In this work, we compare the resolution of V2-V3 and V3-V4 16S rRNA regions for the purposes of estimating microbial community diversity using paired-end Illumina MiSeq reads, and show that the fragment, including V2 and V3 regions, has higher resolution for lower-rank taxa (genera and species). It allows for a more precise distance-based clustering of reads into species-level OTUs. Statistically convergent estimates of the diversity of major species (defined as those that together are covered by 95% of reads) can be achieved at the sample sizes of 10000 to 15000 reads. The relative error of the Shannon index estimate for this condition is lower than 4%.

We present a metagenomic study of Lake Baikal (East Siberia). Two samples obtained from the water column under the ice cover (5 and 20 m deep) in March 2016 have been deep sequenced and the reads assembled to generate metagenome-assembled genomes (MAGs) that are representative of the microbes living in this special environment. Compared with freshwater bodies studied around the world, Lake Baikal had an unusually high fraction of Other groups, such as and , were in proportions similar to those found in other lakes. The genomes (and probably cells) tended to be small, presumably reflecting the extremely oligotrophic and cold prevalent conditions. Baikal microbes are novel lineages recruiting very little from other water bodies and are distantly related to other freshwater microbes. Despite their novelty, they showed the closest relationship to genomes discovered by similar approaches from other freshwater lakes and reservoirs. Some of them were particularly similar to MAGs from the Baltic Sea, which, although it is brackish, connected to the ocean, and much more eutrophic, has similar climatological conditions. Many of the microbes contained rhodopsin genes, indicating that, in spite of the decreased light penetration allowed by the thick ice/snow cover, photoheterotrophy could be widespread in the water column, either because enough light penetrates or because the microbes are already adapted to the summer ice-less conditions. We have found a freshwater SAR11 subtype I/II representative showing striking synteny with strains, as well as a phage infecting the widespread freshwater bacterium Despite the increasing number of metagenomic studies on different freshwater bodies, there is still a missing component in oligotrophic cold lakes suffering from long seasonal frozen cycles. Here, we describe microbial genomes from metagenomic assemblies that appear in the upper water column of Lake Baikal, the largest and deepest freshwater body on Earth. This lake is frozen from January to May, which generates conditions that include an inverted temperature gradient (colder up), decrease in light penetration due to ice, and, especially, snow cover, and oligotrophic conditions more similar to the open-ocean and high-altitude lakes than to other freshwater or brackish systems. As could be expected, most reconstructed genomes are novel lineages distantly related to others in cold environments, like the Baltic Sea and other freshwater lakes. Among them, there was a broad set of streamlined microbes with small genomes/intergenic spacers, including a new nonmarine-like (subtype I/II) genome.

Lake Baikal is the deepest lake in the world. Its depth provides the only bathypelagic (> 1000 m deep) freshwater habitat on Earth and its oxic, ultra-oligotrophic features make it a freshwater counterpart of the deep ocean. Here we have analyzed metagenomes from 1250 and 1350 m deep samples and built 231 metagenome-assembled genomes (MAGs). We detected high fractions of Thaumarchaeota (ca. 20% of 16S rRNA reads) and members of the candidate phyla radiation (CPR) (3-4.5%). Among the MAGs, we obtained ammonia-oxidizing archaea (AOA, Nitrosopumilaceae) and bacteria (AOB, Nitrosomonadaceae), and nitrite-oxidizers (Nitrospirae) indicating very active nitrification. A new clade of freshwater SAR202 Chloroflexi and methanotrophs (Methyloglobulus) were also remarkably abundant, the latter reflecting a possible role of methane oxidation as well. Novel species of streamlined and cosmopolitan bacteria such as Ca. Fonsibacter or acI Actinobacteria were more abundant at the surface but also present in deep waters. Conversely, CPRs, Myxococcales, Chloroflexi, DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota) archaea, or Gammaproteobacteria were found only in bathypelagic samples. We noted various important taxonomic and metabolic differences between deep aphotic region of Lake Baikal and marine waters of similar depth: Betaproteobacteriales, CPR, and DPANN superphylum were only found in bathypelagic Baikal, while Deltaproteobacteria, Gammaproteobacteria, or Alphaproteobacteria prevailed in oceanic samples. The genes mediating ammonia and methane oxidation, aromatic compound degradation, or alkane/methanesulfonate monooxygenases were detected in higher numbers in deep Baikal compared to their oceanic counterparts or its own surface. Overall, depth seems to be less relevant than salinity in configuring the microbial community.