Biology subjects

Filipenko, M.

Publications and source records attributed to Filipenko, M..

6 recordsLinked to original sources

bioRxiv2025

Comparison of biochemical properties of RNases HII from E. coli, Geobacillus stearothermophilus and Thermus thermophilus

RNases H (EC 3.1.26.4) are a family of enzymes participating in removal of ribonucleotides from double-stranded DNA by hydrolyzing their phosphodiester bonds. Despite a long history of research, several important aspects of RNase HII functioning are poorly known including optimal pH, salts and thermal stability. This lack of empirical data hampers the selection of an optimal RNase HII for a specific practical application. In the present study, we compared biochemical properties of previously cloned RNases HII from E. coli, Geobacillus stearothermophilus and Thermus thermophilus: optimal temperature, pH, salts, divalent cofactors, thermal stability and specific activity. As expected, the most thermostable enzyme was Tth RNase HII, and Mg2+ was the most preferential cofactor for all RNases. Gst RNase HII was partially inhibited by K+ ions, while other enzymes did not demonstrate preferences for any salt. The enzymes from E. coli and G. stearothermophilus were typical RNases HII, while Tth RNase HII was a JRNase (junction ribonuclease). All three RNases did not cleave a DNA-RNA3-DNA2-RNA1-DNA/DNA substrate. The presented results will facilitate usage of RNases HII in practical applications and provide a basis for further comparative studies of RNases HII from various organisms.

molecular biology↗

bioRxiv2025

eLaRodON: identification of large genomic rearrangements in Oxford Nanopore sequencing data

Long-read sequencing enables more accurate detection of large genomic rearrangements (LGRs) compared to short-read technologies. However, existing tools for LGR calling continue to evolve and require further optimization. In this study, we present eLaRodON, a novel tool for identifying LGRs in Oxford Nanopore sequencing data. Using publicly available datasets--including Mycobacterium tuberculosis genomes with extensive complete genome references and the human cell line NA12878--we demonstrate that eLaRodON outperforms existing tools (NanoSV, Sniffles2, NanoVar, and SVIM), achieving an AUC of 0.61 (vs. 0.13-0.43) for M. tuberculosis and 0.86 (vs. 0.40-0.72) for the human genome. Validation against gold-standard LGR sets (derived from de novo Flye assemblies and Mauve alignments) confirmed the tools high accuracy. Notably, we identified recurrent false-positive LGR patterns across diverse datasets (M. tuberculosis, NA12878, and {lambda} phage controls). Orthogonal validation by targeted NGS and Sanger sequencing yielded a variant verification rate of 67-100% for different LGR types--including those supported by a single read. These results represent a significant advancement in LGR detection accuracy, with implications for genomic research and clinical applications. eLaRodON is an open-source program, and its code can be freely accessed on GitHub: https://github.com/aakechin/eLaRodON/.

genomics↗

bioRxiv2024

A novel approach for simultaneous detection of structural and single-nucleotide variants based on a combination of chromosome conformation capture and exome sequencing

Effective molecular diagnosis of congenital diseases hinges on comprehensive genomic analysis, traditionally reliant on various methodologies specific to each variant type--whole exome or genome sequencing for single nucleotide variants (SNVs), array CGH for copy-number variants (CNVs), and microscopy for structural variants (SVs). We introduce a novel, integrative approach combining exome sequencing with chromosome conformation capture, termed Exo-C. This method enables the concurrent identification of SNVs in clinically relevant genes and SVs across the genome and allows analysis of heterozygous and mosaic carriers. Enhanced with targeted long-read sequencing, Exo-C evolves into a cost-efficient solution capable of resolving complex SVs at base-pair accuracy. Through several case studies, we demonstrate how Exo-Cs multifaceted application can effectively uncover diverse causative variants and elucidate disease mechanisms in patients with rare disorders.

genomics↗

bioRxiv2023

Expanding the list of sequence-agnostic enzymes for chromatin conformation capture assays with S1 nuclease

This study presents a novel approach for mapping global chromatin interactions using S1 nuclease, a sequence-agnostic enzyme. We develop and outline a protocol that leverages S1 nucleases ability to effectively introduce breaks into both open and closed chromatin regions, allowing for comprehensive profiling of chromatin properties. Our S1 Hi-C method enables the preparation of high-quality Hi-C libraries, marking a significant advancement over previously established DNase I Hi-C protocols. Moreover, S1 nucleases capability to fragment chromatin to mono-nucleosomes suggests the potential for mapping the three-dimensional organization of the genome at high resolution. This methodology holds promise for an improved understanding of chromatin state-dependent activities and may facilitate the development of new genomic methods.

molecular biology↗

bioRxiv2020

NGS-PrimerPlex: high-throughput primer design for multiplex polymerase chain reactions

SummaryMultiplex PCR has multiple applications in molecular biology, including developing new targeted NGS-panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier and extension of existing NGS-panels. The primer design process takes into consideration formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome SNPs overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with median coverage of 97.4% of target regions by at least 30 reads. Availability and implementationNGS-PrimerPlex is open-source and freely available at https://github.com/aakechin/NGS-PrimerPlex/. Contactaa_kechin@niboch.nsc.ru Supplementary informationSupplementary data

bioinformatics↗

bioRxiv2019

Global variability of the human IgG glycome

Immunoglobulin G (IgG) is the most abundant serum antibody and is a key determinant of the humoral immune response. Its structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. IgG N-glycome patterns change with the age of individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is affected by a combination of genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of whole IgG, analysed in 5 different populations totalling 10,482 IgG glycomes, and of IgGs fragment crystallisable region (Fc), analysed in 2,530 samples from 27 populations sampled across the world. We observed that country of residence associates with many N-glycan features and is a strong predictor of monogalactosylation variability. IgG galactosylation also strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators. We found that subjects from developing countries had low IgG galactosylation levels, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to country-specific environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

biochemistry↗