The uppermost water horizon of subglacial Lake Vostok could be microbial DNA-free, as shown by Oxford Nanopore sequencing technology

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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