aariПроблемы Арктики и АнтарктикиArctic and Antarctic Research0555-26482618-6713Государственный научный центр Российской Федерации Арктический и антарктический научно-исследовательский институт10.30758/0555-2648-2024-70-4-554-564aari-671Research ArticleЭКОЛОГИЯ, БИОЦЕНОЛОГИЯ И БИОГЕОГРАФИЯECOLOGY, BIOCENOLOGY AND BIOGEOGRAPHYThe uppermost water horizon of subglacial Lake Vostok could be microbial DNA-free, as shown by Oxford Nanopore sequencing technologyThe uppermost water horizon of subglacial Lake Vostok could be microbial DNA-free, as shown by Oxford Nanopore sequencing technologyhttps://orcid.org/0000-0002-2574-882XBulatS. А.BulatS. A.

Гатчина, Екатеринбург

Sergey A. Bulat

Gatchina, Ekaterinburg

https://orcid.org/0009-0000-0533-7284AnosovaO. I.AnosovaO. I.

Усть-Лабинск

Oksana I. Anosova

Ust-Labinsk

https://orcid.org/0009-0005-0749-3420TsvetkovaА. Yu.TsvetkovaA. Yu.

Гатчина, Санкт-Петербург

Anna Yu. Tsvetkova

Gatchina, St. Petersburg

https://orcid.org/0000-0003-1140-5751ShvetsovА. V.ShvetsovA. V.

Гатчина, Санкт-Петербург

Alexey V. Shvetsov

Gatchina, St. Petersburg

Петербургский институт ядерной физики им. Б.П. Константинова Национального исследовательского центра «Курчатовский институт»; Институт физики и технологии, Уральский федеральный университетРоссияPetersburg Nuclear Physics Institute named after B. P. Konstantinov of National Research Centre “Kurchatov Institute”; Institute of Physics and Technology, Ural Federal UniversityRussian FederationПервый университетский лицей имени Н.И. Лобачевского — филиал Московского государственного университета имени М.В. ЛомоносоваРоссияLobachevsky First University Lyceum, Lomonosov Moscow State University branch campusRussian FederationПетербургский институт ядерной физики им. Б.П. Константинова Национального исследовательского центра «Курчатовский институт»; Санкт-Петербургский политехнический университет Петра ВеликогоРоссияPetersburg Nuclear Physics Institute named after B. P. Konstantinov of National Research Centre “Kurchatov Institute”; Peter the Great St. Petersburg Polytechnic UniversityRussian Federation202421122024704Специальный выпуск Исследования подледникового озера Восток554564Copyright © Bulat S.А., Anosova O.I., Tsvetkova А.Y., Shvetsov А.V., 20242024Bulat S.А., Anosova O.I., Tsvetkova А.Y., Shvetsov А.V.Bulat S.A., Anosova O.I., Tsvetkova A.Y., Shvetsov A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.aaresearch.science/jour/article/view/671

Целью исследования был поиск микробной жизни в подледниковом антарктическом озере Восток путем изучения верхнего слоя воды, попавшей в скважину и замерзшей в ней после того, как озеро было вскрыто. Образец был получен из скважины на глубине 3721 м и состоял изо льда замерзшей озерной воды. Он был тщательно деконтаминирован, расплавлен в чистом помещении, и выделенная геномная ДНК была амплифицирована с использованием вырожденных праймеров, специфичных для области v3-v4 бактериальных генов 16S рРНК. Для секвенирования полученных ампликонов использовали метод Сэнджера и технологию высокопроизводительного секвенирования Oxford Nanopore. Анализ ДНК по методу Сэнджера выявил в общей сложности 16 бактериальных филотипов, из которых только один филотип, 3721v34-24, прошел все критерии на контаминацию. Этот филотип был доминирующим и включал 41,4 % клонов с тремя аллельными вариантами, но остался неклассифицированным, показав 87,7 % сходства с ближайшим таксоном в GenBank Mucilaginibacter daejeonensis NR_041505 из филума Bacteroidota (семейство Sphingobacteriaceae). Технология Oxford Nanopore секвенирования дала 21067 прочтений для образца 3721 м и 3780 прочтений для контроля. Из них 7203 (34 %) и 1988 (53 %) прочтений для образца льда и контроля, соответственно, были классифицированы с аккуратностью 93 %. Для образца 3721 м был идентифицирован 21 бактериальный филотип с численностью таксонов выше 0,5 %. Пятнадцать из них оказались общими с находками по Сэнгеру, а остальные шесть были уникальными, но присутствовали в нанопоровом контроле или оказались очевидными контаминантами. Пятнадцать филотипов, совпадающих с таковыми по Сэнджеру, были определены как контаминанты. Филотип по Сэнджеру 3721v34-24, который считался истинной находкой для воды озера, в нанопоровом секвенировании был обнаружен как в образце льда 3721 м, так и контроле, т. е. был также отнесен к контаминантам. Таким образом, самый верхний горизонт воды в озере Восток может не содержать микробной ДНК. Для прояснения этого вопроса проводятся дальнейшие исследования замерзших в скважине проб воды.

The research aimed to search for microbial life in subglacial Lake Vostok. This was done by examining the uppermost layer of water that entered the borehole and froze after the lake was accessed. The sample was collected from a depth of 3721 m and consisted of water-frozen re-cored ice. It underwent thorough decontamination and was melted successively in cold and cleanroom facilities. Genomic DNA was then isolated and amplified using v3-v4 16S rRNA bacterial gene region-specific degenerate primers. The Sanger method and high-throughput Oxford Nanopore sequencing were used to sequence the amplicons generated. The Sanger DNA analysis revealed 16 bacterial phylotypes, and only one of them, 3721v34-24, met all the contamination criteria. This phylotype was the dominant one, making up 41.4 % of the clones and consisting of three allelic variants. However, it remained unclassified and showed 87.7 % similarity to the closest GenBank entry, Mucilaginibacter daejeonensis NR_041505 of Bacteroidota (family Sphingobacteriaceae). The Oxford Nanopore technology generated 21067 reads for the 3721m sample and 3780 for the control one. Among these, 7203 (34 %) and 1988 (53 %) reads for the ice sample and the control one were classified with 93 % accuracy. For the 3721m sample, 21 bacterial phylotypes were identified with an abundance above 0.5 %. Fifteen were identical to the Sanger findings and identified as contaminants. The remaining six were different, either found in the control Nanopore trial or were apparent contaminants. The discovery of phylotype 3721v34-24 in the lake water by Sanger sequencing was unexpected. However, it was later detected in the 3721m sample and control experiments using nanopore sequencing, indicating it was also a contaminant. Thus, the research suggests that the topmost water layer in Lake Vostok may not contain any microbial DNA. Additional frozen-water samples are currently being analyzed to investigate the issue further.

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The authors declare that there are no conflicts of interest present.