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Biopsy y'amazi (LB) ni igitekerezo kirimo gukundwa cyane mu rwego rw'ubuvuzi bw'ibinyabuzima. Iki gitekerezo gishingiye ahanini ku gupima ibice bya ADN igenda izenguruka hanze y'umubiri (ccfDNA), birekurwa cyane cyane nk'uduce duto nyuma y'urupfu rw'uturemangingo mu bice bitandukanye by'umubiri. Igice gito cy'ibi bice bikomoka ku bice by'amahanga (by'amahanga) cyangwa ku binyabuzima. Mu kazi ka none, twakoresheje iki gitekerezo ku bimera, ubwoko bw'abarinda buzwiho ubushobozi bwo kuyungurura amazi yo mu nyanja. Dukoresha ubushobozi bw'ibimera gukora nk'ibiyungurura karemano kugira ngo dufate ibice bya ADN y'ibidukikije biva ahantu hatandukanye kugira ngo dutange amakuru yerekeye ibinyabuzima by'ibinyabuzima byo ku nkombe z'inyanja. Ibisubizo byacu bigaragaza ko hemolymph y'ibimera irimo uduce twa ADN dutandukanye cyane mu bunini, kuva kuri 1 kugeza kuri 5 kb. Gukurikirana imbunda byagaragaje ko umubare munini w'uduce twa ADN dukomoka ku mikorobe zo mu mahanga. Muri byo, twabonye uduce twa ADN dukomoka kuri bagiteri, archaea, na virusi, harimo na virusi zizwiho kwanduza ubwoko butandukanye bw'ibinyabuzima bikunze kuboneka mu bimera byo ku nkombe z'inyanja. Mu gusoza, ubushakashatsi bwacu bwerekana ko igitekerezo cya LB gikoreshwa ku bimera by’ingurube ari isoko y’ubumenyi bwinshi ariko butaracukumburwa ku bijyanye n’ubusumbane bw’udukoko mu rusobe rw’ibinyabuzima byo ku nkombe z’inyanja.
Ingaruka z'imihindagurikire y'ikirere (CC) ku binyabuzima byo mu nyanja ni ubushakashatsi burimo kwiyongera cyane. Ubushyuhe bw'isi ntibutera gusa ibibazo bikomeye by'umubiri, ahubwo bunatuma habaho ihindagurika ry'ubushyuhe bw'ibinyabuzima byo mu nyanja, bukagira ingaruka ku miterere y'ibinyabuzima byinshi, bigatuma bishakisha imiterere myiza kurushaho [1, 2]. Uretse kugira ingaruka ku binyabuzima bya metazoan, CC ibangamira uburinganire bw'imibanire y'ibinyabuzima n'udukoko. Iyi dysbacteriosis ya mikorobe iteje akaga gakomeye ku binyabuzima byo mu nyanja kuko ituma ibinyabuzima byo mu nyanja byibasirwa n'udukoko twandura [3, 4]. Bikekwa ko SS igira uruhare runini mu rupfu rw'abantu benshi, ari ikibazo gikomeye ku micungire y'ibinyabuzima byo mu nyanja ku isi [5, 6]. Iki ni ikibazo cy'ingenzi bitewe n'ingaruka z'ubukungu, ibidukikije n'imirire y'ibinyabuzima byinshi byo mu nyanja. Ibi ni ukuri cyane cyane ku bivanguwe bituye mu turere two ku mpera y'isi, aho ingaruka za CK ziba vuba kandi zikomeye [6, 7]. Mu by'ukuri, bivanguwe nka Mytilus spp. zikoreshwa cyane mu kugenzura ingaruka za CC ku binyabuzima byo mu nyanja. Ntibitangaje kuba hari umubare munini w’ibipimo by’umubiri byakozwe kugira ngo bigenzure ubuzima bwabyo, akenshi hakoreshejwe uburyo bubiri bukoresha ibipimo by’umubiri bishingiye ku bikorwa bya enzyme cyangwa imikorere y’uturemangingo nk’ubuzima bw’uturemangingo n’ibikorwa bya phagocytic [8]. Ubu buryo burimo kandi gupima ubwinshi bw’ibipimo by’umuvuduko byihariye byirundanya mu ngingo zoroshye nyuma yo kwinjiza amazi menshi yo mu nyanja. Ariko, ubushobozi bwinshi bwo kuyungurura hamwe n’uburyo bwo kuzunguruka amaraso bw’ibice bibiri bitanga amahirwe yo guteza imbere ibipimo by’amaraso bishya hakoreshejwe igitekerezo cya biopsy y’amazi (LB), uburyo bworoshye kandi buto bwo kuvura abarwayi. [9, 10]. Nubwo ubwoko bwinshi bw’ibipimo by’umubiri bushobora kuboneka muri LB y’umuntu, iki gitekerezo gishingiye ahanini ku isesengura rya ADN ry’ibice bya ADN (ccfDNA) biri mu maraso. Mu by’ukuri, kuba ADN iri mu maraso y’umuntu byamenyekanye kuva hagati mu kinyejana cya 20 [11], ariko ni mu myaka ya vuba aha gusa aho uburyo bwo gusesengura bufite ubushobozi bwo gusuzuma ibintu bukoresheje ccfDNA. Kuba hari uduce twa ADN tuzenguruka biterwa nuko ADN ya genomike (nuclear na mitochondrial) irekurwa mu buryo butunguranye nyuma y'urupfu rw'uturemangingo. Ku bantu bafite ubuzima bwiza, igipimo cya ccfDNA ubusanzwe kiba kiri hasi (<10 ng/mL) ariko gishobora kwiyongeraho inshuro 5-10 ku barwayi bafite indwara zitandukanye cyangwa bahura n'ibibazo by'ingutu, bigatera kwangirika kw'ingingo. Ku bantu bafite ubuzima bwiza, igipimo cya ccfDNA ubusanzwe kiba kiri hasi (<10 ng/mL) ariko gishobora kwiyongeraho inshuro 5-10 ku barwayi bafite indwara zitandukanye cyangwa bahura n'ibibazo by'ingutu, bigatera kwangirika kw'ingingo. У здоровых людей маран вккДНК в умеме низкая (<10 нг / ھى) Ku bantu bafite ubuzima bwiza, ubusanzwe ingano ya cccDNA iba iri hasi (<10 ng/mL), ariko ishobora kwiyongeraho inshuro 5-10 ku barwayi bafite indwara zitandukanye cyangwa bafite stress ituma ingingo zabo zangirika.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，从而导致组织损伤。在 健康 个体 中 c ccfdna 的 浓度 较 低 （（ <10 ng / ml） 但 在 各 种 病理 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10Концентрации ccfDNA обохно низкие (<10 нг / كان) у здоровых людей, но могут быть увеличены в 5-10 раз у удов с различными пидологиями или чисом, что при имитит к повреждению тканей. Ubusanzwe igipimo cya ccfDNA kiba kiri hasi (<10 ng/ml) ku bantu bazima, ariko gishobora kwiyongera inshuro 5-10 ku barwayi bafite indwara zitandukanye cyangwa stress, bigatera kwangirika kw'ingingo.Ingano y'uduce twa ccfDNA iratandukanye cyane, ariko akenshi iri hagati ya 150 na 200 bp. [12]. Isesengura rya ccfDNA yikora, ni ukuvuga ccfDNA ikomoka ku turemangingo dusanzwe cyangwa twahinduwe, rishobora gukoreshwa mu kumenya impinduka za genetiki n'iza epigenetike ziri muri genome ya nuclear na/cyangwa mitochondrial, bityo bigafasha abaganga guhitamo uburyo bwihariye bwo kuvura bugamije molekile [13]. Ariko, ccfDNA ishobora kuboneka mu masoko yo mu mahanga nka ccfDNA ikomoka ku turemangingo tw'umwana uri mu nda igihe atwite cyangwa mu ngingo zatewe [14,15,16,17]. ccfDNA kandi ni isoko y'ingenzi y'amakuru yo kumenya niba hari aside nucleic y'ikintu cyandura (cyangwa abanyamahanga), ibyo bikaba bituma hamenyekana indwara zandura zitamenyekanye mu maraso, birinda biopsy y'udusimba twanduye [18]. Ubushakashatsi buherutse bwerekanye ko amaraso y'umuntu afite isoko y'amakuru menshi ashobora gukoreshwa mu kumenya indwara za virusi na bagiteri, kandi ko hafi 1% bya ccfDNA iboneka mu maraso y'umuntu ikomoka mu mahanga [19]. Ubu bushakashatsi bwerekana ko ibinyabuzima biri mu mikorobe zizenguruka mu kinyabuzima bishobora gupimwa hakoreshejwe isesengura rya ccfDNA. Ariko, kugeza vuba aha, iki gitekerezo cyakoreshwaga gusa ku bantu, ndetse no mu zindi nyamaswa zo mu bwonko [20, 21].
Muri iyi nyandiko, dukoresha ubushobozi bwa LB kugira ngo dusesengure ccfDNA ya Aulacomya atra, ubwoko bwo mu majyepfo bukunze kuboneka mu birwa bya Kerguelen byo mu majyepfo, itsinda ry'ibirwa biri hejuru y'ubutaka bunini bwashinzwe mu myaka miliyoni 35 ishize. Dukoresheje uburyo bwo kugerageza bwa in vitro, twasanze ibice bya ADN mu mazi yo mu nyanja bifatwa vuba na mussels bikinjira mu gice cya hemolymph. Gukurikirana imbunda byagaragaje ko hemolymph ccfDNA y'inyama igizwe n'ibice bya ADN byayo kandi bitari ibyayo, harimo bagiteri zihuza n'ibindi bice bya ADN bivuye mu binyabuzima bisanzwe mu birunga byo mu nyanja bikonje. Hemolymph ccfDNA kandi irimo urukurikirane rwa virusi rukomoka kuri virusi zifite ubwoko butandukanye bw'aho zituye. Twanabonye ibice bya ADN bivuye mu nyamaswa nyinshi nka amafi yo mu magufwa, anemone zo mu nyanja, algae n'udukoko. Mu gusoza, ubushakashatsi bwacu bwerekana ko igitekerezo cya LB gishobora gukoreshwa neza ku nyamaswa zo mu nyanja zitagira urutirigongo kugira ngo habeho urutonde rwinshi rwa genome mu binyabuzima byo mu nyanja.
Inyamaswa zo mu bwoko bwa Mytilus platensis (M. platensis) zikuze (55-70 mm z'uburebure) na Aulacomya atra (A. atra) zakusanyijwe mu nkengero z'amabuye zo hagati y'inyanja ya Port-au-France (049°21.235 S, 070°13.490 E .). Ibirwa bya Kerguelen mu Ukuboza 2018. Izindi nyamaswa zo mu bwoko bwa blue mussels zikuze (Mytilus spp.) zavuye ku mucuruzi w'ubucuruzi (PEI Mussel King Inc., Prince Edward Island, Kanada) zishyirwa mu kigega cyuzuyemo umwuka (4°C) kirimo 10–20 L y'amazi y'ubukorikori ya 32‰. (umunyu wo mu nyanja w'ubukorano Reef Crystal, Instant Ocean, Virijiniya, muri Amerika). Kuri buri gerageza, uburebure n'uburemere bw'amasashe ya buri bwoko byapimwe.
Porogaramu yo kwinjira ku buntu kuri iyi gahunda iraboneka kuri interineti (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). Muri make, hemolymph ya LB yakusanyijwe mu mitsi ya abductor nk'uko byasobanuwe [22]. Hemolymph yasobanuwe neza hakoreshejwe centrifugation kuri 1200 × g mu gihe cy'iminota 3, supernatant yashyizwe muri firigo (-20 ° C) kugeza ikoreshejwe. Kugira ngo cfDNA ivanwe kandi isukuzwe, ingero (1.5-2.0 ml) zashongeshejwe kandi zigatunganywa hakoreshejwe NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) hakurikijwe amabwiriza y'uwakoze. ccfDNA yabitswe kuri -80 ° C kugeza igihe isesengura rikomeje. Mu igerageza rimwe na rimwe, ccfDNA yakuwemo kandi isukurwa hakoreshejwe QIAamp DNA Investigator Kit (QIAGEN, Toronto, Ontario, Kanada). ADN isukuwe yapimwe hakoreshejwe ikizamini gisanzwe cya PicoGreen. Ikwirakwizwa ry'ibice bya ccfDNA yakuwemo ryasesenguwe hakoreshejwe electrophoresis ya capillary hakoreshejwe Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) hakoreshejwe Kit ya DNA ifite ubushobozi bwo kuvura indwara nyinshi. Isuzuma ryakozwe hakoreshejwe µl 1 y'icyitegererezo cya ccfDNA hakurikijwe amabwiriza y'uwakoze.
Mu gukurikirana ibice bya hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Kanada) yateguye amasomero ya shotgun akoresheje Illumina DNA Mix kit ya Illumina MiSeq PE75 kit. Hakoreshejwe adaptateri isanzwe (BioO). Amadosiye y'amakuru asanzwe araboneka muri NCBI Sequence Read Archive (SRR8924808 na SRR8924809). Ubwiza bw'ibanze bwo gusoma bwapimwe hakoreshejwe FastQC [23]. Trimmomatic [24] yakoreshejwe mu gukata adaptateri no gusoma nabi. Imbunda za Shotgun zifite impera ebyiri zahujwe na FLASH mu gusoma inshuro ndende hamwe n'aho nibura 20 bp kugira ngo hirindwe ko hadahura [25]. Ibisobanuro byahujwe byashyizwemo BLASTN hakoreshejwe ububiko bw’amakuru bwa NCBI Taxonomy bwa bivalve (agaciro ka e < 1e−3 na 90% homology), kandi gupfuka ibice by’imiterere yoroshye byakozwe hakoreshejwe DUST [26]. Ibisobanuro byahujwe byashyizwemo BLASTN hakoreshejwe ububiko bw’amakuru bwa NCBI Taxonomy bwa bivalve (agaciro ka e < 1e−3 na 90% homology), kandi gupfuka ibice by’imiterere yoroshye byakozwe hakoreshejwe DUST [26]. Объединенные ч чения были аннотирован с помощью BLASTN с использованием басы Кных так такомии двустворч чатх моллюсков NCBI (значение e <1e-3 и 90% tutholии) выполнено с использованием DUST [26]. Ibisobanuro byakusanyijwe hamwe byashyizwemo BLASTN hakoreshejwe ububiko bw’amakuru bwa NCBI bivalve taxonomy (agaciro ka e < 1e-3 na 90% homology), kandi gupfuka ibice by’uruhererekane byoroshye byakozwe hakoreshejwe DUST [26].使用双壳类 NCBI 分类数据库（ e 值 <1e-3 和 90% 同源性）用 AMASASO UST DUST [26] 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 使用 使用 使用 使用 ivumbi [26] 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Объединенные ч чения были аннотирован с помощу BLASTN с использованием так консомической бырыгыных двустворч чатх моллюсков NCBI (значение e <1e-3 и 90% tutологии), с выполнено с использованием DUST [26]. Ibisobanuro byakusanyijwe hamwe byashyizwemo BLASTN hakoreshejwe ububiko bw’amakuru bwa NCBI bivalve taxonomic (agaciro ka e <1e-3 na 90% homology), kandi gupfuka ibice by’uruhererekane byoroshye byakozwe hakoreshejwe DUST [26].Ibisomwa byagabanijwemo amatsinda abiri: ajyanye n'ibice bibiri by'amatsinda (aha byitwa kwisoma) n'ibidafite aho bihuriye (bitanafite aho bihuriye). Amatsinda abiri yakusanyijwe ukwayo hakoreshejwe MEGAHIT kugira ngo hakorwe ibice [27]. Hagati aho, ikwirakwizwa ry'ibipimo bya microbiome by'abandi ryashyizwe mu byiciro hakoreshejwe Kraken2 [28] kandi rigaragazwa mu buryo bw'ishusho n'imbonerahamwe ya Krona pie kuri Galaxy [29, 30]. Kmers nziza zagenwe kuba kmers-59 uhereye ku bushakashatsi bwacu bwa mbere. Hanyuma ibice byihariye byamenyekanye hakoreshejwe uburyo bwa BLASTN (bivalve NCBI database, agaciro ka e < 1e−10 na 60% homology) kugira ngo haboneke ibisobanuro bya nyuma. Hanyuma ibice byihariye byamenyekanye hakoreshejwe uburyo bwa BLASTN (bivalve NCBI database, agaciro ka e < 1e−10 na 60% homology) kugira ngo haboneke ibisobanuro bya nyuma. Затем собственные эштиги были идент фицирован путем сопоставления с BLASTN (Баа нных двустворч матх моллюсков NCBI, значение e <1e-10 и kurдология 60%) для оончательной аннотации. Hanyuma, ibice byihariye byaje kumenyekana hakoreshejwe uburyo bwa BLASTN (ububiko bw'amakuru bwa NCBI bivalve, agaciro ka e <1e-10 na 60% homology) kugira ngo hamenyekane ibisobanuro bya nyuma.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。BLASTN （双壳贝类 NCBI 数据库， e 值 <1e-10 和 60% Затем были идент Фифицированя собственные шәһәртиги для окончательной аннотации путем сопоставления с BLASTN (babaа чныхх NCBI для двустворч чехх моллюсков, значение e <1e-10 и зогол 60%). Hanyuma habonetse ibice byihariye kugira ngo bisobanurwe neza binyuze mu guhuza na BLASTN (ububiko bw'amakuru bwa NCBI bivalve, agaciro ka e <1e-10 na 60% homology). Muri ubwo buryo, ibice by’amatsinda bitari ibyawe byashyizwemo BLASTN (database ya nt NCBI, agaciro ka e < 1e−10 na homology ya 60%). Muri ubwo buryo, ibice by’amatsinda bitari ibyawe byashyizwemo BLASTN (database ya nt NCBI, agaciro ka e < 1e−10 na homology ya 60%). Параллельно чужеродные групповые бастиги были аннотирования с помощью BLASTN (Баа нныхх nt NCBI, значение e <1e-10 и курология 60%). Muri ubwo buryo, amatsinda y’abanyamahanga yashyizwemo BLASTN (database ya NT NCBI, agaciro ka e <1e-10 na 60% homology).BLASTN （nt NCBI 数据库， e 值 <1e-10 和 60% 同源性）注释非自身组重叠群。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 карьертиги, не относящиеся к собственной группе, были аннотирован с помощу BLASTN (باسا كاننىخ nt NCBI, значение e <1e-10 и кидология 60%). Muri ubwo buryo, ibice bitari ibyawe byashyizwemo BLASTN (database ya nt NCBI, agaciro ka e <1e-10 na homology ya 60%). BLASTX yanakorewe ku bintu bitari ibyayo hakoreshejwe poroteyine ya nr na RefSeq NCBI (agaciro ka e < 1e−10 na 60% homology). BLASTX yanakorewe ku bintu bitari ibyayo hakoreshejwe poroteyine ya nr na RefSeq NCBI (agaciro ka e < 1e−10 na 60% homology). BLASTX takже был проведен на несамамостоятельных кстигах с использованием базных белка nr и RefSeq NCBI (значение e <1e-10 и идология 60%). BLASTX yanakorewe ku bintu bitari ibyayo hakoreshejwe poroteyine ya nr na RefSeq NCBI (agaciro ka e < 1e-10 na 60% homology).还使用 nr 和 RefSeq 蛋白 NCBI 数据库对非自身重叠群进行了 BLASTX （e 值 <1e-10 和 60% 同源性）。还使用 nr 和 RefSeq 蛋白 NCBI 数据库对非自身重叠群进行了 BLASTX （e 值 <1e-10 和 60% 同源性）。 BLASTX takже выполняли на несамамостоятельных хезмәттигах с использованием басныны белка nr na RefSeq NCBI (значение e <1e-10 и идология 60%). BLASTX yanakorewe ku bintu bitari ibyayo hakoreshejwe poroteyine ya nr na RefSeq NCBI (agaciro ka e <1e-10 na 60% homology).Ibice bya BLASTN na BLASTX by’ibitari ibyabo bigaragaza ibice bya nyuma (reba dosiye y’inyongera).
Imashini zikoreshwa kuri PCR zigaragara mu mbonerahamwe ya S1. Taq DNA polymerase (Bio Basic Canada, Markham, ON) yakoreshejwe mu kongera imiterere ya ccfDNA. Hakoreshejwe uburyo bukurikira bwo gusubiza: denaturation kuri 95°C mu minota 3, 95°C mu munota 1, gushyiraho ubushyuhe bwo gufunga umunota 1, kurekura kuri 72°C mu munota 1, cycles 35, na 72°C mu minota 10. . Ibicuruzwa bya PCR byatandukanyijwe na electrophoresis muri agarose gels (1.5%) irimo SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Kanada) kuri 95 V.
Inyamaswa zo mu bwoko bwa Mussel (Mytilus spp.) zashyizwe mu mazi yo mu nyanja afite ogisijeni ya mililitiro 500 (32 PSU) mu gihe cy'amasaha 24 kuri 4°C. ADN ya plasmid irimo agace gashyiramo galectin-7 cDNA sequence y'umuntu (NCBI accession nomero L07769) yongewe mu kabari ku gipimo cya nyuma cya 190 μg/μl. Inyamaswa zo mu bwoko bwa Mussel zashyizwe mu buryo bumwe nta kongerwamo ADN ni zo zagenzurwaga. Ikigega cya gatatu cyo kugenzura cyarimo ADN idafite inyamaswa zo mu mazi. Kugira ngo hagenzurwe ubuziranenge bwa ADN mu mazi yo mu nyanja, ingero z'amazi yo mu nyanja (20 μl; inshuro eshatu) zavanywe muri buri kigega mu gihe cyagenwe. Kugira ngo ADN ikurikirane neza, inyamaswa zo mu bwoko bwa LB zakusanyijwe mu bihe byagenwe kandi zigasesengurwa na qPCR na ddPCR. Kubera ko amazi yo mu nyanja afite umunyu mwinshi, aliquots zavanzwe mu mazi meza ya PCR (1:10) mbere y'ibizamini byose bya PCR.
PCR y'amadomo ya digitale (ddPCR) yakozwe hakoreshejwe protocole ya BioRad QX200 (Mississippi, Ontario, Kanada). Koresha imiterere y'ubushyuhe kugira ngo umenye ubushyuhe bwiza (Imbonerahamwe S1). Amadomo yakozwe hakoreshejwe generator ya QX200 drop (BioRad). ddPCR yakozwe ku buryo bukurikira: 95°C mu minota 5, cycles 50 za 95°C mu minota 30 n'ubushyuhe bwa annealing mu munota 1 na 72°C mu minota 30, 4°C mu minota 5 na 90°C mu minota 5. Umubare w'amadomo n'ingaruka nziza (umubare wa kopi / µl) byapimwe hakoreshejwe icyuma gisoma amadomo cya QX200 (BioRad). Ingero zifite amadomo ari munsi ya 10.000 zaranzwe. Igenzura ry'ibishushanyo ntiryakozwe buri gihe ddPCR yakorwaga.
qPCR yakozwe hakoreshejwe Rotor-Gene® 3000 (Corbett Research, Sydney, Ositaraliya) na primers zihariye za LGALS7. PCR zose zakozwe muri 20 µl hakoreshejwe QuantiFast SYBR Green PCR Kit (QIAGEN). qPCR yatangijwe n'ingufu zo gucukura mu minota 15 kuri 95°C, hagakurikiraho cycles 40 kuri 95°C mu masegonda 10, na 60°C mu masegonda 60 hamwe n'ikusanyirizo rimwe ry'amakuru. Imirongo ishongesha yakozwe hakoreshejwe ibipimo bikurikirana kuri 95°C mu masegonda 5, 65°C mu masegonda 60, na 97°C mu mpera ya qPCR. Buri qPCR yakozwe mu byiciro bitatu, uretse ingero zo kugenzura.
Kubera ko imineke izwiho kuyungurura cyane, twabanje gusuzuma niba ishobora kuyungurura no kubika uduce twa ADN turi mu mazi yo mu nyanja. Twari dushishikajwe no kumenya niba utwo duce twikusanyiriza mu buryo bwa lymphatic. Twakemuye iki kibazo mu buryo bw'igerageza dukurikirana iherezo ry'uduce twa ADN dushonga twongewe mu bigega by'ubururu bw'imineke. Kugira ngo tworohereze gukurikirana uduce twa ADN, twakoresheje ADN y'inyamahanga (atari iyayo) irimo gene ya galectin-7 y'umuntu. ddPCR ikurikirana uduce twa ADN ya plasmid mu mazi yo mu nyanja n'imineke. Ibisubizo byacu bigaragaza ko niba ingano y'uduce twa ADN mu mazi yo mu nyanja yagumye ihindagurika uko igihe kigenda (kugeza ku minsi 7) mu gihe imineke idahari, ubwo iyo imineke iri imbere, uru rwego rwayo rwagiye rushira burundu mu masaha 8 (Ishusho ya 1a, b). Uduce twa ADN yo hanze twashoboraga kuboneka byoroshye mu minota 15 mu mazi yo mu mitsi no mu maraso (Ishusho ya 1c). Utwo duce twashoboraga kuboneka kugeza ku masaha 4 nyuma yo guhura natwo. Iki gikorwa cyo kuyungurura ku bijyanye n'uduce twa ADN kigereranywa n'igikorwa cyo kuyungurura cya bagiteri n'ibimera [31]. Ibi bisubizo bigaragaza ko ibimera bishobora kuyungurura no gukusanya ADN y'amahanga mu bice byabyo by'amazi.
Ubwinshi bwa ADN ya plasmide mu mazi yo mu nyanja mu gihe hari (A) cyangwa hatari (B), bipimwe na ddPCR. Muri A, ibisubizo bigaragazwa nk'ijanisha, aho imbibi z'udusanduku zigaragaza 75th na 25th percentile. Umurongo wa logarithmic ushyirwamo ugaragazwa mu ibara ry'umutuku, naho agace gatwikiriwe n'ibara ry'umukara kagaragaza 95% by'icyizere. Muri B, umurongo utukura ugaragaza impuzandengo naho umurongo w'ubururu ugaragaza 95% by'icyizere cy'ubwinshi. C Gukusanya ADN ya plasmide mu maraso y'amaraso n'amazi ya valvular y'amafi mu bihe bitandukanye nyuma yo kongeramo ADN ya plasmide. Ibisubizo bigaragazwa nk'amakopi yuzuye yabonetse/mL (±SE).
Hanyuma, twakoze ubushakashatsi ku nkomoko ya ccfDNA mu missels yakusanyijwe mu birwa bya Kerguelen, itsinda ry’ibirwa bya kure bifite ingaruka nke ku bantu. Muri iyo ntego, cccDNA ikomoka ku missel hemolymphs yakuwemo kandi isukurwa hakoreshejwe uburyo busanzwe bukoreshwa mu gusukura cccDNA y’umuntu [32, 33]. Twasanze impuzandengo y’ibice bya hemolymph ccfDNA mu missels iri muri mikorogarama nke kuri ml hemolymph (reba Imbonerahamwe S2, Amakuru y’inyongera). Uru rwego rw’ibice ni runini cyane ugereranije n’abantu bazima (nanogarama nke kuri mililitiro), ariko mu bihe bidasanzwe, ku barwayi ba kanseri, urwego rwa ccfDNA rushobora kugera kuri mikorogarama nyinshi kuri mililitiro [34, 35]. Isesengura ry’ingano ya hemolymph ccfDNA ryagaragaje ko ibi bice bitandukanye cyane mu bunini, kuva kuri 1000 bp kugeza 1000 bp. kugeza kuri 5000 bp (Ishusho ya 2). Ibisubizo nk'ibyo byabonetse hakoreshejwe QIAamp Investigator Kit ishingiye kuri silica, uburyo bukunze gukoreshwa mu bumenyi bw'ubugenzacyaha mu gutandukanya no gutunganya ADN y'uturemangingo tw'uturemangingo mu buryo bwihuse uhereye ku bipimo bya ADN bike, harimo na ccfDNA [36].
Electrophoregramu ya ccfDNA ihagarariye hemolymph y'inyama. Yakuwemo NucleoSnap Plasma Kit (hejuru) na QIAamp ADN Investigator Kit. B Violin plot yerekana uko hemolymph ccfDNA ikwirakwira (±SE) mu nyamaswa. Imirongo y'umukara n'umutuku igaragaza quartiles zo hagati n'iza mbere n'iza gatatu, uko zikurikirana.
Hafi 1% bya ccfDNA mu bantu no mu nyamaswa z’inkende bifite isoko y’amahanga [21, 37]. Bitewe n’uburyo amaraso agenda asohoka neza bw’ibice bibiri, amazi yo mu nyanja akungahaye kuri mikorobe, ndetse n’ingano ya ccfDNA y’ibice bibiri, twatekereje ko hemolymph ccfDNA y’ibice bibiri ishobora kuba irimo ADN y’udukoko twinshi kandi dutandukanye. Kugira ngo tugerageze iki gitekerezo, twakurikiranye hemolymph ccfDNA iva mu bipimo bya Aulacomya atra byakusanyijwe mu birwa bya Kerguelen, bitanga abasomyi barenga miliyoni 10, 97.6% muri bo bakaba baratsinze igenzura ry’ubuziranenge. Ibyasomwe byashyizwe mu byiciro hakurikijwe amasoko bwite n’atari yo bwite hakoreshejwe ububiko bwa BLASTN na NCBI bivalve (Ishusho ya S1, Amakuru Y’inyongera).
Ku bantu, ADN ya kirimbuzi na mitochondrial ishobora kurekurwa mu maraso [38]. Ariko, muri ubu bushakashatsi, ntibyashobotse gusobanura mu buryo burambuye ADN ya kirimbuzi ya missels, bitewe nuko genome ya A. atra itarakurikiranwa cyangwa ngo isobanurwe. Ariko, twashoboye kumenya ibice byinshi bya ccfDNA by’inkomoko yacu dukoresheje isomero rya bivalve (Ishusho ya S2, Amakuru y’inyongera). Twemeje kandi ko hari ibice bya ADN by’inkomoko yacu binyuze mu kongera PCR kuri izo gene za A. atra zakurikiranywe (Ishusho ya 3). Mu buryo nk’ubwo, bitewe nuko genome ya mitochondrial ya A. atra iboneka mu bubiko rusange, umuntu ashobora kubona ibimenyetso byerekana ko hari ibice bya ccfDNA bya mitochondrial mu maraso ya A. atra. Kuba hari ibice bya ADN bya mitochondrial byemejwe no kongera PCR (Ishusho ya 3).
Utunyangingo twa mitochondrial twagaragaye muri hemolymph ya A. atra (utudomo dutukura - umubare w'imigabane: SRX5705969) na M. platensis (utudomo tw'ubururu - umubare w'imigabane: SRX5705968) twazamuwe na PCR. Ishusho yakuwe muri Breton et al., 2011 B Kongera hemolymph supernatant iva kuri A. atra Bibikwa ku mpapuro za FTA. Koresha icyuma cya mm 3 kugira ngo wongere mu muyoboro wa PCR urimo uruvange rwa PCR.
Bitewe n’uko mikorobe nyinshi ziri mu mazi yo mu nyanja, twabanje kwibanda ku miterere y’uruhererekane rwa ADN za mikorobe muri hemolymph. Kugira ngo tubigereho, dukoresha ingamba ebyiri zitandukanye. Ingamba za mbere zakoresheje Kraken2, gahunda ishingiye kuri algorithm yo gushyira mu byiciro uruhererekane rw’uru ... Uburyo bwa ddPCR bwemeje ko hari ibice bya ADN ya Vibrio muri ccfDNA ya A. atra hemolymph (Ishusho ya 4c) [41]. Kugira ngo haboneke amakuru arambuye ku nkomoko ya bagiteri ya ccfDNA, hafashwe ubundi buryo (Ishusho ya S2, Amakuru y'inyongera). Muri iki kibazo, abasomyi bavanze bateranijwe nk'abasoma bafite impande ebyiri kandi bashyizwe mu byiciro nk'abakomoka kuri self (bivalves) cyangwa abakomoka kuri self hakoreshejwe BLASTN hamwe n'agaciro ka e ka 1e−3 hamwe n'igabanywa rifite homology >90%. Muri iki kibazo, abasomyi bavanze bateranijwe nk'abasoma bafite impande ebyiri kandi bashyizwe mu byiciro nk'abakomoka kuri self (bivalves) cyangwa abakomoka kuri self hakoreshejwe BLASTN hamwe n'agaciro ka e ka 1e−3 hamwe n'igabanywa rifite homology >90%. В этом случае перекрывающиеся чтения были собрани как чтения с парными концами и были мсифицирован как собственные (двустворч мате моллюски) или чужие по происхождению с использове inamaологией> 90%. Muri iki kibazo, inyandiko zihurirana zakusanyijwe nk'inyandiko zisozanya kandi zashyizwe mu byiciro nk'umwimerere (bivalve) cyangwa izitari iz'umwimerere hakoreshejwe agaciro ka BLASTN na e ka 1e-3 kandi zigashyirwa ku murongo urenze 90%.BLASTN 和 1e-3 的 e 值和> 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 末端 读数 ， 使用 使用 使用 使用 使用 使用 1e-3 的 的 值 和> 90% 同源性 的 分类 自身 （双 壳类） 非 自身。。。。。。。。。 В этом случае перекрывающиеся ч чентия были собрани как чтения с парными концами и мсисифицирован как собственные (двустворч мате моллуски) или чобобственные по происхождению с испольчове е с inamaологии> 90%. Muri iki kibazo, inyandiko zihurirana zakusanyijwe nk'inyandiko zisozanya maze zishyirwa mu byiciro nk'izawe (bivalves) cyangwa izitari iz'umwimerere hakoreshejwe agaciro ka e BLASTN na 1e-3 ​​kandi hagaragazwa umubare w'inyuguti >90%.Kubera ko genome ya A. atra itarashyirwa mu byiciro, twakoresheje ingamba zo guteranya za de novo za MEGAHIT Next Generation Sequencing (NGS) assembler. Habonetse ko hari ibice 147.188 by’ibice byakuweho (bivalve) bishingiye ku bivange. Ibi bice byashyizwemo byaturikijwe n’agaciro ka e-values ​​​​ka 1e-10 hakoreshejwe BLASTN na BLASTX. Iyi ngamba yatumye tumenya ibice 482 bitari ibivange biri muri A. atra ccfDNA. Ibice birenga kimwe cya kabiri (57%) by’ibi bice bya ADN byakuwe muri bagiteri, cyane cyane bivuye ku bimera bya gill, harimo n’ibimera bya sulfotrophic, no ku bimera bya gill Solemya velum (Ishusho ya 5).
Ubwinshi bungana ku rwego rw'ubwoko. B Ubudasa bw'uturemangingo tw'udukoko tubiri tw'ingenzi (Firmicutes na Proteobacteria). Iyongera ryihariye rya ddPCR C Vibrio spp. A. Ibice bya gene ya 16S rRNA (ubururu) muri hemolymphe eshatu za atra.
Hasesenguwe ibice 482 byakusanyijwe. Imiterere rusange y'ikwirakwizwa rya taxonomic ry'ibisobanuro bya metagenomic contig (prokaryotes na eukaryotes). B Ikwirakwizwa rirambuye ry'ibice bya ADN bya bagiteri byagaragajwe na BLASTN na BLASTX.
Isesengura rya Kraken2 ryagaragaje kandi ko DNA y’ibimera igizwe n’uduce twa ADN twa kera, harimo uduce twa ADN twa Euryarchaeota (65%), Crenarchaeota (24%), na Thaurmarcheota (11%) (Ishusho ya 6a). Kuba hari uduce twa ADN twakuwe muri Euryarchaeota na Crenarchaeota, twabonetse mbere mu muryango w’udukoko twa mussels zo muri Kaliforuniya, ntibikwiye gutungurana [42]. Nubwo Euryarchaeota ikunze kujyana n’ibihe bikomeye, ubu byagaragaye ko Euryarchaeota na Crenarcheota byombi biri mu duce twa prokaryote dukunze kugaragara mu bidukikije byo mu mazi [43, 44]. Kuba hari udukoko twa methanogenic mu bimera ntibitangaje, bitewe n’amakuru aherutse gutangazwa ku mazi menshi ya methane ava mu mazi yo hasi ku gisenge cya Kerguelen [45] ndetse n’umusaruro wa methane wa mikorobe wagaragaye ku nkombe z’ibirwa bya Kerguelen [46].
Hanyuma twitaye ku bipimo bya virusi za ADN. Uko tubizi neza, iyi ni yo nyigo ya mbere idashingiye ku ntego y’uko virusi iri mu missels ingana. Nkuko byari byitezwe, twabonye uduce twa ADN twa bacteriophages (Caudovirales) (Ishusho ya 6b). Ariko, ADN ya virusi ikunze kugaragara ikomoka kuri phylum ya nucleocytoviruses, izwi kandi nka virus ya DNA nini ya cytoplasmic (NCLDV), ifite genome nini kurusha virusi iyo ari yo yose. Muri iyi phylum, ADN nyinshi zikomoka ku miryango ya Mimimidoviridae (58%) na Poxviridae (21%), aho inyamaswa zo mu bwoko bwa Mimimidoviridae zisanzwe zirimo inyamaswa zo mu mugongo na arthropods, mu gihe igice gito cy’izi ADN kikomoka ku algae izwi. Ifata algae yo mu mazi ya eukaryotic. Utu tunyangingo twakuwe kuri virusi ya Pandora, virusi nini ifite genome nini kurusha gene iyo ari yo yose izwi ya virusi. Igishimishije ni uko ubwoko bw'ibinyabuzima bizwiho kwandura iyi virusi, nk'uko byagaragajwe n'uburyo bwo gukurikirana DNA mu maraso, bwari bunini cyane (Ishusho ya S3, Amakuru y'inyongera). Harimo virusi zandura udukoko nka Baculoviridae na Iridoviridae, ndetse na virusi zandura amoeba, algae n'inyamaswa zo mu mugongo. Twanabonye urutonde rujyanye na genome ya Pithovirus sibericum. Pitoviruses (zizwi kandi nka "zombie viruses") zabanje gukurwa mu duce tw'isi dufite imyaka 30.000 muri Siberiya [47]. Bityo, ibisubizo byacu bihuye na raporo zabanje zigaragaza ko atari ubwoko bwose bwa none bw'izi virusi bwazimye [48] kandi ko izi virusi zishobora kuba ziri mu bidukikije byo mu nyanja biri kure cyane.
Amaherezo, twagerageje kugira ngo turebe niba dushobora kubona uduce twa ADN twavuye mu zindi nyamaswa zifite uturemangingo twinshi. BLASTN na BLASTX habonetse uduce 482 tw’amahanga hamwe n’amasomero ya nt, nr na RefSeq (genomike na poroteyine). Ibisubizo byacu bigaragaza ko mu duce tw’amahanga twa ccfDNA y’inyamaswa zifite uturemangingo twinshi ari yo ADN y’amagufwa y’amagufwa yiganjemo (Ishusho ya 5). Habonetse uduce twa ADN twavuye mu dukoko n’andi moko. Igice kinini cy’uduce twa ADN ntikiramenyekana, birashoboka ko biterwa n’uko umubare munini w’ibinyabuzima byo mu mazi ugaragara mu bubiko bwa genomike ugereranije n’ibinyabuzima byo ku isi [49].
Muri iyi nyandiko, dukoresha igitekerezo cya LB kuri mussels, tuvuga ko hemolymph ccfDNA shot sequencing ishobora gutanga ibisobanuro ku miterere y'ibinyabuzima byo ku nkombe z'inyanja. By'umwihariko, twasanze 1) hemolymph y'inyama igizwe n'ibice byinshi bya ADN binini (~1-5 kb) bizenguruka; 2) ibi bice bya ADN byigenga kandi ntibigenga 3) Mu masoko y'amahanga y'ibi bice bya ADN, twabonye ADN ya bagiteri, ADN ya kera na virusi, ndetse na ADN y'izindi nyamaswa nyinshi; 4) Kwirundanya kw'ibi bice bya ccfDNA by'amahanga muri hemolymph bibaho vuba kandi bigafasha mu kuyungurura imbere mu nyamaswa. Mu gusoza, ubushakashatsi bwacu bwerekana ko igitekerezo cya LB, kugeza ubu cyakoreshejwe cyane cyane mu rwego rwa biomedicine, gikubiyemo isoko y'ubumenyi bwinshi ariko butaracukumburwa bushobora gukoreshwa mu gusobanukirwa neza imikoranire iri hagati y'ubwoko bw'abarinda uruziga n'ibidukikije byabwo.
Uretse inyamaswa zo mu bwoko bwa cat, ccfDNA yagaragaye mu nyamaswa zo mu bwoko bwa cat, harimo imbeba, imbwa, injangwe, n'amafarashi [50, 51, 52]. Ariko, uko tubizi, ubushakashatsi bwacu ni bwo bwa mbere bwagaragaje uburyo ccfDNA iboneka kandi igakurikiranwa mu moko yo mu mazi afite uburyo bwo kuzenguruka mu buryo bufunguye. Ubu buryo bw'umubiri n'ubushobozi bwo kuyungurura bwa mussels, nibura mu gice kimwe, bishobora gusobanura ingano itandukanye y'uduce twa ADN duzenguruka ugereranije n'utundi duce. Ku bantu, uduce twinshi twa ADN duzenguruka mu maraso ni uduce duto dufite ingano iri hagati ya 150 na 200 bp. dufite uburebure bwa 167 bp [34, 53]. Igice gito ariko kinini cy'uduce twa ADN kiri hagati ya 300 na 500 bp, kandi hafi 5% birebire kuruta 900 bp. [54]. Impamvu y’ubu bunini bw’iyi ngano ni uko isoko nyamukuru ya ccfDNA mu maraso iterwa n’urupfu rw’uturemangingo, byaba bitewe n’urupfu rw’uturemangingo cyangwa bitewe no kwangirika kw’uturemangingo tw’amaraso mu bantu bazima cyangwa bitewe no kugabanuka k’uturemangingo tw’uturemangingo mu barwayi ba kanseri (bizwi nka ADN y’uturemangingo izenguruka). Ingano ya ccfDNA ya hemolymph twasanze mu turemangingo yari hagati ya 1000 na 5000 bp, bigaragaza ko ccfDNA y’uturemangingo ifite inkomoko itandukanye. Iki ni igitekerezo cyumvikana, kubera ko turemangingo dufite sisitemu y’imitsi ifunguye kandi tuba mu mazi yo mu nyanja arimo ADN nyinshi ya mikorobe. Mu by’ukuri, igerageza ryacu rya laboratwari rikoresheje ADN y’inyuma ryagaragaje ko turemangingo dukusanya uduce twa ADN mu mazi yo mu nyanja, nibura nyuma y’amasaha make tugabanuka nyuma yo kwinjira mu turemangingo cyangwa kurekurwa cyangwa kubikwa mu bigo bitandukanye. Bitewe n'ubuke bw'uturemangingo (twa prokaryotic na eukaryotic), gukoresha ibice biri mu miyoboro y'amaraso bizagabanya ingano ya ccfDNA iva mu bikomoka ku bintu byihariye ndetse no mu bindi bikomoka ku bindi bidukikije. Dukurikije akamaro k'ubudahangarwa bw'umubiri bwa bivalve n'umubare munini wa phagocytes zizenguruka, twakomeje gutekereza ko na ccfDNA yo mu mahanga ikunzwe cyane mu miyoboro y'amaraso izenguruka ikusanya ADN y'amahanga iyo igeze mu mikorobe cyangwa imyanda y'uturemangingo. Ibyo twakoze byose bigaragaza ko bivalve hemolymph ccfDNA ari ububiko bwihariye bw'amakuru ya molekile kandi bishimangira urwego rwayo nk'ubwoko bw'abarinda.
Amakuru yacu agaragaza ko gukurikirana no gusesengura ibice bya hemolymph ccfDNA bikomoka kuri bagiteri bishobora gutanga amakuru y'ingenzi ku bimera bya bagiteri biyikikije ndetse na bagiteri ziri mu rusobe rw'ibinyabuzima byo mu nyanja. Uburyo bwo gukurikirana amasasu bwagaragaje urukurikirane rwa bagiteri ya commensal A. atra gill itari kuba yarakoreshejwe iyo uburyo busanzwe bwo kumenya rRNA bwa 16S buza gukoreshwa, bitewe n'uburyo bwo gutandukanya ibitabo by'ubushakashatsi. Mu by'ukuri, gukoresha amakuru ya LB yakusanyijwe muri M. platensis mu gice kimwe cya mussel kuri Kerguelen byagaragaje ko imiterere y'udukoko dufitanye isano na bagiteri yari imwe ku bwoko bwa mussel bwombi (Ishusho ya S4, Amakuru y'inyongera). Uku gusa kwa mussel ebyiri zitandukanye mu buryo bw'uturemangingo bishobora kugaragaza imiterere ya bagiteri mu bidukikije bya Kerguelen bikonje, birimo sulfure, n'ibirunga [55, 56, 57, 58]. Urwego rwo hejuru rwa mikorobe zigabanya sulfure rwasobanuwe neza mu gihe cyo gusarura mussels mu turere two ku nkombe turimo ibimera [59], nko ku nkombe za Port-au-France. Ikindi gishoboka ni uko ibimera by’ibimera bishobora kugira ingaruka ku ikwirakwira ry’ibinyabuzima mu buryo butambitse [60, 61]. Hakenewe ubushakashatsi bwinshi kugira ngo hamenyekane isano iri hagati y’ibidukikije byo mu mazi, ubuso bwo hasi mu nyanja, n’imiterere ya bagiteri zihuza mu bimera. Ubu bushakashatsi buracyakomeje.
Uburebure n'ubwinshi bwa hemolymph ccfDNA, uburyo bworoshye bwo kuyisukura, hamwe n'ubwiza bworoshye bwo gutuma ikurikirana vuba ni bimwe mu byiza byinshi byo gukoresha ccfDNA y'inyama mu gusuzuma urusobe rw'ibinyabuzima mu rusobe rw'ibinyabuzima byo ku nkombe z'inyanja. Ubu buryo ni ingirakamaro cyane mu kugaragaza ubwoko bwa virusi (viromes) mu rusobe runaka [62, 63]. Bitandukanye na bagiteri, archaea, na eukaryotes, genome za virusi ntizirimo jeni zibitswe mu buryo bwa phylogenetike nka 16S sequences. Ibisubizo byacu bigaragaza ko ibipimo by'amazi bivuye mu bwoko bw'ibipimo nka mussels bishobora gukoreshwa mu kumenya umubare munini w'uduce twa virusi ya ccfDNA tuzwiho kwanduza abantu basanzwe baba mu rusobe rw'ibinyabuzima byo ku nkombe z'inyanja. Ibi birimo virusi zizwiho kwanduza protozoa, arthropods, udukoko, ibimera, na virusi za bagiteri (urugero, bacteriophages). Ingano nk'iyo yabonetse ubwo twasuzumaga hemolymph ccfDNA virome y'ibimera by'ubururu (M. platensis) byakusanyijwe mu rusobe rumwe rw'ibimera kuri Kerguelen (Imbonerahamwe S2, Amakuru Y'inyongera). Gukurikirana imbunda za ccfDNA mu by’ukuri ni uburyo bushya burimo gutera imbere mu kwiga virome y’abantu cyangwa izindi nyoni [21, 37, 64]. Ubu buryo ni ingirakamaro cyane mu kwiga virusi za ADN zifite imigozi ibiri, kuko nta gene imwe iboneka muri virusi zose za ADN zifite imigozi ibiri, zihagarariye ubwoko bwa virusi butandukanye kandi bunini muri Baltimore [65]. Nubwo inyinshi muri izi virusi zitarashyirwa mu byiciro kandi zishobora kuba zirimo virusi zo mu gice kitazwi na gato cy’isi ya virusi [66], twasanze virome n’ubwoko bwa mussels A. atra na M. platensis biri hagati y’ubwoko bubiri. kimwe (reba ishusho ya S3, andi makuru). Uku gusa ntabwo bitangaje, kuko bishobora kugaragaza kutagira uburyo bwo guhitamo ADN iri mu bidukikije. Ubushakashatsi bw’ejo hazaza hakoreshejwe RNA isukuye burakenewe kugira ngo hamenyekane virome ya RNA.
Mu bushakashatsi bwacu, twakoresheje umuyoboro ukomeye cyane wakuwe mu kazi ka Kowarski na bagenzi be [37], bakoresheje uburyo bwo gukuramo ibice bibiri byashyizwe hamwe mbere na nyuma yo guteranya ccfDNA gakondo, bigatuma habaho umubare munini w'ibipimo bitarashyirwa ku ikarita. Kubwibyo, ntitwakwemeza ko bimwe muri ibi bipimo bitarashyirwa ku ikarita bishobora kuba bigifite inkomoko yabyo, ahanini kubera ko tudafite genome y'ubu bwoko bw'ibimera. Twakoresheje kandi uyu muyoboro kuko twari duhangayikishijwe n'ibimera biri hagati y'ibipimo byihariye n'ibitari ibyabyo ndetse n'uburebure bw'ibipimo byakozwe na Illumina MiSeq PE75. Indi mpamvu ituma ibice byinshi bitarashyirwa ku ikarita ni uko udukoko twinshi two mu mazi, cyane cyane mu turere twa kure nka Kerguelen, tutarashyirwa ku ikarita. Twakoresheje Illumina MiSeq PE75, dutekereza ko uburebure bw'ibice bya ccfDNA busa n'ubwa ccfDNA y'umuntu. Ku bushakashatsi bw'ejo hazaza, bitewe n'ibyavuye mu bushakashatsi bwacu bigaragaza ko hemolymph ccfDNA ifite gusoma igihe kirekire kurusha abantu cyangwa inyamaswa zo mu bwoko bwa ng'ombe, turasaba gukoresha urubuga rwo gukurikirana ibintu rukwiriye ibice birebire bya ccfDNA. Ubu buryo buzoroshya cyane kumenya ibimenyetso byinshi byo gusesengura byimbitse. Kubona urukurikirane rwuzuye rwa genome ya kirimbuzi ya A. atra rutaboneka ubu byanafasha cyane gutandukanya ccfDNA n'inkomoko yayo bwite n'iy'iyindi. Kubera ko ubushakashatsi bwacu bwibanze ku buryo bwo gukoresha igitekerezo cya biopsy y'amazi ku bimera, twizeye ko uko iki gitekerezo kizakoreshwa mu bushakashatsi bw'ejo hazaza, ibikoresho bishya n'imiyoboro bizakorwa kugira ngo byongere ubushobozi bw'ubu buryo bwo kwiga ubwoko butandukanye bw'udukoko two mu bimera.
Nk'ikimenyetso cy'ubuvuzi kidakoreshwa mu kwanduza abantu, urugero rwo hejuru rwa ccfDNA mu maraso y'umuntu rufitanye isano n'indwara zitandukanye, kwangirika kw'ingingo, n'imimerere y'imihangayiko [67,68,69]. Uku kwiyongera gufitanye isano no kurekurwa kw'ibice bya ADN byaturutseho nyuma yo kwangirika kw'ingingo. Twakemuye iki kibazo dukoresheje umuvuduko w'ubushyuhe bukabije, aho imineke yahuye n'ubushyuhe bwa dogere selisiyusi 30 mu gihe gito. Twakoze ubu bushakashatsi ku moko atatu atandukanye ya mineke mu bushakashatsi butatu bwigenga. Ariko, nta mpinduka twabonye ku rugero rwa ccfDNA nyuma yo gushyuha cyane (reba Ishusho S5, andi makuru). Ubu buvumbuzi bushobora gusobanura, nibura igice, ko imineke ifite sisitemu yo kuzenguruka amaraso kandi ikusanya ADN nyinshi z'amahanga bitewe n'imikorere yayo yo kuyungurura. Ku rundi ruhande, imineke, kimwe n'izindi nyamaswa nyinshi zidafite urutirigongo, zishobora kwihanganira kwangirika kw'ingingo bitewe n'imihangayiko, bityo bikagabanya kurekurwa kwa ccfDNA mu maraso yazo [70, 71].
Kugeza ubu, isesengura rya ADN ku binyabuzima mu bidukikije by’amazi ryibanze cyane cyane kuri metabarcoding ya ADN (eDNA) ku bidukikije. Ariko, ubu buryo bukunze kugarukira mu isesengura ry’ibinyabuzima iyo hakoreshejwe primers. Gukoresha uburyo bwo gutondeka shotgun bigabanya imbogamizi za PCR no guhitamo primers mu buryo bunyuranye. Bityo, mu buryo bumwe, uburyo bwacu bwegereye uburyo bwo gutondeka shotgun bwa eDNA bugezweho, bushobora gukurikirana ADN yacitsemo ibice no gusesengura hafi ya byose [72, 73]. Ariko, hari ibibazo byinshi by’ingenzi bitandukanya LB n’uburyo busanzwe bwa eDNA. Birumvikana ko itandukaniro rikomeye riri hagati ya eDNA na LB ari ugukoresha filter hosts karemano. Gukoresha amoko yo mu mazi nka sponge na bivalves (Dresseina spp.) nk’akayunguruzo karemano mu kwiga eDNA byatangajwe [74, 75]. Ariko, ubushakashatsi bwa Dreissena bwakoresheje biopsy y’inyama zakuwemo ADN. Isesengura rya ccfDNA iva muri LB ntirisaba biopsy y'inyama, ibikoresho byihariye ndetse rimwe na rimwe bihenze hamwe n'ibikoresho bifitanye isano na eDNA cyangwa biopsy y'inyama. Mu by'ukuri, duherutse gutangaza ko ccfDNA iva muri LB ishobora kubikwa no gusesengurwa hifashishijwe FTA nta gukonjesha, ibyo bikaba ari ikibazo gikomeye ku bushakashatsi mu turere twa kure [76]. Gukura ccfDNA mu biopsies by'amazi nabyo biroroshye kandi bitanga ADN nziza yo gukurikirana shotgun no gusesengura PCR. Ibi ni inyungu ikomeye ukurikije zimwe mu mbogamizi za tekiniki zijyanye no gusesengura eDNA [77]. Uburyo bworoshye kandi buhendutse bwo gukoresha uburyo bwo gupima ibintu nabwo bukwiriye cyane cyane muri gahunda zo kugenzura igihe kirekire. Uretse ubushobozi bwazo bwo kuyungurura bwinshi, ikindi kintu kizwi cyane cya bivalves ni imiterere ya mucopolysaccharide mu mucus wazo, ituma virusi ziyongera [78, 79]. Ibi bituma bivalves iba filter nziza karemano yo kugaragaza urusobe rw'ibinyabuzima n'ingaruka z'imihindagurikire y'ikirere mu rusobe runaka rwo mu mazi. Nubwo kuba hari ibice bya ADN bikomoka ku murwayi bishobora gufatwa nk'imbogamizi ku buryo ugereranije na eDNA, ikiguzi cyo kugira ccfDNA nk'iyo ugereranije na eDNA kirasobanuka icyarimwe bitewe n'amakuru menshi aboneka ku bushakashatsi bw'ubuzima. Ibi birimo kuba hari uruhererekane rwa virusi rwinjijwe muri genome y'umurwayi. Ibi ni ingenzi cyane ku missel, bitewe n'uko hari virusi zandurira mu maraso mu buryo butambitse muri bivalve [80, 81]. Indi nyungu ya LB kurusha eDNA ni uko ikoresha imikorere ya phagocytic y'uturemangingo tw'amaraso tuzenguruka muri hemolymph, ikangiza mikorobe (n'uturemangingo twazo). Phagocytosis ni yo nshingano nyamukuru y'uturemangingo tw'amaraso muri bivalve [82]. Hanyuma, ubu buryo bukoresha ubushobozi bwinshi bwo kuyungurura bwa mussels (impuzandengo ya 1.5 l / h y'amazi yo mu nyanja) n'inzira y'iminsi ibiri izenguruka, byongera uruvange rw'amazi atandukanye yo mu nyanja, bigatuma hafatwa heterologous eDNA. [83, 84]. Bityo, isesengura rya ccfDNA y'inyama ni inzira ishimishije bitewe n'ingaruka z'imirire, ubukungu, n'ibidukikije by'inyama. Kimwe n'isesengura rya LB ryakusanyijwe n'abantu, ubu buryo bunatanga amahirwe yo gupima impinduka za genetiki na epigenetic muri ADN y'umuntu mu rwego rwo gusubiza ibintu byo hanze. Urugero, ikoranabuhanga ryo gukurikirana imiterere y'ibisekuru bya gatatu rishobora gutekerezwaho kugira ngo rikore isesengura rya methylation ya genome muri ccfDNA gakondo hakoreshejwe uburyo bwa nanopore. Ubu buryo bugomba koroshywa n'uko uburebure bw'ibice bya ccfDNA y'inyama bujyanye neza n'uburyo bwo gukurikirana imiterere y'inyama bwasomewe igihe kirekire bwemerera isesengura rya methylation ya ADN ya genome kuva ku buryo bumwe bwo gukurikirana imiterere y'inyama bitabaye ngombwa ko habaho impinduka mu binyabutabire.85,86] Ibi ni ibintu bishimishije, kuko byagaragaye ko imiterere ya methylation ya ADN igaragaza igisubizo ku bibazo by'ibidukikije kandi bigakomeza mu bisekuru byinshi. Kubwibyo, bishobora gutanga ubumenyi bw'ingirakamaro ku buryo bw'ibanze bugenga igisubizo nyuma yo guhura n'imihindagurikire y'ikirere cyangwa imyuka mibi [87]. Ariko, ikoreshwa rya LB ntabwo ridafite imbogamizi. Birumvikana ko ibi bisaba ko habaho ubwoko bw'ibipimo mu rusobe rw'ibinyabuzima. Nkuko byavuzwe haruguru, gukoresha LB mu gusuzuma urusobe rw'ibinyabuzima mu rusobe runaka bisaba kandi uburyo bukomeye bwo gusesengura imiterere y'ibinyabuzima bushingiye ku binyabuzima, bukita ku kuba hari ibice bya ADN bikomoka aho byaturutse. Ikindi kibazo gikomeye ni ukuboneka kwa genome zishingiye ku binyabuzima byo mu mazi. Hifujwe ko ingamba nka Marine Mammal Genomes Project n'umushinga wa Fish10k uherutse gushyirwaho [88] bizoroshya isesengura nk'iryo mu gihe kizaza. Gukoresha igitekerezo cya LB ku binyabuzima bigaburira ibinyabuzima byo mu mazi bijyana n'iterambere rigezweho mu ikoranabuhanga ryo gukurikirana ibintu, bigatuma biba byiza cyane mu iterambere rya multi-ohm biomarkers kugira ngo bitange amakuru y'ingenzi ku buzima bw'aho bituye mu mazi mu rwego rwo guhangana n'ibibazo by'ibidukikije.
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