Single RNA Molecules Research Articles

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria tenuissima.

In this study, a novel positive-sense single-stranded RNA (+ ssRNA) mycovirus, Alternaria tenuissima mitovirus 1 (AtMV1), was identified in Alternaria tenuissima strain YQ-2-1, a phytopathogenic fungus causing leaf blight on muskmelon. The genome of AtMV1 is a single RNA molecule that is 3013 nt in length with an A + U content of 66.58% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a 313-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular mass of 35.48 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5' and 3' untranslated regions were predicted to fold into stem-loop and panhandle secondary structures. The results of a BLASTp search revealed that the amino acid (aa) sequence of RdRp of AtMV1 shared the highest sequence similarity (51.04% identity) with that of Sichuan mito-like virus 30, a member of the genus Duamitovirus within the family Mitoviridae. Phylogenetic analysis based on the aa sequence of the RdRp suggested that AtMV1 is a novel member of the genus Duamitovirus. To our knowledge, this is the first report of the complete genome sequence of a new mitovirus infecting A. tenuissima.

Regulation of KLRC and Ceacam gene expression by miR-141 supports cell proliferation and metastasis in cervical cancer cells

IntroductionMicroRNAs (miRNAs) are single RNA molecules that act as global regulators of gene expression in mammalian cells and thus constitute attractive targets in treating cancer. Here we aimed to investigate the possible involvement of miRNA-141 (miR-141) in cervical cancer and to identify its potential targets in cervical cancer cell lines.MethodsThe level of miR-141 in HeLa and C-33A cells has been assessed using the quantitative real-time PCR (qRT-PCR). A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HeLa cells. The protein profile of killer-like receptor C1 (KLRC1), KLRC3, carcinoembryonic antigen‐related cell adhesion molecule 3 (CAM3), and CAM6 was investigated in HeLa cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced cytokines from transfected HeLa cells.ResultsThe expression of miR-141 significantly increased in HeLa and C-33A cells compared to the normal cervical HCK1T cell line. Transfection of HeLa cells with an inhibitor, antagonist miR-141, showed a potent effect on cancer cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HeLa cells overexpressed miR-141 provided a hundred of downregulated genes, including KLRC1, KLRC3, CAM3, and CAM6. KLRC1 and KLRC3 expression profiles markedly depleted in HeLa cells transfected with miR-141 overexpression accompanied by decreasing interleukin 8 (IL-8), indicating the role of miR-141 in avoiding programmed cells death in HeLa cells. Likewise, CAM3 and CAM6 expression reduced markedly in miR-141 transduced cells accompanied by an increasing level of transforming growth factor beta (TGF-β), indicating the impact of miR-141 in cancer cell migration. The IntaRNA program and miRWalk were used to check the direct interaction and potential binding sites between miR-141 and identified genes. Based on this, the seeding regions of each potential target was cloned upstream of the luciferase reporter gene in the pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HeLa cells pre-transfected with miR-141 overexpression vector, while increasing enormously in cells pre-transfected with miR-141 specific inhibitor.ConclusionTogether, these data uncover an efficient miR-141-based mechanism that supports cervical cancer progression and identifies miR-141 as a credible therapeutic target.

Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

Single-molecule live-cell RNA imaging with CRISPR-Csm.

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Complete genome sequence and phylogenetic analysis of a newly discovered fusagra-like virus infecting Carica papaya in Ecuador.

A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae.

NanoMUD: Profiling of pseudouridine and N1-methylpseudouridine using Oxford Nanopore direct RNA sequencing

Nanopore direct RNA sequencing provided a promising solution for unraveling the landscapes of modifications on single RNA molecules. Here, we proposed NanoMUD, a computational framework for predicting the RNA pseudouridine modification (Ψ) and its methylated analog N1-methylpseudouridine (m1Ψ), which have critical application in mRNA vaccination, at single-base and single-molecule resolution from direct RNA sequencing data. Electric signal features were fed into a bidirectional LSTM neural network to achieve improved accuracy and predictive capabilities. Motif-specific models (NNUNN, N = A, C, U or G) were trained based on features extracted from designed dataset and achieved superior performance on molecule-level modification prediction (Ψ models: min AUC = 0.86, max AUC = 0.99; m1Ψ models: min AUC = 0.87, max AUC = 0.99). We then aggregated read-level predictions for site stoichiometry estimation. Given the observed sequence-dependent bias in model performance, we trained regression models based on the distribution of modification probabilities for sites with known stoichiometry. The distribution-based site stoichiometry estimation method allows unbiased comparison between different contexts. To demonstrate the feasibility of our work, three case studies on both in vitro and in vivo transcribed RNAs were presented. NanoMUD will make a powerful tool to facilitate the research on modified therapeutic IVT RNAs and provides useful insight to the landscape and stoichiometry of pseudouridine and N1-pseudouridine on in vivo transcribed RNA species.

Ribosome decision graphs for the representation of eukaryotic RNA translation complexity.

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, within both annotated protein-coding and noncoding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term ribosome decision graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the latter "translons." Nondeterministic events, such as initiation, reinitiation, selenocysteine insertion, or ribosomal frameshifting, are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions and for analyzing genetic variation and quantitative genome-wide data on translation for characterization of regulatory modulators of translation.

Detecting m6A at single-molecular resolution via direct RNA sequencing and realistic training data.

Direct RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. Here, we report on the synthesis of such samples and the development of a bespoke algorithm, mAFiA (m6A Finding Algorithm), that accurately detects single m6A nucleotides in both synthetic RNAs and natural mRNA on single read level. Our approach uncovers distinct modification patterns in single molecules that would appear identical at the ensemble level. Compared to existing methods, mAFiA also demonstrates improved accuracy in measuring site-level m6A stoichiometry in biological samples.

SARS-CoV-2 viral liver aggregates and scarce parenchymal infection implicate systemic disease as a driver of abnormal liver function.

Liver function tests (LFTs) are elevated in >50% of hospitalized individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with increased enzyme levels correlating with a more severe COVID-19 course. Despite these observations, evaluations of viral presence within liver parenchyma and viral impact on liver function remain controversial. Our work is a comprehensive immunopathological evaluation of liver tissue from 33 patients with severe, and ultimately fatal, cases of SARS-CoV-2 infection. Coupled with clinical data, we reveal the absence of SARS-CoV-2 infection in cholangiocytes and hepatocytes despite dramatic systemic viral presence. Critically, we identify significant focal viral sinusoidal aggregates in 2/33 patients and single viral RNA molecules circulating in the hepatic sinusoids of 15/33 patients. Utilizing co-immunofluorescence, focal viral liver aggregates in patients with COVID-19 were colocalized to platelet and fibrin clots, indicating the presence of virus-containing sinusoidal microthrombi. Furthermore, this patient cohort, from the initial months of the COVID-19 pandemic, demonstrates a general downtrend of LFTs over the course of the study timeline and serves as a remarkable historical time point of unattenuated viral replication within patients. Together, our findings indicate that elevated LFTs found in our patient cohort are not due to direct viral parenchymal infection with SARS-CoV-2 but rather likely a consequence of systemic complications of COVID-19. This work aids in the clinical treatment considerations of patients with SARS-CoV-2 as therapies for these patients may be considered in terms of their direct drug hepatotoxity rather than worsening hepatic function due to direct infection.

Chromogenic Visualization of Single RNA Molecules In Situ with Duplex Capability by Rolling Circle Amplification with Alkaline Phosphatase.

Visualizing spatial patterns of gene expression by optical microscopy at single-molecule resolution represents a long-standing challenge for imaging and molecular engineering technologies. In this study, we developed a method for visualizing mRNA with duplex capability by optical microscopy using rolling circle amplification with streptavidin-modified alkaline phosphatase (SA-ALP) to provide highly selective and sensitive RNA detection. ALP-based RNA detection provides comparable sensitivity and specificity to fluorescence-based in situ assays and similar performance to the current RNAscope technique for single-molecule RNA detection, but with improved ease of operation. This versatile and relatively user-friendly method of single-molecule RNA visualization can also overcome common problems of background interference. Our findings show that the red spots generated by the Fast Red staining in situ are readily quantified by image analysis, even in samples with heavy melanin deposition, supporting the clinical translation of this assay to improve diagnostic assays for recalcitrant tissues. This system was adaptable for duplex assays with multiple probes and multiple stains, which is ALP with horseradish peroxidase to produce red and brown signals to simultaneously visualize two different RNA targets. The duplex assay can be successfully applied to quantify mRNA expression from two genes in situ within single cells and multiple cell types. With the advantages of high sensitivity and low hardware requirements, this versatile and user-friendly method of RNA visualization may enable low-resource institutions to conduct previously inaccessible diagnostic or research questions about the in situ expression and distribution of RNAs at single-molecule resolution.

Visualizing and counting single RNA molecules in intact plant tissues.

Visualizing and counting single RNA molecules in intact plant tissues.

Coordination of RNA modifications in the brain and beyond.

Gene expression regulation is a critical process throughout the body, especially in the nervous system. One mechanism by which biological systems regulate gene expression is via enzyme-mediated RNA modifications, also known as epitranscriptomic regulation. RNA modifications, which have been found on nearly all RNA species across all domains of life, are chemically diverse covalent modifications of RNA nucleotides and represent a robust and rapid mechanism for the regulation of gene expression. Although numerous studies have been conducted regarding the impact that single modifications in single RNA molecules have on gene expression, emerging evidence highlights potential crosstalk between and coordination of modifications across RNA species. These potential coordination axes of RNA modifications have emerged as a new direction in the field of epitranscriptomic research. In this review, we will highlight several examples of gene regulation via RNA modification in the nervous system, followed by a summary of the current state of the field of RNA modification coordination axes. In doing so, we aim to inspire the field to gain a deeper understanding of the roles of RNA modifications and coordination of these modifications in the nervous system.

SARS-CoV-2 infection activates the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes pathway in the lung: a review

The infection of COVID-19 is directly linked to the destruction of lung epithelial cells, and the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) system has been implicated in the pathology of respiratory infections. This study aimed to systematize the relationship between the pathophysiology of COVID-19 and the cGAS-STING system’s activation in the lungs. Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is an RNA virus that belongs to the Coronaviridae family whose genetic material is produced by a single positive RNA molecule (RNA+). The cGAS-STING signaling pathway has emerged as a key mediator of injury caused by infection and cellular or tissue stress. The cGAS-STING cyclic pathway is part of innate immunity and is activated from cytosolic DNA responses present in newly formed syncytia, by cell-to-cell fusion, in target of angiotensin-converting enzyme 2 (ACE2) expression and SARS-CoV-2 Spike protein. Although this pathway is canonically understood to be responsive to both pathogen-derived and host-derived DNA, it has been demonstrated to cross-communicate with the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). cGAS-STING activation is significant to interferon production, mainly type-I interferons (IFN-I), in a SARS-CoV-2 infection scenario, indicating a major antiviral role of the cGAS-STING pathway. It was identified that in SARS-CoV-2 the cGAS-STING axis is activated, but the inflammatory response could be specific for nuclear factor-κB (NF-κB) in infected cells, and that this axis is potentiated by a cytokine storm produced by the immune system’s cells.

Real-time tracking of transcription-translation coupling.

In prokaryotic cells, transcription and translation occur in the same compartment, enabling a functional interaction between both gene expression machineries. Several studies, including recent high-resolution structures, demonstrate a physical link that establishes coupling between both machineries either mediated by direct interactions between the RNAP and the ribosomeor by factor-mediated interactions via the transcription factors NusA and NusG. In contrast, recent work challenges the notion that transcription and translation are generally coupled. Here, to understand what promotes coupling and how it is regulated, we reconstituted in vitro the complete active transcription-translation coupling system and developed multicolor single-molecule fluorescence microscopy assays to track in real-time how single RNA molecules are actively transcribed (Duss et al., Cell, 2019) and simultaneously translated by the ribosome. Furthermore, by site-specifically labeling the ribosome and the RNAP, we can detect the direct physical interaction between both machineries. Our data shows that physical coupling between RNAP and the ribosome can be transient and depends on the mRNA length between both machineries. We also detect different coupled states while the polymerase moves away from the ribosome, likely representing the different states found by cryoEM (Wang & Ebright, Science, 2020; Webster & Weixlbaumer, Science, 2020; O’Reilly & Rappsilber, Science, 2020). RNAP and the ribosome can also remain physically coupled even if the transcription machinery moves more than 100 nucleotides away from the ribosome suggesting that physical and functional coupling can be unrelated. Overall, by tracking the conformation and interaction dynamics of different macromolecular machines over time, our methods allow us to investigate how different macromolecular machines cooperate to fine-tune gene expression.

Direct observation of RNA structure and dynamics in repeat-RNA assemblies using single molecule fluorescence microscopy.

Huntington's disease (HD), a fatal neurodegeneration, results from somatic expansions of CAG repeats in exon-1 of the Huntingtin (HTT) gene. Genome-wide association studies (GWAS) implicate age of onset (AO) of HD to strongly depend on the length of uninterrupted CAG repeats with certain variations in flanking sequences acting as an AO enhancer or an AO delayer. Stable, GC-rich hairpins formed by expanded CAG repeats in the HTT-mRNAs are thought to trigger dysregulated protein sequestration and aberrant assembly of cytoplasmic and nuclear repeat-RNA-protein (RNP) aggregates. While aggregation is observed to be stochastic in cells, the mechanism of how repeat-RNA aggregate assembly is initiated remains unknown. Specifically, it remains unclear how the extended hairpin structures interact intermolecularly to aggregate and to what extents proteins contribute to RNA-RNA aggregation. Furthermore, it is unknown how sequence variations to the HTT-mRNA affects its aggregation propensities. To address these aspects of repeat-RNP aggregate assembly, we use in vitro transcribed HTT exon-1 RNA containing normal, expanded and interrupted CAG repeats to form repeat-RNA assemblies in vitro. Using a single molecule fluorescence microscopy method, we monitor, in real time, the dynamics and structural changes of single HTT RNA molecules bound to phase separated HTT RNA assemblies. Additionally, we probe how the RNA binding dynamics are affected by HTT-RNA flanking sequences.This study aims to provide mechanistic insights into how expanded repeat-RNP aggregate assembly is initiated in order to understand how physical interactions between triplet-repeat containing RNAs contributes to the assembly of macromolecular repeat-RNP structures implicated in neuronal toxicity. The outcome from the proposed study has potential to develop novel therapeutic target(s) for designing new approaches to prevent aberrant RNP foci formation.

Pseudoknots in RNA Structure Prediction.

RNA molecules play active roles in the cell and are important for numerous applications in biotechnology and medicine. The function of an RNA molecule stems from its structure. RNA structure determination is time consuming, challenging, and expensive using experimental methods. Thus, much research has been directed at RNA structure prediction through computational means. Many of these methods focus primarily on the secondary structure of the molecule, ignoring the possibility of pseudoknotted structures. However, pseudoknots are known to play functional roles in many RNA molecules or in their method of interaction with other molecules. Improving the accuracy and efficiency of computational methods that predict pseudoknots is an ongoing challenge for single RNA molecules, RNA-RNA interactions, and RNA-protein interactions. To improve the accuracy of prediction, many methods focus on specific applications while restricting the length and the class of the pseudoknotted structures they can identify. In recent years, computational methods for structure prediction have begun to catch up with the impressive developments seen in biotechnology. Here, we provide a non-comprehensive overview of available pseudoknot prediction methods and their best-use cases. © 2023 Wiley Periodicals LLC.

FRET probe for detecting two mutations in one EGFR mRNA.

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes.

Researchers have used RNA in situ hybridization to detect the presence of RNA in cells and tissues for approximately 50years. The recent development of a method capable of visualizing a single RNA molecule by utilizing tiled fluorescently labeled oligonucleotide probes that together produce a diffraction-limited spot has greatly increased the resolution of this technique, allowing for the precise determination of subcellular RNA localization and relative abundance. Here, we present our method for single molecule RNA fluorescence in situ hybridization (smFISH) in whole mount Drosophila testes and discuss how we have utilized this method to better understand the expression pattern of the highly unusual Y-linked genes.

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection.

While injection molding is becoming the fabrication modality of choice for high-scale production of microfluidic devices, especially those used for in vitro diagnostics, its translation into the growing area of nanofluidics (structures with at least one dimension <100 nm) has not been well established. Another prevailing issue with injection molding is the high startup costs and the relatively long time between device iterations making it in many cases impractical for device prototyping. We report, for the first time, functional nanofluidic devices with dimensions of critical structures below 30 nm fabricated by injection molding for the manipulation, identification, and detection of single molecules. UV-resin molds replicated from Si masters served as mold inserts, negating the need for generating Ni-mold inserts via electroplating. Using assembled devices with a cover plate via hybrid thermal fusion bonding, we demonstrated two functional thermoplastic nanofluidic devices. The first device consisted of dual in-plane nanopores placed at either end of a nanochannel and was used to detect and identify single ribonucleotide monophosphate molecules via resistive pulse sensing and obtain the effective mobility of the molecule through nanoscale electrophoresis to allow its identification. The second device demonstrated selective binding of a single RNA molecule to a solid phase bioreactor decorated with a processive exoribonuclease, XRN1. Our results provide a simple path towards the use of injection molding for device prototyping in the development stage of any nanofluidic or even microfluidic application, through which rapid scale-up is made possible by transitioning from prototyping to high throughput production using conventional Ni mold inserts.

Personalized medicine: the role of sequencing technologies in diagnostics, prediction and selection of treatment of monogenous and multifactorial diseases

The review highlights various methods for deciphering the nucleotide sequence (sequencing) of nucleic acids and their importance for the implementation of the three main principles of personalized medicine: prevention, predictability and personalization. The review, along with its own practical examples, considers three generations of sequencing technologies: 1) sequencing of cloned or amplified DNA fragments according to Sanger and its analogues; 2) massive parallel sequencing of DNA libraries with short reads (NGS); and 3) sequencing of single molecules of DNA and RNA with long reads. The methods of whole genome, whole exome, targeted, RNA sequencing and sequencing based on chromatin immunoprecipitation are also discussed. The advantages and limitations of the above methods for diagnosing monogenic and oncological diseases, as well as for identifying risk factors and predicting the course of socially significant multifactorial diseases are discussed. Using examples from clinical practice, algorithms for the application and selection of sequencing technologies are demonstrated. As a result of the use of sequencing technologies, it has now become possible to determine the molecular mechanism of the development of monogenic, orphan and multifactorial diseases, the knowledge of which is necessary for personalized patient therapy. In science, these technologies paved the way for international genome projects — the Human Genome Project, the HapMap, 1000 Genomes Project, the Personalized Genome Project, etc.