virus o nā mussels. E like me ka mea i manaʻo ʻia, ua loaʻa iā mākou nā ʻāpana DNA o nā bacteriophages (Caudovirales) (Kiʻi 6b). Eia naʻe, ʻo ka DNA viral maʻamau loa mai kahi phylum o nā nucleocytoviruses, i ʻike ʻia hoʻi ʻo ka nuclear cytoplasmic large DNA virus (NCLDV), nona ka genome nui loa o nā virus āpau. I loko o kēia phylum, ʻo ka hapa nui o nā moʻo DNA no nā ʻohana Mimimidoviridae (58%) a me Poxviridae (21%), nona nā kikowaena kūlohelohe e komo pū ana me nā vertebrates a me nā arthropods, ʻoiai he hapa liʻiliʻi o kēia mau moʻo DNA no nā algae virological i ʻike ʻia. Hoʻopilikia i nā algae eukaryotic kai. Ua loaʻa pū nā moʻo mai ka virus Pandora, ka virus nunui me ka nui o ka genome nui loa o nā genera viral i ʻike ʻia. ʻO ka mea hoihoi, ʻo ka laulā o nā kikowaena i ʻike ʻia ua loaʻa i ka virus, e like me ka mea i hoʻoholo ʻia e ka hemolymph ccfDNA sequencing, he nui loa ia (Kiʻi S3, ʻIke Hoʻohui). Hoʻokomo pū ʻia me nā virus e hoʻopilikia i nā pepeke e like me Baculoviridae a me Iridoviridae, a me nā virus e hoʻopilikia i ka amoeba, algae a me nā vertebrates. Ua loaʻa pū iā mākou nā moʻo e kūlike ana i ka genome Pithovirus sibericum. Ua hoʻokaʻawale mua ʻia nā Pitoviruses (i ʻike ʻia hoʻi ʻo "zombie viruses") mai ka permafrost 30,000 makahiki ma Siberia [47]. No laila, kūlike kā mākou mau hopena me nā hōʻike mua e hōʻike ana ʻaʻole i pau nā ʻano hou o kēia mau virus [48] a aia paha kēia mau virus i nā ʻōnaehana kai subarctic mamao.
ʻO ka mea hope loa, ua hoʻāʻo mākou e ʻike inā hiki iā mākou ke loaʻa nā ʻāpana DNA mai nā holoholona multicellular ʻē aʻe. Ua ʻike ʻia he 482 mau contigs haole e BLASTN lāua ʻo BLASTX me nā hale waihona puke nt, nr a me RefSeq (genomic a me ka protein). Hōʻike kā mākou mau hopena i waena o nā ʻāpana haole o ccfDNA o nā holoholona multicellular, ʻo ka DNA o nā iwi iwi ka mea nui (Kiʻi 5). Ua loaʻa pū nā ʻāpana DNA mai nā pepeke a me nā ʻano ʻē aʻe. ʻAʻole i ʻike ʻia kahi hapa nui o nā ʻāpana DNA, malia paha ma muli o ka hōʻike ʻole ʻia o ka nui o nā ʻano kai i loko o nā waihona genomic i hoʻohālikelike ʻia me nā ʻano terrestrial [49].
Ma kēia pepa, hoʻopili mākou i ke kumumanaʻo LB i nā mussels, me ka hoʻopaʻapaʻa ʻana e hiki i ka hemolymph ccfDNA shot sequencing ke hāʻawi i ka ʻike i ka haku mele ʻana o nā kaiaola kai kai. Ma ke ʻano kūikawā, ua ʻike mākou 1) loaʻa i ka mussel hemolymph nā ʻano kiʻekiʻe (nā pae microgram) o nā ʻāpana DNA e kaapuni nui ana (~1-5 kb); 2) he kūʻokoʻa a kūʻokoʻa ʻole kēia mau ʻāpana DNA 3) Ma waena o nā kumu haole o kēia mau ʻāpana DNA, ua ʻike mākou i ka bacterial, archaeal a me ka viral DNA, a me ka DNA o nā holoholona multicellular ʻē aʻe; 4) ʻO ka hōʻiliʻili ʻana o kēia mau ʻāpana ccfDNA haole i loko o ka hemolymph e hana wikiwiki a hāʻawi i ka hana kānana kūloko o nā mussels. I ka hopena, hōʻike kā mākou noiʻi ʻana ʻo ke kumumanaʻo o LB, ka mea i hoʻohana nui ʻia ma ke kahua o ka biomedicine, e hoʻopili ana i kahi kumu waiwai akā ʻaʻole i ʻimi ʻia o ka ʻike e hiki ke hoʻohana ʻia e hoʻomaopopo maikaʻi i ka pilina ma waena o nā ʻano sentinel a me ko lākou kaiapuni.
Ma waho aʻe o nā primates, ua hōʻike ʻia ka hoʻokaʻawale ʻana o ka ccfDNA i nā mammals, me nā ʻiole, nā ʻīlio, nā pōpoki, a me nā lio [50, 51, 52]. Eia naʻe, i ko mākou ʻike, ʻo kā mākou noiʻi ka mea mua e hōʻike i ka ʻike ʻana a me ke kaʻina ʻana o ka ccfDNA i nā ʻano kai me kahi ʻōnaehana kahe ākea. ʻO kēia hiʻohiʻona anatomical a me ka hiki ke kānana o nā mussels, ma ka liʻiliʻi ma kahi hapa, e wehewehe i nā ʻano nui like ʻole o nā ʻāpana DNA e kahe ana i hoʻohālikelike ʻia me nā ʻano ʻē aʻe. I loko o ke kanaka, ʻo ka hapa nui o nā ʻāpana DNA e kahe ana i loko o ke koko he mau ʻāpana liʻiliʻi e kū ana i ka nui mai 150 a 200 bp. me ka piko kiʻekiʻe loa o 167 bp [34, 53]. ʻO kahi hapa liʻiliʻi akā koʻikoʻi o nā ʻāpana DNA ma waena o 300 a me 500 bp ka nui, a ma kahi o 5% ʻoi aku ka lōʻihi ma mua o 900 bp. [54]. ʻO ke kumu o kēia hoʻolaha nui ʻana, ʻo ia ke kumu nui o ka ccfDNA i loko o ka plasma ma muli o ka make ʻana o nā cell, ma muli paha o ka make ʻana o nā cell a i ʻole ma muli o ka necrosis o nā cell hematopoietic e kaapuni ana i nā kānaka olakino a i ʻole ma muli o ka apoptosis o nā cell tumor i nā maʻi maʻi kanesa (i ʻike ʻia ʻo ka circulating tumor DNA). , ctDNA). ʻO ka hoʻolaha nui ʻana o ka hemolymph ccfDNA a mākou i loaʻa ai i loko o nā mussels mai 1000 a 5000 bp, e hōʻike ana he kumu ʻokoʻa ko ka mussel ccfDNA. He kuhiakau kūpono kēia, ʻoiai he ʻōnaehana vascular semi-open ko nā mussels a noho i nā wahi kai kai e loaʻa ana nā kiʻekiʻe o ka microbial genomic DNA. ʻO ka ʻoiaʻiʻo, ua hōʻike kā mākou mau hoʻokolohua hale hana e hoʻohana ana i ka DNA exogenous e hōʻiliʻili nā mussels i nā ʻāpana DNA i loko o ka wai kai, ma ka liʻiliʻi ma hope o kekahi mau hola ua hoʻohaʻahaʻa ʻia lākou ma hope o ka lawe ʻana o ka cellular a/a i ʻole e hoʻokuʻu ʻia a/a i ʻole mālama ʻia i nā hui like ʻole. Ma muli o ka laha ʻole o nā cell (ʻo prokaryotic a me eukaryotic), ʻo ka hoʻohana ʻana i nā compartments intravalvular e hōʻemi i ka nui o ka ccfDNA mai nā kumu ponoʻī a me nā kumu haole. Ke noʻonoʻo nei i ke koʻikoʻi o ka pale ʻana o ka bivalve a me ka nui o nā phagocytes e kaʻapuni ana, ua kuhi hou mākou ua hoʻonui ʻia nā ccfDNA haole i nā phagocytes e kaʻapuni ana e hōʻiliʻili ana i ka DNA haole ma hope o ka ʻai ʻana i nā microorganism a/a i ʻole nā ​​​​​​ʻōpala cellular. I ka hui pū ʻia, hōʻike kā mākou mau hopena he waihona kū hoʻokahi ka bivalve hemolymph ccfDNA o ka ʻike molekala a hoʻoikaika i ko lākou kūlana ma ke ʻano he ʻano kiaʻi.
Hōʻike kā mākou ʻikepili e hiki i ka hoʻonohonoho ʻana a me ka nānā ʻana i nā ʻāpana hemolymph ccfDNA i loaʻa mai ka bacteria ke hāʻawi i ka ʻike koʻikoʻi e pili ana i ka flora bacteria host a me nā bacteria i loaʻa i loko o ka ʻōnaehana kai a puni. Ua hōʻike nā ʻano hana hoʻonohonoho pana i nā moʻo o ka bacteria commensal A. atra gill i hala ʻole inā ua hoʻohana ʻia nā ʻano ʻike ʻana o 16S rRNA maʻamau, ma muli o kahi ʻāpana o kahi manaʻo waihona puke kuhikuhi. ʻO ka ʻoiaʻiʻo, ʻo kā mākou hoʻohana ʻana i ka ʻikepili LB i hōʻiliʻili ʻia mai M. platensis ma ka papa mussel like ma Kerguelen ua hōʻike ʻia ua like ka haku mele ʻana o nā symbionts bacteria e pili ana i ka gill no nā ʻano mussel ʻelua (Kiʻi S4, ʻIke Hoʻohui). ʻO kēia ʻano like o ʻelua mussels like ʻole e hōʻike paha i ka haku mele ʻana o nā kaiāulu bacteria i loko o nā waihona anu, sulfurous, a me ka lua pele o Kerguelen [55, 56, 57, 58]. Ua wehewehe maikaʻi ʻia nā pae kiʻekiʻe o nā microorganisms hoʻemi sulfur i ka wā e ʻohi ai i nā mussels mai nā wahi kahakai bioturbated [59], e like me ke kahakai o Port-au-France. ʻO kekahi mea hiki ke hoʻopilikia ʻia ka flora mussel commensal e ka hoʻoili ʻana ma ke ʻano ākea [60, 61]. Pono ka noiʻi hou aku e hoʻoholo i ka pilina ma waena o ke kaiapuni kai, ka ʻilikai, a me ke ʻano o nā bacteria symbiotic i loko o nā mussels. Ke hoʻomau nei kēia mau haʻawina i kēia manawa.
ʻO ka lōʻihi a me ka nui o ka hemolymph ccfDNA, kona maʻalahi o ka hoʻomaʻemaʻe ʻana, a me ke kūlana kiʻekiʻe e ʻae i ka hoʻonohonoho wikiwiki ʻana i ka shotgun kekahi o nā pono he nui o ka hoʻohana ʻana i ka mussel ccfDNA e loiloi i ka biodiversity i nā kaiaola kai kai. He kūpono loa kēia ʻano hana no ke ʻano ʻana i nā kaiāulu viral (viromes) i loko o kahi kaiaola i hāʻawi ʻia [62, 63]. ʻAʻole like me ka bacteria, archaea, a me eukaryotes, ʻaʻole i loaʻa i nā genomes viral nā genes i mālama ʻia e like me nā moʻo 16S. Hōʻike kā mākou mau hopena e hiki ke hoʻohana ʻia nā biopsies wai mai nā ʻano hōʻailona e like me nā mussels e ʻike i nā helu nui o nā ʻāpana virus ccfDNA i ʻike ʻia e hoʻopili i nā mea hoʻokipa e noho maʻamau i nā kaiaola kai kai. Hoʻokomo pū kēia me nā virus i ʻike ʻia e hoʻopili i nā protozoa, arthropods, nā pepeke, nā mea kanu, a me nā virus bacteria (e.g., bacteriophages). Ua loaʻa kahi hoʻolaha like i ka wā i nānā ai mākou i ka hemolymph ccfDNA virome o nā mussels polū (M. platensis) i hōʻiliʻili ʻia ma ka papa mussel like ma Kerguelen (Papa S2, ʻIke Hoʻohui). ʻO ke kaʻina hana Shotgun o ccfDNA he ala hou ia e loaʻa ana ka ikaika i ke aʻo ʻana i ka virome o nā kānaka a i ʻole nā ​​ʻano ʻē aʻe [21, 37, 64]. He mea pono loa kēia ala no ke aʻo ʻana i nā virus DNA pālua, ʻoiai ʻaʻohe gene hoʻokahi i mālama ʻia ma waena o nā virus DNA pālua āpau, e hōʻike ana i ka papa like ʻole a ākea o nā virus ma Baltimore [65]. ʻOiai ʻo ka hapa nui o kēia mau virus e noho mau ana i ka hoʻokaʻawale ʻole ʻia a hiki ke hoʻokomo i nā virus mai kahi ʻāpana i ʻike ʻole ʻia o ka honua viral [66], ua ʻike mākou ʻo nā virome a me nā pae kikowaena o nā mussels A. atra lāua ʻo M. platensis e hāʻule ma waena o nā ʻano ʻelua. like (e nānā i ke kiʻi S3, ʻike hou aku). ʻAʻole he mea kupanaha kēia ʻano like, no ka mea e hōʻike ana paha i ka nele o ke koho ʻana i ka lawe ʻana o ka DNA i loaʻa i ke kaiapuni. Pono nā haʻawina e hiki mai ana e hoʻohana ana i ka RNA i hoʻomaʻemaʻe ʻia e wehewehe i ka RNA virome.
Ma kā mākou noiʻi ʻana, ua hoʻohana mākou i kahi pipeline paʻa loa i hoʻololi ʻia mai ka hana a Kowarski a me nā hoa hana [37], nāna i hoʻohana i ka holoi ʻana i ʻelua ʻanuʻu o nā heluhelu i hui pū ʻia a me nā contigs ma mua a ma hope o ka hōʻuluʻulu ʻana o ka ccfDNA maoli, e hopena ana i kahi hapa nui o nā heluhelu i hoʻopaʻa ʻole ʻia. No laila, ʻaʻole hiki iā mākou ke kāpae i kekahi o kēia mau heluhelu i hoʻopaʻa ʻole ʻia e loaʻa ana i ko lākou kumu ponoʻī, ʻoiai ʻaʻohe o mākou genome kuhikuhi no kēia ʻano mussel. Ua hoʻohana pū mākou i kēia pipeline no ka mea ua hopohopo mākou e pili ana i nā chimera ma waena o nā heluhelu ponoʻī a me nā heluhelu ʻole a me nā lōʻihi heluhelu i hana ʻia e ka Illumina MiSeq PE75. ʻO kekahi kumu no ka hapa nui o nā heluhelu i hoʻopaʻa ʻole ʻia, ʻo ia ka nui o nā microbes kai, ʻoi aku hoʻi ma nā wahi mamao e like me Kerguelen, ʻaʻole i hoʻopaʻa inoa ʻia. Ua hoʻohana mākou iā Illumina MiSeq PE75, me ka manaʻo he like ka lōʻihi o nā ʻāpana ccfDNA me ka ccfDNA kanaka. No nā haʻawina e hiki mai ana, ma muli o kā mākou mau hopena e hōʻike ana he lōʻihi ka heluhelu ʻana o ka hemolymph ccfDNA ma mua o nā kānaka a/a i ʻole nā ​​mammals, ke paipai nei mākou e hoʻohana i kahi kahua sequencing i kūpono no nā ʻāpana ccfDNA lōʻihi. E maʻalahi kēia hana i ka ʻike ʻana i nā hōʻailona hou aʻe no ka nānā hohonu. ʻO ka loaʻa ʻana o ka moʻo genome nuclear A. atra piha i loaʻa ʻole i kēia manawa e kōkua nui hoʻi i ka hoʻokaʻawale ʻana o ccfDNA mai nā kumu ponoʻī a me nā kumu ʻole ponoʻī. No ka mea ua kālele kā mākou noiʻi ʻana i ka hiki ke hoʻopili i ke kumumanaʻo o ka biopsy wai i nā mussels, ke manaʻolana nei mākou e hoʻomohala ʻia nā mea hana hou a me nā pipelines e hoʻonui i ka hiki o kēia ʻano hana e aʻo ai i ka ʻokoʻa microbial o nā mussels. kaiaola kai.
Ma ke ʻano he biomarker lapaʻau ʻaʻole invasive, pili nā pae plasma kanaka kiʻekiʻe o ka ccfDNA me nā maʻi like ʻole, ka hōʻino ʻana o nā ʻiʻo, a me nā kūlana kaumaha [67,68,69]. Pili kēia hoʻonui ʻana me ka hoʻokuʻu ʻana o nā ʻāpana DNA o kona kumu ponoʻī ma hope o ka hōʻino ʻana o nā ʻiʻo. Ua hoʻoponopono mākou i kēia pilikia me ka hoʻohana ʻana i ke kaumaha wela koʻikoʻi, kahi i hōʻike pōkole ʻia ai nā mussels i ka mahana o 30 °C. Ua hana mākou i kēia loiloi ma luna o ʻekolu mau ʻano mussels like ʻole i ʻekolu mau hoʻokolohua kūʻokoʻa. Eia naʻe, ʻaʻole mākou i ʻike i kekahi loli i nā pae ccfDNA ma hope o ke kaumaha wela koʻikoʻi (e nānā i ke Kiʻi S5, ʻike hou aku). Hiki i kēia ʻike ke wehewehe, ma ka liʻiliʻi ma kahi hapa, ʻo ka ʻoiaʻiʻo he ʻōnaehana kahe koko hapa-hāmama ko nā mussels a hōʻiliʻili i nā nui o ka DNA haole ma muli o kā lākou hana kānana kiʻekiʻe. Ma ka ʻaoʻao ʻē aʻe, ʻoi aku ka paʻakikī o nā mussels, e like me nā invertebrates he nui, i ka hōʻino ʻana o nā ʻiʻo i hoʻokumu ʻia e ke kaumaha, no laila e kaupalena ana i ka hoʻokuʻu ʻana o ccfDNA i loko o kā lākou hemolymph [70, 71].
A hiki i kēia lā, ua kālele nui ka loiloi DNA o ka biodiversity i loko o nā kaiaolaola wai i ka metabarcoding DNA kaiapuni (eDNA). Eia nō naʻe, ua kaupalena pinepine ʻia kēia ʻano hana i ka loiloi biodiversity i ka wā e hoʻohana ʻia ai nā primers. ʻO ka hoʻohana ʻana i ka shotgun sequencing e pale aku i nā palena o PCR a me ke koho biased o nā set primer. No laila, ma kekahi ʻano, ʻoi aku ka kokoke o kā mākou ʻano hana i ke ʻano hana sequencing eDNA Shotgun kiʻekiʻe i hoʻohana hou ʻia, hiki ke hoʻonohonoho pololei i ka DNA i ʻoki ʻia a kālailai i ka aneane o nā meaola āpau [72, 73]. Eia nō naʻe, aia kekahi mau pilikia kumu e hoʻokaʻawale iā LB mai nā ʻano eDNA maʻamau. ʻOiaʻiʻo, ʻo ka ʻokoʻa nui ma waena o eDNA a me LB ka hoʻohana ʻana i nā kikowaena kānana kūlohelohe. Ua hōʻike ʻia ka hoʻohana ʻana i nā ʻano kai e like me nā sponges a me nā bivalves (Dresseina spp.) ma ke ʻano he kānana kūlohelohe no ke aʻo ʻana i ka eDNA [74, 75]. Eia nō naʻe, ua hoʻohana ka haʻawina a Dreissena i nā biopsies kiko mai kahi i unuhi ʻia ai ka DNA. ʻAʻole koi ka loiloi o ccfDNA mai LB i ka biopsy kiko, nā lako hana kūikawā a i kekahi manawa pipiʻi a me nā logistics e pili ana me ka eDNA a i ʻole ka biopsy kiko. ʻOiaʻiʻo, ua hōʻike koke mākou e hiki ke mālama ʻia a kālailai ʻia ka ccfDNA mai LB me ke kākoʻo FTA me ka ʻole o ka mālama ʻana i kahi kaulahao anu, he pilikia nui ia no ka noiʻi ʻana ma nā wahi mamao [76]. He maʻalahi hoʻi ka unuhi ʻana o ccfDNA mai nā biopsies wai a hāʻawi i ka DNA kiʻekiʻe no ka shotgun sequencing a me ka nānā ʻana o PCR. He pōmaikaʻi nui kēia i hāʻawi ʻia i kekahi o nā palena loea e pili ana me ka nānā ʻana o eDNA [77]. ʻO ka maʻalahi a me ke kumukūʻai haʻahaʻa o ke ʻano laʻana he kūpono loa ia no nā papahana nānā lōʻihi. Ma waho aʻe o ko lākou hiki ke kānana kiʻekiʻe, ʻo kekahi hiʻohiʻona kaulana o nā bivalves ʻo ia ka mucopolysaccharide kemika o kā lākou mucus, kahi e paipai ai i ka omo ʻana o nā virus [78, 79]. ʻO kēia ka mea e hoʻolilo ai i nā bivalves i kānana kūlohelohe kūpono no ka wehewehe ʻana i ka biodiversity a me ka hopena o ka loli o ke aniau i loko o kahi ʻōnaehana wai i hāʻawi ʻia. ʻOiai hiki ke ʻike ʻia ka loaʻa ʻana o nā ʻāpana DNA i loaʻa mai ka host ma ke ʻano he palena o ke ʻano i hoʻohālikelike ʻia me eDNA, ʻo ke kumukūʻai e pili ana me ka loaʻa ʻana o ia ʻano ccfDNA maoli i hoʻohālikelike ʻia me eDNA e hoʻomaopopo like ʻia no ka nui o ka ʻike i loaʻa no nā haʻawina olakino. offset host. ʻO kēia ka loaʻa ʻana o nā moʻo viral i hoʻohui ʻia i loko o ka genome o ka host host. He mea nui loa kēia no nā mussels, i hāʻawi ʻia i ke alo o nā retroviruses leukemic i hoʻoili ʻia ma ka pae i loko o nā bivalves [80, 81]. ʻO kekahi pono o LB ma luna o eDNA ʻo ia ka hoʻohana ʻana i ka hana phagocytic o nā cell koko e kahe ana i loko o ka hemolymph, kahi e hoʻopuni ai i nā microorganism (a me kā lākou genomes). ʻO Phagocytosis ka hana nui o nā cell koko i loko o nā bivalves [82]. ʻO ka hope loa, hoʻohana ke ʻano hana i ka hiki ke kānana kiʻekiʻe o nā mussels (awelika 1.5 l/h o ka wai kai) a me ke kahe ʻana o ʻelua lā, e hoʻonui ai i ka hui ʻana o nā papa like ʻole o ka wai kai, e ʻae ana i ka hopu ʻana o ka eDNA heterologous. [83, 84]. No laila, ʻo ka nānā ʻana o ka mussel ccfDNA he ala hoihoi ia i hāʻawi ʻia i nā hopena meaʻai, hoʻokele waiwai, a me ke kaiapuni o nā mussels. E like me ka nānā ʻana o LB i hōʻiliʻili ʻia mai nā kānaka, wehe pū kēia ʻano hana i ka hiki ke ana i nā loli genetic a me epigenetic i ka DNA host ma ka pane ʻana i nā mea exogenous. No ka laʻana, hiki ke noʻonoʻo ʻia nā ʻenehana hoʻonohonoho hanauna ʻekolu e hana i ka loiloi methylation genome-ākea i loko o ka ccfDNA maoli me ka hoʻohana ʻana i ka hoʻonohonoho nanopore. Pono e hoʻomaʻalahi ʻia kēia kaʻina hana e ka ʻoiaʻiʻo ʻo ka lōʻihi o nā ʻāpana mussel ccfDNA e kūpono loa me nā kahua hoʻonohonoho heluhelu lōʻihi e ʻae i ka loiloi methylation DNA genome-ākea mai kahi holo hoʻonohonoho hoʻokahi me ka ʻole o ka pono no nā hoʻololi kemika.85,86] He mea hoihoi kēia, no ka mea, ua hōʻike ʻia e hōʻike ana nā ʻano methylation DNA i kahi pane i ke kaumaha o ke kaiapuni a hoʻomau i nā hanauna he nui. No laila, hiki iā ia ke hāʻawi i ka ʻike waiwai i nā ʻano hana kumu e hoʻomalu ana i ka pane ma hope o ka hōʻike ʻana i ka loli o ke aniau a i ʻole nā ​​​​mea haumia [87]. Eia naʻe, ʻaʻole me nā palena ʻole ka hoʻohana ʻana o LB. ʻAʻole pono e ʻōlelo, pono kēia i ke alo o nā ʻano hōʻailona i loko o ka ʻōnaehana ola. E like me ka mea i ʻōlelo ʻia ma luna, ʻo ka hoʻohana ʻana iā LB e loiloi i ka biodiversity o kahi ʻōnaehana ola i hāʻawi ʻia e pono ai kahi pipeline bioinformatics koʻikoʻi e noʻonoʻo ana i ke alo o nā ʻāpana DNA mai ke kumu. ʻO kekahi pilikia nui ka loaʻa ʻana o nā genomes kuhikuhi no nā ʻano kai. Ua manaʻolana ʻia e kōkua nā hana e like me ka Marine Mammal Genomes Project a me ka papahana Fish10k i hoʻokumu hou ʻia [88] i ia ʻano loiloi i ka wā e hiki mai ana. ʻO ka hoʻopili ʻana o ka manaʻo LB i nā meaola hānai kānana kai e kūlike pū me nā holomua hou loa i ka ʻenehana sequencing, e kūpono ana no ka hoʻomohala ʻana i nā biomarkers multi-ohm e hāʻawi i ka ʻike koʻikoʻi e pili ana i ke olakino o nā wahi noho kai e pane ana i ke kaumaha o ke kaiapuni.
Ua waiho ʻia ka ʻikepili hoʻonohonoho genome i loko o ka NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 ma lalo o Bioprojects SRR8924808.
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