The oceans are still the most underexplored part of Earth. If diversity of large organisms in the Arctic Ocean has been in many ways investigated, research on the diversity of “Arctic” microorganisms is lacking. One of the problems, apart from inaccessibility, is obtaining high-quality microbial DNA: when working with natural samples, DNA often degrades — it is obtained in low quantities and can contain inhibitors interfering with sequencing.

As of today, there are many methods for isolating microbial DNA, but they are not directly optimized for marine samples. A lack of an understandable, proven methodology for this task slows down the study of marine microorganisms — at the same time, it is these studies that foster the search for new genes responsible for the synthesis of antibiotics or “genetic editors” enzymes. A research team from Skoltech, Moscow State University, and other leading scientific organizations, led by the head of the Laboratory of Metagenome Analysis Artem Isaev, identified the most effective kits for DNA isolation from different types of marine samples and presented results in a new paper in the Scientific Reports journal.

The waters of the northern seas of Russia have remained unexplored for a long time. “This is a hard-to-reach area, but very diverse, so we focused specifically on the Pacific and Arctic Oceans. Together with the Marine Research Center of Moscow State University, we organized expeditions and collected three types of samples: sea water and sediments, as well as samples of invertebrates,” said the leading author of the work Alina Demkina, a senior technician at Skoltech’s Laboratory of Metagenome Analysis.

According to the researchers, although metagenomic research is actively developing, there are still no common criteria and standards for working with marine samples. “Natural samples are very specific. Our goal was to isolate high-quality DNA, allowing long-reads sequencing. It turned out that there is no standard technique suitable for each type of marine sample. Creating it from scratch is a very long, expensive, and time-consuming task, so we focused on ready-made commercial kits and checked which of them could produce the best-quality result for different types of marine samples,” shared Darya Slonova, a co-author and a junior research scientist at Skoltech’s Laboratory of Metagenome Analysis.

The researchers tested eight DNA isolation kits. For each combination of the kit and sample, they measured the amount of purified DNA, the degree of its fragmentation, the presence of PCR-inhibiting pollutants, the admixture of eukaryotic DNA, alpha diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicon sequencing. Scientists have also identified the so-called “kitomes” — a composition of microbial taxa that may be present in the reagents used to isolate DNA. This is especially important, since the waters of the northern seas are quite poor in microbes, and therefore even a small admixture of contaminating DNA can affect the analysis.

“The article gives recommendations to scientists on which methods are best suited for isolation of high-quality DNA from marine samples,” continued Alina Demkina. “Researchers will learn which kits are suitable for each specific type of sample, depending on the goals of their work,” Darya Slonova supported.

 

Scheme of the study. Source: Benchmarking DNA isolation methods for marine metagenomics.

The research was supported by grant No. 075-10-2021-114 titled “Atlas of Microbial Communities of the Russian Federation” from the Ministry of Science and Higher Education of Russia and grant No. 22-14-00004 from the Russian Science Foundation titled “Search and Characterization of New Bacterial Immunity Systems and Viral Anti-Restriction Proteins.”

The Atlas of Microbial Communities of the Russian Federation is a large project of Skoltech’s Laboratory of Metagenome Analysis and Moscow State University. It seeks to study the diversity of microorganisms in Russia. It involves not only scientists, but a wide range of people — school and university students, and volunteers. Researchers organize expeditions and collect soil and water samples in hard-to-reach places, isolate metagenomic DNA (DNA of all microorganisms in the samples) and search for new antibiotics, isolating special biosynthetic clusters from metagenomes.

 

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