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ʻO ka biopsy wai (LB) kahi manaʻo e loaʻa wikiwiki nei ka kaulana i ka ʻoihana biomedical.Hoʻokumu nui ʻia ka manaʻo ma ka ʻike ʻana i nā ʻāpana o ka extracellular DNA (ccfDNA), ka mea i hoʻokuʻu nui ʻia ma ke ʻano he mau ʻāpana liʻiliʻi ma hope o ka make ʻana o ke kelepona i nā ʻano kikoʻī.ʻO kahi hapa liʻiliʻi o kēia mau ʻāpana mai nā ʻiʻo a i ʻole nā mea ola ʻē aʻe.Ma ka hana o kēia manawa, ua hoʻohana mākou i kēia manaʻo i nā ʻiʻo, he ʻano mea mālama i ʻike ʻia no ko lākou hiki ke kānana wai kai kiʻekiʻe.Hoʻohana mākou i ka hiki o nā ʻū e hana ma ke ʻano he kānana kūlohelohe no ka hopu ʻana i nā ʻāpana DNA kaiapuni mai nā kumu like ʻole e hāʻawi i ka ʻike e pili ana i ke ʻano olaola o nā kaiaola kai.Hōʻike kā mākou hopena i ka hemolymph mussel i loko o nā ʻāpana DNA e ʻokoʻa loa ka nui, mai 1 a 5 kb.Ua hōʻike ʻia ke kaʻina hana pana i ka nui o nā ʻāpana DNA no ka microbial kumu ʻē aʻe.Ma waena o lākou, ua ʻike mākou i nā ʻāpana DNA mai bacteria, archaea, a me nā maʻi maʻi, me nā maʻi maʻi i ʻike ʻia e hoʻopilikia i nā ʻano mea like ʻole i loaʻa ma nā kaiaola kai kai.I ka hopena, hōʻike kā mākou haʻawina ʻo ka manaʻo o LB i hoʻopili ʻia i nā mussels he kumu waiwai akā ʻaʻole i ʻike ʻia e pili ana i ka ʻokoʻa microbial i nā kaiaola kai moana.
ʻO ka hopena o ka hoʻololi ʻana i ke aniau (CC) i ka biodiversity o nā kaiaola kai he wahi e ulu wikiwiki ana o ka noiʻi.ʻO ka hoʻomehana honua ʻaʻole wale ke kumu o ke koʻikoʻi koʻikoʻi koʻikoʻi, akā ke koi pū nei i nā palena evolutionary o ke kūpaʻa wela o nā mea ola kai, e pili 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 ke ʻano like ʻole o nā metazoans, hoʻopau ʻo CC i ke koena paʻakikī o ka pilina o ka host-microbial.ʻO kēia microbial dysbacteriosis he mea hoʻoweliweli koʻikoʻi i nā kaiaola kai ʻoiai ʻoi aku ka maʻalahi o nā meaola kai i nā pathogens infectious [3, 4].Manaʻo ʻia he kuleana koʻikoʻi ka SS i ka make nui, he pilikia koʻikoʻi ia no ka hoʻokele ʻana i nā kaiaola kai honua [5, 6].He mea koʻikoʻi kēia ma muli o ka hopena hoʻokele waiwai, kaiaola a me ka meaʻai o nā ʻano kai he nui.He ʻoiaʻiʻo kēia no nā bivalves e noho ana ma nā ʻāpana polar, kahi i ʻoi aku ka wikiwiki a me ka paʻakikī o nā hopena o CK [6, 7].ʻO ka ʻoiaʻiʻo, ʻo nā bivalves e like me Mytilus spp.hoʻohana nui ʻia e nānā i nā hopena o CC ma nā kaiaola kai.ʻAʻole ia he mea kupanaha, ua hoʻomohala ʻia kahi helu nui o nā biomarkers e nānā i ko lākou olakino, e hoʻohana pinepine ana i kahi ala ʻelua-papa e pili ana i nā biomarkers hana e pili ana i ka hana enzymatic a i ʻole nā hana kelepona e like me ka cell viability a me ka hana phagocytic [8].Hoʻokomo pū ʻia kēia mau ʻano i ke ana ʻana o ka neʻe ʻana o nā hōʻailona kaomi kikoʻī e hōʻiliʻili ai i loko o nā ʻiʻo palupalu ma hope o ka lawe ʻana i ka nui o ka wai kai.Eia nō naʻe, ʻo ka hiki ke kānana kiʻekiʻe a me ka semi-open circulatory system o nā bivalves e hāʻawi i kahi manawa e hoʻomohala ai i nā biomarkers hemolymph hou me ka hoʻohana ʻana i ka manaʻo o ka biopsy wai (LB), kahi ala maʻalahi a liʻiliʻi i ka hoʻokele maʻi.nā laʻana koko [9, 10].ʻOiai hiki ke loaʻa kekahi mau ʻano molekole kaʻapuni i loko o ke kanaka LB, ua hoʻokumu mua ʻia kēia manaʻo ma ka nānā ʻana i ke kaʻina hana DNA o nā ʻāpana DNA extracellular (ccfDNA) i loko o ka plasma.ʻO kaʻoiaʻiʻo, uaʻikeʻia ka heleʻana o ka DNA circulating i loko o ka plasma kanaka mai ka waena o ka 20th century [11], akā i nā makahiki i hala iho nei,ʻo ka hikiʻana mai o nāʻano hana hoʻonohonoho kiʻekiʻe i alakaʻiʻia i ka hōʻailona maʻi ma muli o ccfDNA.ʻO ka loaʻa ʻana o kēia mau ʻāpana DNA e holo nei ma muli o ka hoʻokuʻu passive o ka genomic DNA (nuklea a me ka mitochondrial) ma hope o ka make ʻana o ke kelepona. I loko o nā kānaka olakino, he haʻahaʻa ka nui o ka ccfDNA (<10 ng/mL) akā hiki ke hoʻonui ʻia e 5-10 mau manawa i ka poʻe maʻi e loaʻa ana i nā pathologies like ʻole a i ʻole i ke koʻikoʻi, e hopena i ka pōʻino o ke kino. I loko o nā kānaka olakino, he haʻahaʻa ka nui o ka ccfDNA (<10 ng/mL) akā hiki ke hoʻonui ʻia e 5-10 mau manawa i ka poʻe maʻi e loaʻa ana i nā pathologies like ʻole a i ʻole i ke koʻikoʻi, e hopena i ka pōʻino o ke kino. У здоровых людей концентрация вккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 раз ухличон или подвергающихся стрессу, приводящему к повреждению тканей. I ka poʻe olakino, ʻoi aku ka haʻahaʻa o ka cccDNA (<10 ng/mL), akā hiki ke hoʻonui ʻia e 5-10 mau manawa i ka poʻe maʻi me nā maʻi maʻi like ʻole a i ʻole ke kaumaha e alakaʻi i ka pōʻino o ke kino.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受受受压加倍,从而导致组织损伤。在 健康 个体 中 , ccfdna 的 浓度 较 低 ((<10 ng/ml) 但 在 各 种 病理 或 承叛 或 承叛加 5-10 , 从而 组织。。 损伤 损伤 损伤 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 раз у плич ями или стрессом, что приводит к повреждению тканей. He haʻahaʻa ka nui o ka ccfDNA (<10 ng/ml) i nā kānaka olakino, akā hiki ke hoʻonui ʻia i 5-10-fold i nā poʻe maʻi me nā pathologies like ʻole a i ʻole koʻikoʻi, e hopena i ka pōʻino o ke kino.He ʻokoʻa ka nui o nā ʻāpana ccfDNA, akā maʻamau, mai 150 a 200 bp.[12].ʻO ka nānāʻana i ka ccfDNA i loaʻa iā ia iho,ʻo ia hoʻi, ccfDNA mai nā pūnaewele hoʻokipa maʻamau a i hoʻololiʻia paha, hiki ke hoʻohanaʻia no kaʻikeʻana i nā hoʻololi genetic a me nā epigenetic i loaʻa i loko o ka nuklea a / a iʻole ka mitochondrial genome, ma laila e kōkua ai i nā kauka e koho i nā molecular-targeted therapies [13].Eia naʻe, hiki ke loaʻa ka ccfDNA mai nā kumu'ē aʻe e like me ccfDNA mai nā pūpū fetal i ka wā o ka hāpaiʻana a iʻole mai nā'ōpū i hoʻololiʻia [14,15,16,17].ʻO ka ccfDNA kekahi kumu nui o ka ʻike no ka ʻike ʻana i ka loaʻa ʻana o nā waikawa nucleic o kahi mea maʻi infectious (haole), e ʻae ai i ka ʻike non-invasive o nā maʻi ākea ʻaʻole i ʻike ʻia e nā moʻomeheu koko, e pale ana i ka biopsy invasive o ka ʻiʻo maʻi [18].Ua hōʻike maoli nā haʻawina hou i ke koko o ke kanaka he kumu waiwai o ka ʻike e hiki ke hoʻohana ʻia e ʻike i nā maʻi maʻi viral a me nā maʻi bacterial, a ma kahi o 1% o ka ccfDNA i loaʻa i loko o ka plasma kanaka no ka haole [19].Hōʻike kēia mau haʻawina e hiki ke loiloi ʻia ke ʻano like ʻole o ka microbiome kaapuni o ke kino me ka hoʻohana ʻana i ka loiloi ccfDNA.Eia naʻe, a hiki i kēia manawa, ua hoʻohana wale ʻia kēia manaʻo i nā kānaka a, i ka liʻiliʻi liʻiliʻi, i nā vertebrates ʻē aʻe [20, 21].
Ma ka pepa o kēia manawa, hoʻohana mākou i ka mana LB no ke kālailai ʻana i ka ccfDNA o Aulacomya atra, he ʻano ʻano hema i ʻike mau ʻia ma nā mokupuni ʻo Kerguelen subantarctic, he hui o nā mokupuni ma luna o kahi pāpū nui i kūkulu ʻia he 35 miliona mau makahiki i hala.huaʻi pele.Me ka hoʻohana ʻana i kahi ʻōnaehana hoʻokolohua in vitro, ʻike mākou ua lawe koke ʻia nā ʻāpana DNA i loko o ke kai e nā mussels a komo i ke keʻena hemolymph.Ua hōʻike ʻia ke kaʻina pū pana i ka mussel hemolymph ccfDNA i loko o nā ʻāpana DNA o kona kumu ponoʻī a ʻaʻole ponoʻī, me nā hua bacteria symbiotic a me nā ʻāpana DNA mai nā biomes maʻamau o nā kaiaola kai moana lua pele anu.Loaʻa i ka Hemolymph ccfDNA nā kaʻina viral i loaʻa mai nā maʻi me nā pae hoʻokipa like ʻole.Ua ʻike pū mākou i nā ʻāpana DNA mai nā holoholona multicellular e like me nā iʻa iwi, nā anemone kai, nā algae a me nā pepeke.I ka hopena, hōʻike kā mākou haʻawina e hiki ke hoʻohana pono ʻia ka manaʻo LB i nā invertebrates moana e hana i kahi repertoire genomic waiwai i nā kaiaola kai.
ʻO nā mākua (55-70 mm ka lōʻihi) Mytilus platensis (M. platensis) a me Aulacomya atra (A. atra) i hōʻiliʻili ʻia mai nā kahakai pōhaku intertidal o Port-au-France (049°21.235 S, 070°13.490 E.).ʻO nā mokupuni ʻo Kerguelen i Dekemaba 2018. Ua loaʻa mai nā ʻūlū polū makua ʻē aʻe (Mytilus spp.) mai kahi mea kūʻai aku (PEI Mussel King Inc., Prince Edward Island, Kanada) a waiho ʻia i loko o kahi pahu i hoʻopaʻa ʻia (4°C) aerated tank i loaʻa ka 10-20 L o 32‰ brine artificial.(paʻakai kai hana ʻia ʻo Reef Crystal, Instant Ocean, Virginia, USA).No kēlā me kēia hoʻokolohua, ua ana ʻia ka lōʻihi a me ke kaumaha o kēlā me kēia pūpū.
Loaʻa ka protocol open access no kēia polokalamu ma ka pūnaewele (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).I ka pōkole, ua hōʻiliʻili ʻia ʻo LB hemolymph mai nā ʻiʻo abductor e like me ka mea i wehewehe ʻia [22].Ua wehewehe ʻia ka hemolymph e ka centrifugation ma 1200 × g no 3 mau minuke, ua maloʻo ka supernatant (-20 ° C) a hiki i ka hoʻohana ʻana.No ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe ʻana i ka cfDNA, ua hoʻoheheʻe ʻia nā laʻana (1.5-2.0 ml) a hoʻohana ʻia me ka NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) e like me nā ʻōlelo a ka mea hana.Ua mālama ʻia ka ccfDNA ma -80°C a hiki i ka nānā hou ʻana.Ma kekahi mau hoʻokolohua, ua hoʻokaʻawale ʻia a hoʻomaʻemaʻe ʻia ka ccfDNA me ka hoʻohana ʻana i ka QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Canada).Ua helu ʻia ka DNA i hoʻomaʻemaʻe ʻia me ka hoʻohana ʻana i kahi hōʻike PicoGreen maʻamau.Ua kālailai ʻia ka māhele ʻāpana o ka ccfDNA i hoʻokaʻawale ʻia e ka electrophoresis capillary me ka hoʻohana ʻana i kahi bioanalyzer Agilent 2100 (Agilent Technologies Inc., Santa Clara, CA) me ka hoʻohana ʻana i kahi High Sensitivity DNA Kit.Hana ʻia ka hoʻāʻo me ka hoʻohana ʻana i ka 1 µl o ka laʻana ccfDNA e like me nā ʻōlelo a ka mea hana.
No ke kaʻina ʻana o nā ʻāpana hemolymph ccfDNA, ua hoʻomākaukau ʻo Génome Québec (Montreal, Quebec, Kanada) i nā hale waihona pū pana me ka hoʻohana ʻana i ka pahu Illumina DNA Mix o ka pahu Illumina MiSeq PE75.Ua hoʻohana ʻia kahi mea hoʻopili maʻamau (BioO).Loaʻa nā faila ʻikepili maka mai ka NCBI Sequence Read Archive (SRR8924808 a me SRR8924809).Ua loiloi ʻia ka maikaʻi heluhelu maʻamau me ka hoʻohana ʻana iā FastQC [23].Ua hoʻohana ʻia ʻo Trimmomatic [24] no ka hoʻopili ʻana i nā mea hoʻopili a me nā heluhelu maikaʻi ʻole.Ua hoʻohui ʻia ʻo FLASH i kahi heluhelu lōʻihi lōʻihi me ka liʻiliʻi o 20 bp e pale aku i nā mismatches [25]. Hoʻopili ʻia nā heluhelu i hoʻohui ʻia me BLASTN me ka hoʻohana ʻana i kahi waihona NCBI Taxonomy bivalve (e waiwai <1e−3 a me 90% homology), a ua hana ʻia ka masking o nā kaʻina haʻahaʻa haʻahaʻa me ka DUST [26]. Hoʻopili ʻia nā heluhelu i hoʻohui ʻia me BLASTN me ka hoʻohana ʻana i kahi waihona NCBI Taxonomy bivalve (e waiwai <1e−3 a me 90% homology), a ua hana ʻia ka masking o nā kaʻina haʻahaʻa haʻahaʻa me ka DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN с использованием базы данных таксономии двустворчорчослылюм 3 и 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ua helu ʻia nā heluhelu pooled me BLASTN me ka hoʻohana ʻana i ka NCBI bivalve taxonomy database (e waiwai <1e-3 a me 90% homology), a ua hana ʻia ka masking sequence haʻahaʻa me ka DUST [26].使用双壳类NCBI 分类数据库(e 值< 1e-3 和90% 同源性)用BLASTN 注释合并的读敽的读敽,并使盻并并使直复杂度序列的掩蔽。使用 双 壳类 ncbi 分类 (((<1e-3 和 90% 同源) 用 用 用 注释 合并 读数 , 读数 , 6用复杂度 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 掩蔽蔽Объединенные чтения были аннотированы с помощью BLASTN с использованием таксономической базы данных двозленичермча <1e-3 и 90% гомологии), а маскирование последовательностей низкой сложности было выполнено с использованием DUST [26]. Ua helu ʻia nā heluhelu pooled me BLASTN me ka hoʻohana ʻana i ka NCBI bivalve taxonomic database (e waiwai <1e-3 a me 90% homology), a ua hana ʻia ka masking sequence haʻahaʻa me ka hoʻohana ʻana i ka DUST [26].Ua māhele ʻia nā heluhelu i ʻelua pūʻulu: pili i nā kaʻina bivalve (i kapa ʻia ʻo ia ka heluhelu ponoʻī) a me ka pili ʻole (heluhelu ʻole ponoʻī).ʻElua mau pūʻulu i hui pū ʻia me ka hoʻohana ʻana i ka MEGAHIT e hana i nā contigs [27].I kēia manawa, ua hoʻokaʻawale ʻia ka ʻauhau ʻauhau o nā microbiome malihini e hoʻohana ana iā Kraken2 [28] a hōʻike ʻia i ke kiʻi kiʻi e kahi pakuhi Krona pie ma Galaxy [29, 30].Ua hoʻoholo ʻia nā kmers maikaʻi loa he kmers-59 mai kā mākou mau hoʻokolohua mua. Ua ʻike ʻia nā contigs ponoʻī e ka alignment me BLASTN (bivalve NCBI database, e waiwai <1e−10 a me 60% homology) no kahi hōʻike hope. Ua ʻike ʻia nā contigs ponoʻī e ka alignment me BLASTN (bivalve NCBI database, e waiwai <1e−10 a me 60% homology) no kahi hōʻike hope. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN (база данных двустворчатых моллюбиск, 1 меч ология 60%) для окончательной аннотации. A laila ʻike ʻia nā contigs ponoʻī ma ka hoʻohālikelike ʻana me BLASTN (NCBI bivalve database, e waiwai <1e-10 a me 60% homology) no ka hōʻike hope.BLASTN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 хдмун оллюсков, значение e <1e-10 и гомология 60%). A laila ua ʻike ʻia nā contigs ponoʻī no ka hōʻike hope loa ma ka hoʻohālikelike ʻana me BLASTN (NCBI bivalve database, e waiwai <1e-10 a me 60% homology). Ma ke ʻano like, ua hōʻike ʻia nā contigs nonself me BLASTN (nt NCBI database, e waiwai <1e−10 a me 60% homology). Ma ke ʻano like, ua hōʻike ʻia nā contigs nonself me BLASTN (nt NCBI database, e waiwai <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 like, ua helu ʻia nā contigs hui haole me BLASTN (NT NCBI database, e waiwai <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 даNCных 1 неглех nt 1 огия 60%). Ma ka like, ua hōʻike ʻia nā contigs hui ʻole me BLASTN (nt NCBI database, e waiwai <1e-10 a me 60% homology). Ua mālama ʻia ʻo BLASTX ma nā contigs nonself me ka hoʻohana ʻana i ka nr a me RefSeq protein NCBI databases (e waiwai <1e−10 a me 60% homology). Ua mālama ʻia ʻo BLASTX ma nā contigs nonself me ka hoʻohana ʻana i ka nr a me RefSeq protein NCBI databases (e waiwai <1e−10 a me 60% homology). BLASTX также был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI (значогение e <1e%)-10 e <1e. Ua hana ʻia ʻo BLASTX ma nā contigs non-self me ka hoʻohana ʻana i ka nr a me RefSeq NCBI protein database (e waiwai <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 pū ʻia ʻo BLASTX ma nā contigs non-self me ka hoʻohana ʻana i ka nr a me RefSeq NCBI protein databases (e waiwai <1e-10 a me 60% homology).ʻO nā loko BLASTN a me BLASTX o non-self-contigs e hōʻike ana i nā contigs hope loa (e ʻike i ka faila hoʻohui).
Hoʻopaʻa ʻia nā mea mua i hoʻohana ʻia no PCR ma ka Papa S1.Ua hoʻohana ʻia ʻo Taq DNA polymerase (Bio Basic Canada, Markham, ON) no ka hoʻonui ʻana i nā genes target ccfDNA.Ua hoʻohana ʻia kēia mau ʻano hopena: denaturation ma 95°C no 3 mau minuke, 95°C no 1 minuke, hoʻonoho i ka mahana annealing no 1 minuke, elongation ma 72°C no 1 minuke, 35 mau pōʻaiapuni, a hope loa 72°C i loko o 10 mau minuke..Ua hoʻokaʻawale ʻia nā huahana PCR e ka electrophoresis i nā gels agarose (1.5%) i loaʻa iā SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Canada) ma 95 V.
Ua hoʻomaʻamaʻa ʻia nā ʻū (Mytilus spp.) i loko o 500 ml wai kai i hoʻonaʻauao ʻia (32 PSU) no 24 mau hola ma 4°C.ʻO ka DNA Plasmid i loaʻa kahi mea hoʻokomo e hoʻopili ana i ke kaʻina cDNA galectin-7 kanaka (NCBI accession number L07769) i hoʻohui ʻia i ka vial i ka hopena hope o 190 μg / μl.ʻO nā mussels i hoʻomoʻa ʻia ma lalo o nā kūlana like me ka hoʻohui ʻole ʻana i ka DNA ʻo ia ka mana.ʻO ke kolu o ka pahu hoʻomalu i loaʻa ka DNA me ka ʻole o nā ʻū.No ka nānā ʻana i ka maikaʻi o ka DNA i loko o ka wai kai, lawe ʻia nā laʻana wai kai (20 μl; ʻekolu repetitions) mai kēlā me kēia pahu i ka manawa i kuhikuhi ʻia.No ka plasmid DNA traceability, LB mussels ua ʻohi ʻia i nā manawa i hōʻike ʻia a nānā ʻia e qPCR a me ddPCR.Ma muli o ke kiʻekiʻe o ka paʻakai o ka wai kai, ua hoʻoheheʻe ʻia nā aliquot i ka wai maikaʻi PCR (1:10) ma mua o nā hoʻokolohua PCR āpau.
Ua hana ʻia ʻo Digital droplet PCR (ddPCR) me ka hoʻohana ʻana i ka protocol BioRad QX200 (Mississauga, Ontario, Canada).E hoʻohana i ka ʻike wela no ka hoʻoholo ʻana i ka wela maikaʻi loa (Pakaukau S1).Ua hana ʻia nā hāʻule me ka hoʻohana ʻana i kahi mea hana hāʻule QX200 (BioRad).Ua hana ʻia ka ddPCR penei: 95 ° C no 5 min, 50 pōʻai 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 hāʻule a me nā hopena maikaʻi (ka helu o nā kope/µl) me ka hoʻohana ʻana i kahi mea heluhelu hāʻule QX200 (BioRad).Ua hōʻole ʻia nā laʻana me ka liʻiliʻi o 10,000 droplets.ʻAʻole i hoʻokō ʻia ka mana hoʻohālike i kēlā me kēia manawa i holo ai ka ddPCR.
Ua hana ʻia ʻo qPCR me ka hoʻohana ʻana i ka Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) a me LGALS7 mau mea kikoʻī.Hana ʻia nā PCR quantitative a pau ma 20 µl me ka QuantiFast SYBR Green PCR Kit (QIAGEN).Ua hoʻomaka ʻia ka qPCR me kahi incubation 15 min ma 95 ° C a ukali ʻia e 40 cycles ma 95 ° C no 10 kekona a ma 60 ° C no 60 kekona me hoʻokahi hōʻiliʻili ʻikepili.Hoʻokumu ʻia nā ʻāpana hoʻoheheʻe me ka hoʻohana ʻana i nā ana holomua 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 i triplicate, koe wale no nā laʻana mana.
No ka mea, ʻike ʻia ka mussel no ka nui o ka kānana kānana, ua noiʻi mua mākou inā hiki iā lākou ke kānana a mālama i nā ʻāpana DNA i loko o ke kai.Ua hoihoi pū mākou i ka hōʻiliʻili ʻana o kēia mau ʻāpana i kā lākou ʻōnaehana lymphatic semi-open.Ua hoʻoholo mākou i kēia pilikia me ka hoʻokolohua ʻana ma ka ʻimi ʻana i ka hopena o nā ʻāpana DNA hiki ke hoʻohui ʻia i nā pahu mussel uliuli.No ka hoʻomaʻamaʻa ʻana i nā ʻāpana DNA, ua hoʻohana mākou i ka DNA plasmid haole (ʻaʻole iā ʻoe iho) i loaʻa ka gene galectin-7 kanaka.ʻIke ʻo ddPCR i nā ʻāpana DNA plasmid i loko o ke kai a me nā ʻū.Hōʻike kā mākou mau hualoaʻa inā mau ka nui o nā ʻāpana DNA i loko o ka wai kai i ka manawa (a hiki i nā lā 7) i ka loaʻa ʻole o nā mussels, a laila ma ke alo o nā ʻōpala ua aneane nalo loa kēia pae i loko o nā hola 8 (Fig. 1a,b).Ua ʻike maʻalahi nā ʻāpana o ka DNA exogenous i loko o 15 min i loko o ka wai intravalvular a me ka hemolymph (Fig. 1c).Hiki ke ʻike ʻia kēia mau ʻāpana a hiki i 4 mau hola ma hope o ka hoʻolaha ʻana.Hoʻohālikelike ʻia kēia hana kānana e pili ana i nā ʻāpana DNA me ka hana kānana o nā bacteria a me nā algae [31].Hōʻike kēia mau hualoaʻa e hiki i nā mussels ke kānana a hōʻiliʻili i ka DNA haole i loko o kā lākou mau keʻena wai.
ʻO ka pilina pili o ka DNA plasmid i loko o ka wai kai ma ke alo (A) a i ʻole (B) o nā mussels, ana ʻia e ddPCR.Ma A, hōʻike ʻia nā hopena ma ke ʻano he pākēneka, me nā palena o nā pahu e hōʻike ana i ka 75th a me 25th percentile.Hōʻike ʻia ka ʻōkuhi logarithmic kūpono i ka ʻulaʻula, a ʻo ka wahi i uhi ʻia i ka hinahina e hōʻike ana i ka 95% o ka manawa hilinaʻi.Ma B, hōʻike ka laina ʻulaʻula i ka mean a ʻo ka laina polū e hōʻike ana i ka manawa hilinaʻi 95% no ka noʻonoʻo.C Ka hōʻiliʻili ʻana o ka DNA plasmid i ka hemolymph a me ka wai valvular o nā mussels i nā manawa like ʻole ma hope o ka hoʻohui ʻana o ka plasmid DNA.Hōʻike ʻia nā hopena ma ke ʻano he kope piha i ʻike ʻia/mL (± SE).
Ma hope aʻe, ua noiʻi mākou i ke kumu o ka ccfDNA i nā mussel i hōʻiliʻili ʻia mai nā moena mussel ma nā mokupuni ʻo Kerguelen, kahi pūʻulu mamao o nā mokupuni me ka mana anthropogenic palena ʻole.No kēia kumu, ua hoʻokaʻawale ʻia a hoʻomaʻemaʻe ʻia ka cccDNA mai nā hemolymph mussel e nā ʻano hana maʻamau e hoʻomaʻemaʻe i ka cccDNA kanaka [32, 33].Ua ʻike mākou ʻo ka awelika hemolymph ccfDNA i loko o nā mussels i loko o nā micrograms haʻahaʻa no kēlā me kēia ml hemolymph range (e nānā i ka Papa S2, ʻIke Hoʻohui).ʻOi aku ka nui o kēia ʻano o ka ʻike ma mua o ka poʻe olakino (nā nanograms haʻahaʻa i kēlā me kēia milliliter), akā i nā hihia koʻikoʻi, i nā maʻi maʻi maʻi, hiki ke kiʻekiʻe o ka ccfDNA ke piʻi i kekahi mau micrograms no ka milliliter [34, 35].Ua hōʻike ʻia kahi loiloi o ka puʻunaue nui o ka hemolymph ccfDNA he ʻokoʻa ka nui o kēia mau ʻāpana, mai ka 1000 bp a i ka 1000 bp.a hiki i 5000 bp (Fig. 2).Ua loaʻa nā hopena like me ka hoʻohana ʻana i ka silica-based QIAamp Investigator Kit, kahi ʻano hana maʻamau i ka ʻepekema forensic e hoʻokaʻawale wikiwiki a hoʻomaʻemaʻe i ka DNA genomic mai nā haʻawina DNA haʻahaʻa haʻahaʻa, me ka ccfDNA [36].
Lunamakaainana ccfDNA electrophoregram o mussel hemolymph.Wehe ʻia me NucleoSnap Plasma Kit (luna) a me QIAamp DNA Investigator Kit.B ʻO ka ʻāpana violin e hōʻike ana i ka puʻunaue ʻana o ka hemolymph ccfDNA ʻona (± SE) i loko o nā ʻū.ʻO nā laina ʻeleʻele a me ka ʻulaʻula e hōʻike ana i ka median a me ka quartile mua a me ke kolu.
Ma kahi o 1% o ccfDNA i loko o nā kānaka a me nā primates he kumu haole [21, 37].Hāʻawi ʻia i ka ʻōnaehana kaʻapuni semi-open o nā bivalves, ka wai kai waiwai nui i ka microbial, a me ka puʻunaue nui o ka mussel ccfDNA, manaʻo mākou e loaʻa paha i ka mussel hemolymph ccfDNA kahi punawai waiwai a ʻokoʻa o ka DNA microbial.No ka hoʻāʻo ʻana i kēia kuhiakau, ua hoʻokaʻina mākou i ka hemolymph ccfDNA mai nā laʻana o Aulacomya atra i hōʻiliʻili ʻia mai nā mokupuni ʻo Kerguelen, e loaʻa ana ma luna o 10 miliona mau heluhelu, 97.6% o ia mea i hala i ka mana maikaʻi.Hoʻokaʻawale ʻia nā heluhelu e like me nā kumu ponoʻī a me nā kumu ʻole me ka hoʻohana ʻana i ka BLASTN a me NCBI bivalve databases (Fig. S1, ʻIke Hoʻohui).
I loko o nā kānaka, hiki ke hoʻokuʻu ʻia ka DNA nuklea a me ka mitochondrial i loko o ke kahe koko [38].Eia nō naʻe, i kēia haʻawina, ʻaʻole hiki ke wehewehe kikoʻī i ka DNA genomic nuklea o nā mussels, ʻoiai ʻaʻole i hoʻonohonoho ʻia a wehewehe ʻia ka genome A. atra.Eia naʻe, ua hiki iā mākou ke ʻike i kekahi mau ʻāpana ccfDNA o ko mākou kumu ponoʻī me ka hoʻohana ʻana i ka waihona bivalve (Fig. S2, ʻIke Hoʻohui).Ua hōʻoia pū mākou i ka loaʻa ʻana o nā ʻāpana DNA o ko mākou kumu ponoʻī ma o ka hoʻonui ʻia ʻana o PCR o kēlā mau genes A. atra i hoʻonohonoho ʻia (Fig. 3).Pēlā nō, no ka loaʻa ʻana o ka mitochondrial genome o A. atra i loko o nā ʻikepili lehulehu, hiki i kekahi ke ʻike i nā hōʻike no ka loaʻa ʻana o nā ʻāpana mitochondrial ccfDNA i ka hemolymph o A. atra.Ua hōʻoia ʻia ka hele ʻana o nā ʻāpana DNA mitochondrial e ka PCR amplification (Fig. 3).
Loaʻa nā ʻano genes mitochondrial i ka hemolymph o A. atra (nā kiko ʻulaʻula - helu helu: SRX5705969) a me M. platensis (nā kiko uliuli - helu helu: SRX5705968) i hoʻonui ʻia e PCR.Kiʻi i hoʻololi ʻia mai Breton et al., 2011 B Hoʻonui ʻia o ka hemolymph supernatant mai A. atra i mālama ʻia ma ka pepa FTA.E hoʻohana i ka punch 3 mm e hoʻohui pololei i ka paipu PCR i loko o ka hui PCR.
Ma muli o ka nui o ka microbial content i loko o ka wai kai, ua nānā mua mākou i ke ʻano o ka microbial DNA sequences i hemolymph.No ka hana ʻana i kēia, hoʻohana mākou i ʻelua mau hoʻolālā like ʻole.ʻO ka hoʻolālā mua i hoʻohana iā Kraken2, kahi papahana hoʻonohonoho hoʻonohonoho hoʻonohonoho algorithm e hiki ke ʻike i nā kaʻina microbial me ka pololei e like me BLAST a me nā mea hana ʻē aʻe [28].ʻOi aku ma mua o 6719 heluhelu i hoʻoholo ʻia he kumu bacteria, ʻoiai ʻo 124 a me 64 mai archaea a me nā maʻi maʻi (Fig. 4).ʻO nā ʻāpana DNA bacterial nui loa he Firmicutes (46%), Proteobacteria (27%), a me Bacteroidetes (17%) (Fig. 4a).Ua kūlike kēia māhele me nā haʻawina mua o ka microbiome mussel blue moana [39, 40].ʻO Gammaproteobacteria ka papa nui o Proteobacteria (44%), me nā Vibrionales he nui (Fig. 4b).Ua hōʻoia ke ala ddPCR i ka hiki ʻana o nā ʻāpana Vibrio DNA i ka ccfDNA o A. atra hemolymph (Fig. 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 (Fig. S2, ʻIke Hoʻohui). I kēia hihia, heluhelu ʻia ua ʻākoakoa ʻia nā overlapped e like me ka heluhelu ʻana i ka lua-hope a ua helu ʻia ʻo ia iho (bivalves) a i ʻole kumu kumu ʻole me ka hoʻohana ʻana i ka BLASTN a me kahi waiwai e o 1e−3 a me kahi ʻoki me ka> 90% homology. I kēia hihia, heluhelu ʻia ua ʻākoakoa ʻia nā overlapped e like me ka heluhelu ʻana i ka lua-hope a ua helu ʻia ʻo ia iho (bivalves) a i ʻole kumu kumu ʻole me ka hoʻohana ʻana i ka 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 какдлуныество юски) или чужие по происхождению с использованием BLASTN и значения e 1e-3 и отсечения с гомологией> 90%. I kēia hihia, ua ʻohi ʻia nā mea heluhelu e like me nā heluhelu i hoʻopau ʻia a ua helu ʻia ma ke ʻano he kamaʻāina (bivalve) a i ʻole kumu maoli me ka hoʻohana ʻana i ka BLASTN a me ka waiwai e o 1e-3 a ʻoki ʻia me >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 会咐>90% 我为为自身(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9 和 1 % 和 1%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 собствент и) или несобственные по происхождению с использованием значений e BLASTN и 1e-3 и порога гомологии> 90%. I kēia hihia, ua hōʻiliʻili ʻia nā mea heluhelu e like me nā heluhelu i hoʻopau ʻia a hoʻokaʻawale ʻia ma ke ʻano he ponoʻī (bivalves) a i ʻole kumu maoli me ka hoʻohana ʻana i nā waiwai e BLASTN a me 1e-3 a me kahi paepae homology>90%.No ka mea ʻaʻole i hoʻonohonoho ʻia ka genome A. atra, ua hoʻohana mākou i ka hoʻolālā hui de novo o ka MEGAHIT Next Generation Sequencing (NGS) assembler.Ua ʻike ʻia he 147,188 contigs he hilinaʻi (bivalves) o ke kumu.A laila ua pahū ʻia kēia mau contigs me nā e-waiwai o 1e-10 me ka hoʻohana ʻana i BLASTN a me BLASTX.Ua ʻae kēia hoʻolālā iā mākou e ʻike i nā ʻāpana non-bivalve 482 i loaʻa ma A. atra ccfDNA.ʻOi aku ma mua o ka hapalua (57%) o kēia mau ʻāpana DNA i loaʻa mai ka bacteria, ka nui mai nā hōʻailona gill, me nā hōʻailona sulfotrophic, a mai nā hōʻailona gill Solemya velum (Fig. 5).
Ka nui pili ma ka pae ʻano.B Ka ʻokoʻa microbial o ʻelua phyla nui (Firmicutes a me Proteobacteria).ʻO ka hoʻonui ʻana o ka ddPCR C Vibrio spp.A. Nā ʻāpana o ka 16S rRNA gene (uliuli) i ʻekolu atra hemolymphs.
He 482 mau contigs i hōʻiliʻili ʻia.ʻO ka moʻolelo maʻamau o ka puʻunaue ʻauhau 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 ʻia ka loiloi Kraken2 aia i loko o ka mussel ccfDNA nā ʻāpana DNA archaeal, me nā ʻāpana DNA o Euryarchaeota (65%), Crenarchaeota (24%), a me Thaurmarcheota (11%) (Fig. 6a).ʻO ka loaʻa ʻana o nā ʻāpana DNA i loaʻa mai Euryarchaeota a me Crenarchaeota, i ʻike mua ʻia i loko o ke kaiāulu microbial o Californian mussels, ʻaʻole ia he mea kupanaha [42].ʻOiai pili pinepine ʻia ʻo Euryarchaeota me nā kūlana koʻikoʻi, ua ʻike ʻia i kēia manawa ʻo Euryarchaeota a me Crenarcheota kekahi o nā prokaryotes maʻamau i loko o ke kaiapuni cryogenic moana [43, 44].ʻAʻole he mea kupanaha ka loaʻa ʻana o nā microorganisms methanogenic i loko o nā mussels, no ka mea, ua hōʻike ʻia nā hōʻike hou e pili ana i ka nui o ka methane leaks mai lalo mai ma ka Plateau Kerguelen [45] a me ka hana ʻana o ka microbial methane i ʻike ʻia ma ke kahakai o nā mokupuni ʻo Kerguelen [46].
Ua neʻe ko mākou manaʻo i ka heluhelu ʻana mai nā maʻi DNA.I ka maikaʻi o ko mākou ʻike, ʻo kēia ka noiʻi mua ʻana i waho o ka maʻi maʻi o nā ʻū.E like me ka mea i manaʻo ʻia, ua loaʻa iā mākou nā ʻāpana DNA o bacteriophages (Caudovirales) (Fig. 6b).Eia nō naʻe, loaʻa mai ka DNA viral maʻamau mai kahi phylum o nucleocytoviruses, i ʻike pū ʻia ʻo ka nuclear cytoplasmic large DNA virus (NCLDV), nona ka genome nui loa o kēlā me kēia maʻi.Ma loko o kēia phylum, ʻo ka hapa nui o nā kaʻina DNA no nā ʻohana Mimimidoviridae (58%) a me Poxviridae (21%), nona nā pūʻali kūlohelohe i nā vertebrates a me nā arthropods, ʻoiai ʻo kahi hapa liʻiliʻi o kēia mau kaʻina DNA no nā algae virological i ʻike ʻia.Hoʻopilikia i nā limu eukaryotic moana.Loaʻa ʻia nā kaʻina mai ka maʻi Pandora, ka maʻi pilikua me ka nui genome nui o nā genera viral i ʻike ʻia.ʻO ka mea hoihoi, ʻo ka laulā o nā pūʻali i ʻike ʻia ua loaʻa i ka maʻi maʻi, e like me ka mea i hoʻoholo ʻia e ka hemolymph ccfDNA sequencing, ua ʻano nui (Figure S3, ʻIke Hoʻohui).Loaʻa iā ia nā maʻi maʻi e hoʻopili ai i nā pepeke e like me Baculoviridae a me Iridoviridae, a me nā maʻi maʻi e hoʻopili ai i ka amoeba, algae a me nā vertebrates.Ua ʻike pū mākou i nā kaʻina e pili ana i ka genome Pithovirus sibericum.Ua hoʻokaʻawale mua ʻia nā Pitoviruses (i ʻike ʻia ʻo "zombie viruses") mai 30,000 mau makahiki permafrost ma Siberia [47].No laila, ua kūlike kā mākou hopena me nā hōʻike mua e hōʻike ana ʻaʻole pau nā ʻano ʻano hou o kēia mau maʻi maʻi [48] a aia paha kēia mau maʻi i loko o nā kaiaola kai subarctic mamao.
ʻO ka hope, ua hoʻāʻo mākou e ʻike inā hiki iā mākou ke ʻike i nā ʻāpana DNA mai nā holoholona multicellular ʻē aʻe.Ua ʻike ʻia he 482 contigs haole e BLASTN a me 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 ka ccfDNA o nā holoholona multicellular DNA o nā iwi iwi i mua (Fig. 5).Ua ʻike pū ʻia nā ʻāpana DNA mai nā iniseti a me nā ʻano ʻē aʻe.ʻAʻole i ʻike ʻia kahi hapa nui o nā ʻāpana DNA, ma muli paha o ka hōʻike ʻole ʻana o ka nui o nā ʻano kai i loko o nā waihona genomic i hoʻohālikelike ʻia me nā ʻano honua [49].
Ma ka pepa o kēia manawa, hoʻohana mākou i ka manaʻo LB i nā ʻiʻo, me ka hoʻopaʻapaʻa ʻana e hiki i ka hemolymph ccfDNA shot sequencing ke hāʻawi i ka ʻike i ke ʻano o nā kaiaola kai moana.ʻO ka mea nui, ua ʻike mākou he 1) mussel hemolymph i loaʻa i nā ʻano kiʻekiʻe kiʻekiʻe (mau microgram pae) o nā ʻāpana DNA nui (~1-5 kb) kaʻapuni;2) He kūʻokoʻa a kūʻokoʻa ʻole kēia mau ʻāpana DNA.4) ʻO ka hōʻiliʻili ʻana o kēia mau ʻāpana ccfDNA haole i loko o ka hemolymph hiki koke a kōkua i ka hana kānana kūloko o nā ʻū.I ka hopena, hōʻike kā mākou noiʻi ʻana i ka manaʻo o LB, ka mea i hoʻohana nui ʻia ma ke kahua o ka biomedicine, hoʻopili i kahi kumu waiwai akā ʻike ʻole ʻia e hiki ke hoʻohana ʻia e hoʻomaopopo maikaʻi i ka pilina ma waena o nā ʻano sentinel a me ko lākou kaiapuni.
Ma kahi o nā primates, ua hōʻike ʻia ka hoʻokaʻawale ʻana o ccfDNA i nā mammals, me nā ʻiole, nā ʻīlio, nā pōpoki, a me nā lio [50, 51, 52].Eia nō naʻe, i ko mākou ʻike, ʻo kā mākou haʻawina ka mea mua e hōʻike i ka ʻike a me ka hoʻonohonoho ʻana o ccfDNA i nā ʻano kai me kahi ʻōnaehana hāmama.Hiki i kēia hiʻohiʻona anatomical a me ka hiki ke kānana ʻana o nā mussels, ma ka liʻiliʻi loa, e wehewehe i nā ʻano nui like ʻole o ka ʻāpana DNA e hoʻohālikelike ʻia me nā ʻano 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 ka nui mai 150 a 200 bp.me ka piko kiʻekiʻe o 167 bp [34, 53].ʻO kahi hapa liʻiliʻi akā nui o nā ʻāpana DNA ma waena o 300 a 500 bp ka nui, a ma kahi o 5% ka lōʻihi ma mua o 900 bp.[54].ʻO ke kumu o kēia puʻunaue nui ʻo ia ke kumu nui o ka ccfDNA i loko o ka plasma ma muli o ka make cell, ma muli o ka make cell a i ʻole ma muli o ka necrosis o ka holo ʻana o nā cell hematopoietic i nā poʻe olakino a i ʻole ma muli o ka apoptosis o nā pūnana tumo i nā maʻi maʻi maʻi (i ʻike ʻia ʻo DNA circulating tumor)., ctDNA).ʻO ka puʻunaue nui o ka hemolymph ccfDNA i loaʻa iā mākou ma nā mussels mai 1000 a 5000 bp, e manaʻo ana he kumu ʻokoʻa ka mussel ccfDNA.He kuhiakau kūpono kēia, no ka mea, he semi-open vascular system ka mussels a noho i loko o nā kaiaola kai i loaʻa nā kiʻekiʻe o ka microbial genomic DNA.ʻO ka ʻoiaʻiʻo, ua hōʻike kā mākou mau hoʻokolohua hoʻokolohua me ka hoʻohana ʻana 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 e hoʻohaʻahaʻa ʻia ma hope o ka lawe ʻia ʻana o ke kelepona a/a i hoʻokuʻu ʻia a mālama ʻia i loko o nā hui like ʻole.Hāʻawi ʻia i ka liʻiliʻi o nā cell (prokaryotic a me eukaryotic), ʻo ka hoʻohana ʻana i nā keʻena intravalvular e hōʻemi i ka nui o ka ccfDNA mai nā kumu ponoʻī a me nā kumu haole.No ka noʻonoʻo ʻana i ke koʻikoʻi o ka bivalve innate immunity a me ka nui o nā phagocytes e holo nei, ua manaʻo hou mākou ua hoʻonui ʻia ka ccfDNA haole i nā phagocytes e hōʻiliʻili ana i ka DNA haole ma ka ʻai ʻana i nā microorganisms a/a i ʻole nā ʻōpala pūnaewele.Hoʻohui pū ʻia, hōʻike kā mākou mau hopena i ka bivalve hemolymph ccfDNA kahi waihona kūʻokoʻa o ka ʻike molekala a hoʻoikaika i ko lākou kūlana ma ke ʻano he ʻano mea mālama.
Hōʻike kā mākou ʻikepili i ka hoʻopili ʻana a me ka nānā ʻana i nā ʻāpana hemolymph ccfDNA i loaʻa mai i ka bacteria e hiki ke hāʻawi i ka ʻike koʻikoʻi e pili ana i ka flora bacterial host a me ka bacteria i loaʻa i loko o ka kaiaola kai a puni.Ua hōʻike ʻia nā ʻenehana hoʻopaʻa kiʻi ʻana i nā kaʻina o ka bacteria commensal A. atra gill i nalo inā i hoʻohana ʻia nā ʻano hana ʻike 16S rRNA maʻamau, ma muli o ke ʻano o ka waihona puke.ʻO ka ʻoiaʻiʻo, ʻo kā mākou hoʻohana ʻana i ka ʻikepili LB i hōʻiliʻili ʻia mai M. platensis i loko o ka papa mussel ma Kerguelen i hōʻike ʻia he like ke ʻano o nā symbionts bacteria e pili ana i ka gill no nā ʻano mussel ʻelua (Fig. S4, ʻIke Hoʻohui).Hiki ke hoʻohālikelike kēia ʻano like o ʻelua mau ʻano ʻano ʻano like ʻole i ke ʻano o nā kaiāulu bacteria i ke anuanu, sulfurous, a me ka lua pele o Kerguelen [55, 56, 57, 58].Ua wehewehe maikaʻi ʻia ka pae kiʻekiʻe o nā microorganisms hoʻemi i ka sulfur i ka wā e ʻohi ai i nā ʻū mai nā wahi kai bioturbated [59], e like me ke kahakai o Port-au-France.ʻO kekahi mea ʻē aʻe e pili ana i ka flora mussel commensal e ka hoʻouna ʻana i ka pae [60, 61].Pono e noiʻi hou aʻe no ka hoʻoholo ʻana i ka pilina ma waena o ke kaiapuni moana, ka ʻili o ke kai, a me ka hoʻohui ʻana o nā hua bacteria symbiotic i loko o nā ʻū.Ke hoʻomau nei kēia mau haʻawina i kēia manawa.
ʻO ka lōʻihi a me ka noʻonoʻo ʻana o ka hemolymph ccfDNA, kona maʻalahi o ka hoʻomaʻemaʻe ʻana, a me ke ʻano kiʻekiʻe e ʻae ai i ka hoʻopili wikiwiki ʻana i ka pū pana pua kekahi o nā mea maikaʻi loa o ka hoʻohana ʻana i ka mussel ccfDNA e loiloi i ke ʻano biodiversity i nā kaiaola kai moana.ʻOi aku ka maikaʻi o kēia ala no ka ʻike ʻana i nā kaiāulu viral (virome) i loko o kahi kaiaola i hāʻawi ʻia [62, 63].ʻAʻole like me ka bacteria, archaea, a me nā eukaryotes, ʻaʻole i loaʻa i nā genome viral nā genes phylogenetically conserved e like me 16S sequences.Hōʻike kā mākou mau hopena e hiki ke hoʻohana ʻia nā biopsies wai mai nā ʻano mea hōʻike e like me nā ʻūhā no ka ʻike ʻana i ka nui o nā ʻāpana virus ccfDNA i ʻike ʻia e hoʻopilikia i nā pūʻali e noho maʻamau i nā kaiaola kai kai.Loaʻa kēia i nā maʻi maʻi i ʻike ʻia e hoʻopilikia i ka protozoa, nā arthropods, nā inike, nā mea kanu, a me nā maʻi maʻi bacteria (e laʻa, bacteriophages).Ua ʻike ʻia ka puʻunaue like i ka wā i nānā ai mākou i ka hemolymph ccfDNA virome o nā ʻūlū polū (M. platensis) i hōʻiliʻili ʻia ma ka papa mussel ma Kerguelen (Papa S2, ʻIke Hoʻohui).ʻO ke kaʻina hana pana o ccfDNA he ala hou e loaʻa ai ka manawa i ka noiʻi ʻana i ka virome o nā kānaka a i ʻole nā mea ʻē aʻe [21, 37, 64].He mea maikaʻi loa kēia ala no ke aʻo ʻana i nā maʻi maʻi DNA kaulua, no ka mea, ʻaʻohe gene i mālama ʻia i waena o nā maʻi maʻi DNA pālua ʻelua, e hōʻike ana i ka ʻano like ʻole a ākea o nā maʻi ma Baltimore [65].ʻOiai ʻo ka hapa nui o kēia mau maʻi maʻi ʻaʻole i hoʻokaʻawale ʻia a hiki ke komo i nā maʻi mai kahi ʻāpana ʻike ʻole ʻia o ka honua viral [66], ua ʻike mākou ua hāʻule nā viromes a me nā pae hoʻokipa o nā mussels A. atra a me M. platensis 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 i ka hemahema o ke koho ʻana i ka lawe ʻana i ka DNA i loko o ke kaiapuni.Pono nā haʻawina e hiki mai ana e hoʻohana ana i ka RNA i hoʻomaʻemaʻe ʻia i kēia manawa e ʻike i ka RNA virome.
Ma kā mākou noiʻi ʻana, ua hoʻohana mākou i kahi pipeline koʻikoʻi loa i hoʻololi ʻia mai ka hana a Kowarski a me nā hoa hana [37], nāna i hoʻohana i ʻelua mau ʻanuʻu i ka holoi ʻana i nā heluhelu i hoʻohui ʻia a me nā contigs ma mua a ma hope o ka hui ʻana o ka ccfDNA maoli, e hopena i ka helu kiʻekiʻe o ka heluhelu ʻole ʻia.No laila, ʻaʻole hiki iā mākou ke hōʻole i ka loaʻa ʻana o kekahi o kēia mau heluhelu i palapala ʻole ʻia, no ka mea 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 chimeras ma waena o ka heluhelu ponoʻī a me ka heluhelu ʻole a me nā lōʻihi heluhelu i hana ʻia e ka Illumina MiSeq PE75.ʻO ke kumu ʻē aʻe o ka hapa nui o nā heluhelu ʻike ʻole ʻia ʻo ia ka hapa nui o nā microbes moana, ʻoi loa ma nā wahi mamao e like me Kerguelen, ʻaʻole i hōʻike ʻia.Ua hoʻohana mākou iā Illumina MiSeq PE75, me ka manaʻo e like ka lōʻihi o ka ʻāpana ccfDNA me ka ccfDNA kanaka.No nā haʻawina e hiki mai ana, i hāʻawi ʻia i kā mākou mau hopena e hōʻike ana he lōʻihi ka heluhelu ʻana o ka hemolymph ccfDNA ma mua o ke kanaka a/a i ʻole nā mammals, manaʻo mākou e hoʻohana i kahi papa kuhikuhi i kūpono no nā ʻāpana ccfDNA lōʻihi.E maʻalahi kēia hana i ka ʻike ʻana i nā hōʻailona hou aʻe no ka nānā hohonu ʻana.ʻO ka loaʻa ʻana o ke kaʻina genome nuklea A. atra i loaʻa ʻole i kēia manawa e maʻalahi hoʻi i ka hoʻokae ʻana i ka ccfDNA mai nā kumu ponoʻī a me nā kumu ʻole ponoʻī.Ma muli o ka nānā ʻana o kā mākou noiʻi i ka hiki ke hoʻohana i ka manaʻo o ka biopsy wai i nā mussels, ke manaʻolana nei mākou e like me ka hoʻohana ʻia ʻana o kēia manaʻo i ka noiʻi e hiki mai ana, e hoʻomohala ʻia nā mea hana hou a me nā pipeline e hoʻonui i ka hiki o kēia ʻano e aʻo ai i ka ʻokoʻa microbial o nā mussels.kaiaolaola moana.
Ma keʻano he biomarker clinical non-invasive, kiʻekiʻe nā kiʻekiʻe plasma kanaka o ccfDNA e pili ana me nā maʻi likeʻole, kaʻino kino, a me nā kūlana kaumaha [67,68,69].Hoʻopili ʻia kēia piʻi ʻana me ka hoʻokuʻu ʻana i nā ʻāpana DNA o kona kumu ponoʻī ma hope o ka pōʻino o ke kino.Ua hoʻoponopono mākou i kēia pilikia me ka hoʻohana ʻana i ke koʻikoʻi wela, kahi i hōʻike pōkole ʻia ai nā mussel i kahi mahana o 30 °C.Ua hana mākou i kēia loiloi ma nā ʻano ʻano ʻano ʻano ʻokoʻa ʻekolu i loko o ʻekolu mau hoʻokolohua kūʻokoʻa.Akā naʻe, ʻaʻole i loaʻa iā mākou kekahi hoʻololi i nā pae ccfDNA ma hope o ke koʻikoʻi wela (e nānā i ke Kiʻi S5, ʻike hou aku).Hiki i kēia ʻike ke wehewehe, ma ka liʻiliʻi loa, i ka ʻoiaʻiʻo o ka mussels he semi-open circulatory system a hōʻiliʻili nui i nā DNA haole ma muli o kā lākou hana kānana kiʻekiʻe.Ma ka ʻaoʻao ʻē aʻe, ʻoi aku ka paʻa ʻana o nā mussels, e like me ka nui o nā invertebrates, i ka hōʻino ʻana o ka ʻiʻo i hoʻoulu ʻia i ke koʻikoʻi, a laila e kaupalena ʻia ka hoʻokuʻu ʻana o ccfDNA i ko lākou hemolymph [70, 71].
I kēia lā, ua kālele nui ʻia ka nānā ʻana o DNA o ka biodiversity i loko o nā kaiaola wai i ka metabarcoding DNA kaiapuni (eDNA).Eia nō naʻe, kaupalena ʻia kēia ʻano hana i ka loiloi biodiversity ke hoʻohana ʻia nā primers.ʻO ka hoʻohana ʻana i ke kaʻina pana pana e hoʻopuni i nā palena o ka PCR a me ke koho ʻana o nā pūʻulu mua.No laila, ma kahi ʻano, ʻoi aku ka pili o kā mākou ala i ke ʻano kiʻekiʻe i hoʻohana ʻia eDNA Shotgun sequencing method, hiki iā ia ke hoʻonohonoho pololei i ka DNA fragmented a nānā i nā mea ola āpau [72, 73].Eia nō naʻe, aia kekahi mau pilikia koʻikoʻi e hoʻokaʻawale i ka LB mai nā ala eDNA maʻamau.ʻOiaʻiʻo, ʻo ka ʻokoʻa nui ma waena o eDNA a me LB ka hoʻohana ʻana i nā pūʻali 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 naʻe, ua hoʻohana ʻo Dreissena i nā biopsies kiko kahi i lawe ʻia ai ka DNA.ʻAʻole koi ka nānā ʻana i ka ccfDNA mai LB i ka biopsy kiko, nā lako kūikawā a i kekahi manawa pipiʻi a me nā logistic pili me ka eDNA a i ʻole biopsy kiko.ʻO kaʻoiaʻiʻo, ua hōʻike koke mākou e hiki ke mālama a nānā ʻia ka ccfDNA mai LB me ke kākoʻo FTA me ka mālama ʻole ʻana i ke kaulahao anu, ʻo ia ka paʻakikī nui no ka noiʻi ʻana ma nā wahi mamao [76].He maʻalahi hoʻi ka unuhi ʻana o ccfDNA mai nā wai biopsies a hāʻawi i ka DNA kiʻekiʻe kiʻekiʻe no ke kaʻina pana pana a me ka nānā ʻana PCR.He pōmaikaʻi nui kēia i hāʻawi ʻia i kekahi o nā palena ʻenehana e pili ana i ka nānā ʻana eDNA [77].ʻO ka maʻalahi a me ke kumu kūʻai haʻahaʻa o ke ʻano hoʻohālike e kūpono hoʻi no nā papahana nānā lōʻihi.Ma waho aʻe o ko lākou hiki ke kānana kiʻekiʻe, ʻo kahi hiʻohiʻona kaulana ʻē aʻe o nā bivalves ʻo ia ka mucopolysaccharide kemika o kā lākou mucus, e hāpai ana i ka absorption o nā maʻi [78, 79].Hoʻolilo kēia i nā bivalves i kānana kūlohelohe kūpono no ka ʻike ʻana i ke ʻano like ʻole a me ka hopena o ka hoʻololi ʻana i ke aniau ma kahi kaiaola wai i hāʻawi ʻia.ʻOiai hiki ke ʻike ʻia ka loaʻa ʻana o nā ʻāpana DNA i loaʻa i ka host ma ke ʻano he palena o ke ʻano i hoʻohālikelike ʻia me ka eDNA, ʻo ke kumukūʻai e pili ana i ka loaʻa ʻana o ia ʻano ccfDNA maoli i hoʻohālikelike ʻia me eDNA hiki ke hoʻomaopopo ʻia no ka nui o ka ʻike i loaʻa no nā haʻawina olakino.offset host.Hoʻopili kēia i ka hele ʻana o nā kaʻina viral i hoʻohui ʻia i loko o ka genome o ka host host.He mea koʻikoʻi kēia no nā mussels, no ka loaʻa ʻana o nā retroviruses leukemic i hoʻouna ʻia ma ka pae i loko o nā bivalves [80, 81].ʻO kekahi pōmaikaʻi ʻē aʻe o LB ma luna o ka eDNA, ʻo ia ka hoʻohana ʻana i ka hana phagocytic o ke kahe ʻana i nā ʻāpana koko i loko o ka hemolymph, kahi e hoʻopili ai i nā microorganism (a me kā lākou genomes).ʻO ka Phagocytosis ka hana nui o nā keʻena koko i nā bivalves [82].ʻO ka mea hope loa, hoʻohana ke ala i ka hiki ke kānana kiʻekiʻe o nā mussels (awelika 1.5 l/h o ka wai kai) a me ʻelua lā kaʻahele, e hoʻonui ai i ka hui ʻana o nā ʻāpana like ʻole o ke kai, e ʻae ai i ka hopu ʻana i ka eDNA heterologous.[83, 84].No laila, ʻo ka ʻimi ʻana i ka mussel ccfDNA he ala hoihoi i hāʻawi ʻia i ka hopena o ka ʻai, hoʻokele waiwai, a me ke kaiapuni o nā ʻiʻo.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 i ka pane ʻana i nā mea exogenous.No ka laʻana, hiki ke noʻonoʻo ʻia nā ʻenehana sequencing ʻekolu e hana i ka loiloi methylation genome-wide ma ka ccfDNA maoli me ka hoʻohana ʻana i ke kaʻina nanopore.Pono e maʻalahi kēia kaʻina hana ma ka ʻoiaʻiʻo o ka lōʻihi o nā ʻāpana mussel ccfDNA i kūpono me nā papa hoʻonohonoho lōʻihi heluhelu e hiki ai i ka genome-wide DNA methylation analysis mai kahi holo hoʻokahi me ka ʻole o ka hoʻololi ʻana i nā kemika.No laila, hiki iā ia ke hāʻawi i ka ʻike koʻikoʻi i nā ʻōnaehana kumu e hoʻokele ana i ka pane ma hope o ka ʻike ʻana i ka hoʻololi ʻana i ke aniau a i ʻole nā pollutants [87].Eia naʻe, ʻaʻole i kaupalena ʻia ka hoʻohana ʻana iā LB.ʻAʻole pono e ʻōlelo, pono kēia i ke ʻano o nā ʻano mea hōʻike i loko o ke kaiaola.E like me ka mea i ʻōlelo ʻia ma luna, me ka hoʻohana ʻana i ka LB no ka loiloi ʻana i ka biodiversity o kahi kaiaola i hāʻawi ʻia, pono pū kekahi pipeline bioinformatics ikaika e noʻonoʻo i ka loaʻa ʻana o nā ʻāpana DNA mai ke kumu.ʻO kekahi pilikia nui ʻo ka loaʻa ʻana o nā genome kuhikuhi no nā ʻano kai.Manaʻolana ʻia nā hana e like me ka Marine Mammal Genomes Project a me ka papahana Fish10k i hoʻokumu hou ʻia nei [88] e hoʻomaʻamaʻa i kēlā ʻano loiloi i ka wā e hiki mai ana.ʻO ka hoʻohana ʻana i ka manaʻo LB i nā mea kānana-feeding organisms ua kūpono pū me nā holomua hou loa i ka ʻenehana sequencing, e kūpono ana i 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 i ka pane ʻana i ke koʻikoʻi o ke kaiapuni.
Ua waiho ʻia ka ʻikepili sequencing genome ma ka NCBI Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/SRR8924808 ma lalo o Bioprojects SRR8924808.
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Ka manawa hoʻouna: ʻAukake-14-2022