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[MIS03-05] NO CLASSIFIED MICROBIAL FINDS IN THE SUBGLACIAL ANTARCTIC LAKE VOSTOK: SANGER FINDS IN THE OXFORD NANOPORE RE-SEQUENCING
Keywords:Antarctica, Subglacial Lake Vostok, Extremophiles, Contamination, Nanopore sequencing
Introduction
The objective was to search for microbial life in the subglacial freshwater Antarctic Lake Vostok by analyzing the uppermost water layer that entered the borehole following the successful lake unsealing at a depth of 3769m from the surface. The samples included the drillbit frozen and re-cored borehole-frozen water ice. The study aimed to re-evaluate previously microbial finds obtained with Sanger sequencing with the high throughput Oxford Nanopore sequencing technology.
Lake Vostok is a giant (270 x 70 km, 15800 km2 area), deep (up to 1.3km) freshwater liquid body buried in a graben beneath a 4-km thick East Antarctic Ice Sheet with the temperature near ice melting point (around -2.5oC) under 400 bar pressure. It is exceptionally oligotrophic and poor in primary chemical ions contents (comparable with surface snow), under the high dissolved oxygen tension (in the range of 320 – 1300 mg/L), with no light, and sealed from the surface biota about 15 Ma ago [1].
Previous Sanger Results
Water-frozen samples were shown to feature very dilute cell concentrations - from 167 to 38 cells per ml. Until recently, the 16S rRNA gene Sanger sequencing came up with four bacterial phylotypes, and all passed numerous contamination criteria. Two phylotypes were reported before [2] - hitherto unknown and phylogenetically unclassified phylotype w123-10 likely belonging to Parcubacteria Candidatus Adlerbacteria and 3429v3-4 showing below-genus level (93.5%) similarity with Herminiimonas glaciei of Oxalobacteraceae (Beta-Proteobacteria). The third find (phylotype 3698v46-27) proved to be conspecific with several species of Marinilactobacillus of Carnobacteriaceae (Firmicutes) featuring very similar 16S rRNA genes. Among them is M. piezotolerans isolated from a 4.15m deep sub-seafloor sediment core collected at 4790.7m deep Nankai Trough. The latest fourth find (phylotype 3721v34-24) remained again unclassified – 87.7% (below family-level) similarity with Mucilaginibacter daejeonensis NR_041505 of Bacteroidetes (Sphingobacteriaceae). All four bacterial phylotypes might represent ingenious microbial communities in the subglacial Lake Vostok.
Nanopore Results
To clarify the above-identified finds (Marinilactobacillus species), the ice-frozen water core 3698m was retested in high throughput nanopore sequencing using the same gDNA but another much broader v3-v4 region 16S rRNA gene amplicons. In the given study, the ice core counterpart 3715m (no finds with Sanger) was included along with nanopore controls (sham DNA isolation/negative PCR and nanopore reagents). It is worse to notice the unclassified/unidentified Sanger finds it seems impossible to retest in nanopore sequencing since they come in the unclassified pool.
The amplicons for the 3698m ice core generated about 240000 classified reads (61%) (trim barcodes and high-accuracy base-calling), for 3715m – about 178000 classified reads (61%), and the controls – 62500 classified reads (54%). As a result, plenty of identified species were found overlapping all three samples (two of ice and one of control), indicating their contaminant status. The Marinilactobacillus species were represented by six reads in the 3698m sample, by three reads in the 3715m sample, and as a surprise, by six reads in control. This could mean that the Sanger find Marinilactobacillus species in a 3698m water-frozen ice core is a false result.
Conclusion
When studying dilute very low biomass ice/snow samples for microbial contents, the core should be taken in interpreting the findings. The new high throughput Oxford Nanopore sequencing technology provides confident proof for classified finds. Nevertheless, three previously recorded unclassified bacterial phylotypes might represent ingenious cell populations in the subglacial Lake Vostok. The clarification of their status is a challenge to work on.
The reported study was partly funded by RFBR research project No.20-55-12006.
References
[1] Bulat et Petit 2022 In: Gargaud M. et al. (eds) Encyclopedia of Astrobiology. Springer, Berlin, Heidelberg. Pp. 1-7. https://doi.org/10.1007/978-3-642-27833-4_1765-3
[2] Bulat 2016 Phil Trans Royal Soc A Math Phys Eng Sci 374 (2059) 20140292
The objective was to search for microbial life in the subglacial freshwater Antarctic Lake Vostok by analyzing the uppermost water layer that entered the borehole following the successful lake unsealing at a depth of 3769m from the surface. The samples included the drillbit frozen and re-cored borehole-frozen water ice. The study aimed to re-evaluate previously microbial finds obtained with Sanger sequencing with the high throughput Oxford Nanopore sequencing technology.
Lake Vostok is a giant (270 x 70 km, 15800 km2 area), deep (up to 1.3km) freshwater liquid body buried in a graben beneath a 4-km thick East Antarctic Ice Sheet with the temperature near ice melting point (around -2.5oC) under 400 bar pressure. It is exceptionally oligotrophic and poor in primary chemical ions contents (comparable with surface snow), under the high dissolved oxygen tension (in the range of 320 – 1300 mg/L), with no light, and sealed from the surface biota about 15 Ma ago [1].
Previous Sanger Results
Water-frozen samples were shown to feature very dilute cell concentrations - from 167 to 38 cells per ml. Until recently, the 16S rRNA gene Sanger sequencing came up with four bacterial phylotypes, and all passed numerous contamination criteria. Two phylotypes were reported before [2] - hitherto unknown and phylogenetically unclassified phylotype w123-10 likely belonging to Parcubacteria Candidatus Adlerbacteria and 3429v3-4 showing below-genus level (93.5%) similarity with Herminiimonas glaciei of Oxalobacteraceae (Beta-Proteobacteria). The third find (phylotype 3698v46-27) proved to be conspecific with several species of Marinilactobacillus of Carnobacteriaceae (Firmicutes) featuring very similar 16S rRNA genes. Among them is M. piezotolerans isolated from a 4.15m deep sub-seafloor sediment core collected at 4790.7m deep Nankai Trough. The latest fourth find (phylotype 3721v34-24) remained again unclassified – 87.7% (below family-level) similarity with Mucilaginibacter daejeonensis NR_041505 of Bacteroidetes (Sphingobacteriaceae). All four bacterial phylotypes might represent ingenious microbial communities in the subglacial Lake Vostok.
Nanopore Results
To clarify the above-identified finds (Marinilactobacillus species), the ice-frozen water core 3698m was retested in high throughput nanopore sequencing using the same gDNA but another much broader v3-v4 region 16S rRNA gene amplicons. In the given study, the ice core counterpart 3715m (no finds with Sanger) was included along with nanopore controls (sham DNA isolation/negative PCR and nanopore reagents). It is worse to notice the unclassified/unidentified Sanger finds it seems impossible to retest in nanopore sequencing since they come in the unclassified pool.
The amplicons for the 3698m ice core generated about 240000 classified reads (61%) (trim barcodes and high-accuracy base-calling), for 3715m – about 178000 classified reads (61%), and the controls – 62500 classified reads (54%). As a result, plenty of identified species were found overlapping all three samples (two of ice and one of control), indicating their contaminant status. The Marinilactobacillus species were represented by six reads in the 3698m sample, by three reads in the 3715m sample, and as a surprise, by six reads in control. This could mean that the Sanger find Marinilactobacillus species in a 3698m water-frozen ice core is a false result.
Conclusion
When studying dilute very low biomass ice/snow samples for microbial contents, the core should be taken in interpreting the findings. The new high throughput Oxford Nanopore sequencing technology provides confident proof for classified finds. Nevertheless, three previously recorded unclassified bacterial phylotypes might represent ingenious cell populations in the subglacial Lake Vostok. The clarification of their status is a challenge to work on.
The reported study was partly funded by RFBR research project No.20-55-12006.
References
[1] Bulat et Petit 2022 In: Gargaud M. et al. (eds) Encyclopedia of Astrobiology. Springer, Berlin, Heidelberg. Pp. 1-7. https://doi.org/10.1007/978-3-642-27833-4_1765-3
[2] Bulat 2016 Phil Trans Royal Soc A Math Phys Eng Sci 374 (2059) 20140292