Sequencing Methods Research Articles

Sequencing and Analysis of Wolbachia Strains from A and B Supergroups Detected in Sylvatic Mosquitoes from Brazil

Wolbachia are endosymbiotic bacteria that infect a wide range of arthropods and filarial nematodes, often manipulating host reproduction. The efficacy of Wolbachia-based interventions for dengue and chikungunya control has been validated through numerous field studies in recent years. This study aimed to investigate the diversity and prevalence of Wolbachia infections in sylvatic mosquitoes from two locations in Recife, Brazil. Multiple mosquito species were screened for Wolbachia using both target marker gene amplification coupled with Sanger sequencing and whole-genome sequencing (WGS) approaches. Phylogenetic analyses were conducted to classify Wolbachia strains into supergroups and assess their evolutionary relationships. Results revealed the presence of Wolbachia in eleven mosquito species examined, with different infection rates. Both supergroups A and B of Wolbachia strains were identified, with Aedes albopictus showing co-infection by both supergroups through the WGS approach. We also detected indirect evidence of Wolbachia horizontal transmission among mosquitoes and other distant host orders. This study provides valuable insights into the distribution and diversity of Wolbachia in sylvatic mosquitoes from Brazil and adds new important data about Wolbachia detection through target marker gene amplicon coupled with Sanger sequencing and WGS methods, highlighting its complementarity to ascertain the presence of Wolbachia in mosquito samples.

A novel approach to detecting microduplication in split hand/foot malformation type 3 at the single-cell level: SHFM as a case study

BackgroundSplit hand/foot malformation (SHFM) is a congenital limb deficiency characterized by missing or shortened central digits. Several gene loci have been associated with SHFM. Identifying microduplications at the single-cell level is challenging in clinical practice, and traditional detection methods may lead to misdiagnoses in embryos and pregnant women.ResultsIn this research, we utilized a low cell count and whole-genome amplification products to employ single nucleotide polymorphism arrays, next-generation sequencing, and third-generation sequencing methods to detect copy number variants of microduplications in a SHFM3 case with limited DNA. Additionally, Karyomapping and combined linkage analysis were conducted to validate the results.ConclusionsThis study establishes a new strategy for identifying microduplications or microdeletions at the single-cell level in clinical preimplantation genetic testing, enhancing the efficiency and accuracy of diagnosing microduplication or microdeletion diseases during IVF-PGT and prenatal diagnosis.

Detecting significant expression patterns in single-cell and spatial transcriptomics with a flexible computational approach

Gene expression data holds the potential to shed light on multiple biological processes at once. However, data analysis methods for single cell sequencing mostly focus on finding cell clusters or the principal progression line of the data. Data analysis for spatial transcriptomics mostly addresses clustering and finding spatially variable genes. Existing data analysis methods are effective in finding the main data features, but they might miss less pronounced, albeit significant, processes, possibly involving a subset of the samples. In this work we present SPIRAL: Significant Process InfeRence ALgorithm. SPIRAL is based on Gaussian statistics to detect all statistically significant biological processes in single cell, bulk and spatial transcriptomics data. The algorithm outputs a list of structures, each defined by a set of genes working simultaneously in a specific population of cells. SPIRAL is unique in its flexibility: the structures are constructed by selecting subsets of genes and cells based on statistically significant and consistent differential expression. Every gene and every cell may be part of one structure, more or none. SPIRAL also provides several visual representations of structures and pathway enrichment information. We validated the statistical soundness of SPIRAL on synthetic datasets and applied it to single cell, spatial and bulk RNA-sequencing datasets. SPIRAL is available at https://spiral.technion.ac.il/.

Vaginal Microbiome and the Risk of Preterm Birth in Women Living With HIV: A Scoping Review.

There are sparse data on the role of thevaginal microbiome (VMB) in pregnancy among pregnant women living with HIV (PWLWH) and its association with spontaneous preterm birth (sPTB). We conducted a scoping review to assess associations between vaginal microbiota and sPTB among PWLWH. Three studies were included, representing a total of 180 PWLWH out of 652 total pregnancies. All studies used modern DNA sequencing methods (16S rRNA amplification, metagenomics, or metatranscriptomics). PWLWH had higher VMB richness and diversity compared to HIV-uninfected pregnant women and higher sPTB rates in two of three studies. A higher proportion of sPTB among PWLWH was observed in those with Lactobacillus-deficient, anaerobe-dominant vaginal microbiota. In two of three studies, higher concentrations of vaginal inflammation markers were associated with increased VMB richness and diversity. HIV status was independently associated with sPTB. It is unclear if increased vaginal microbial diversity among PWLWH or increased vaginal inflammation contributes more to PTB, but HIV does appear to alter the VMB in pregnant individuals and may also affect PTB rates in microbiome-independent pathways. Given the limited number of studies, heterogeneity in sample size, sample collection methods, and inconsistent results it is difficult to causally link HIV, VMB, inflammatory cytokines, and sPTB.

Methodologies for the detection and sequencing of the epigenetic-like oxidative DNA modification, 8-oxo-7,8-dihydroguanine.

The human genome is constantly threatened by endogenous and environmental DNA damaging agents that can induce a variety of chemically modified DNA lesions including 8-oxo-7,8-dihydroguanine (OG). Increasing evidence has indicated that OG is not only a biomarker for oxidative DNA damage but also a novel epigenetic-like modification involved in regulation of gene expression in mammalian cells. Here we summarize the recent progress in OG research focusing on the following points: (i) the mechanism of OG production in organisms and its biological consequences in cells, (ii) the accurate identification of OG in low-abundance genomes and complex biological backgrounds, (iii) the development of OG sequencing methods. These studies will be helpful for further understanding of the molecular mechanisms of OG-induced mutagenesis and its potential roles in human development and diseases such as cancer.

First De Novo genome assembly and characterization of Gaultheria prostrata

Gaultheria Kalm ex L. (Ericaceae), a type of evergreen shrub, known as a natural source of methyl salicylate, possesses rich germplasm resources, strong habitat adaptability, significant ornamental value, and noteworthy pharmacological activities. However, due to the paucity of whole genomic information, genetically deep research in these areas remains limited. Consequently, we intend to obtain genome data through high-throughput sequencing, gene annotation, flow cytometry, transcription factors prediction and genetic marker analysis for a representative species of this genus, with Gaultheria prostrata selected for our study. In this study, we preliminarily obtained the genome of G. prostrata through next-generation sequencing methods. Utilizing 47.94 Gb of high-quality sequence data (108.95× coverage), assembled into 114,436 scaffolds, with an N50 length of 33,667 bp. The genome size assembled by SOAPdenovo, approximately 417 Mb, corresponded closely to predictions by flow cytometry (440 Mb) and k-mer analysis (447 Mb). The genome integrity was evaluated using BUSCO with 91%. The heterozygosity ratio was 0.159%, the GC content was 38.85%, and the repetitive regions encompassed over 34.6% of the genome. A total of 26,497 protein-coding genes have been predicted and annotated across Nr, Swissprot, GO, KEGG, and Pfam databases. Among these, 14,377 and 2,387 genes received functional annotation in Nr and Swissprot, respectively; 21,895, 24,424, and 22,330 genes were similarly annotated in GO, KEGG, and Pfam. Moreover, A total of 279,785 SSRs were identified and 345,270 primers for these SSRs were designed. Within the various nucleotide types of SSRs, AG/CT and AAG/CTT constituted the predominant dinucleotide and trinucleotide repeat types in G. prostrata. In addition, 1,395 transcription factors (TFs) from 75 TF families, 462 transcription regulators (TRs) from 33 TR families and 840 protein kinase (PKs) from 118 PK families were identified in this genome. We also performed phylogenetic analyses of G. prostrata and related species, including estimation of divergence times and expansion and contraction analyses, followed by positive selection analyses of orthologous gene pairs of G. prostrata and its close relative Vaccinium corymbosum. These results provide a reference for in-depth study of genus Gaultheria, contributing to future functional and comparative genomics analyses and providing supporting data for the development of molecular markers.

Effect of Vermicompost Application on the Soil Microbial Community Structure and Fruit Quality in Melon (Cucumis melo)

Melon (Cucumas melon) is widely cultivated and popular because of its quality value and unique flavor. However, the continuous cropping of melons in greenhouses has various negative effects on the soil environment, melon growth, and quality. Recently, farmers have utilized organic fertilization, especially vermicompost, for melons to resist the harmful effects of continuous cropping. A field experiment was conducted to explore the effects of vermicompost on soil microbes and melon fruit quality via high throughput sequencing and chemical sequencing methods. The results showed that the application of vermicompost decreased (p < 0.05) soil pH and increased organic matter, available phosphorus, biomass, urease, catalase, peroxidase, and alkaline phosphatase. A total of 3447 bacterial and 718 fungal operational taxonomic units were identified in all soil samples. Application of vermicompost decreased (p < 0.05) the relative abundances of Acidobacteriota, Gemmatimonadota, Actinobacteriota, and unclassified and increased the relative abundance of Planctomycetota. Compared with the control soil, vermicompost application resulted in significantly higher bacterial Chao indices and a significantly lower Chao index under vermicompost of 60 t ha−1 based on farmers’ normal fertilizer and significantly lower diversity under vermicompost of 90 t ha−1. Otherwise, vermicompost application increased the photosynthetic rate and chlorophyll content of melon leaves and increased the total sugar, soluble solids, vitamin C, soluble protein, and organic acid contents of melon. The results of redundancy analysis indicated that Proteobacteria exhibited a positive correlation with soil ammonium nitrogen (AN) and pH, while showing a negative association with soil available phosphorus and organic matter. Spearman’s correlation analysis revealed that both total sugar content and central soluble solid content in melon had a significant positive correlation (p < 0.05) with Patescibacteria. This study demonstrates that the application of vermicompost alters the microbial community structure in melon cultivation, enhancing fruit quality; this not only promotes a healthier soil ecosystem but also contributes to sustainable and productive practices in melon farming.

Case report: The first known case of male retroperitoneal mesonephric-like adenocarcinoma

AimWe aimed to analyze the clinico-pathological and molecular features of mesonephric-like adenocarcinoma (MLA) to enhance understanding of this tumor type.MethodsThis is the first case of MLA occurring in the retroperitoneum of a male patient. Clinico-pathological and molecular characteristics were analyzed, and the relevant literature was reviewed.ResultsA 65-year-old elderly male was admitted to the hospital with mild bilateral dull pain in the lumbar region for more than 1 month, accompanied by a feeling of dysuria. CT tomography revealed a retroperitoneal tumor. While tumor immuno-histochemistry was positive for CK, CK7, Vimentin, PAX-8, CD10, GATA-3, EMA, and CR to varying degrees, it was negative for P53, WT-1, HMB45, MelanA, CD117, DOG-1, CD34, S-100, ER, PR, AR, CEA, α-inhibin and TTF-1. Ki67 index was <10% in most areas and was approximately 30% in the hotspot areas in the glandular ductal region. Molecular detection (Next-generation sequencing method, 425-gene panel from NanjingShihe Gene Biotechnology Co., Ltd. for targeted DNA enrichment): No clinically significant variants detected. The final pathological diagnosis was a retroperitoneal malignant tumor consistent with a well-moderately differentiated MLA.ConclusionMLA in the retroperitoneum of men has not been reported yet. The diverse morphology and unclear molecular characteristics of this tumor mandate careful diagnosis for good clinical outcomes.

Bla SED-1 beta-lactamase-producing Citrobacter sedlakii isolated from horses and genomic comparison with human-derived isolates.

We aim to detect beta-lactamase-producing Citrobacter sedlakii from horses and compare the genomic characteristics with isolates from humans.Methods and Result: We characterized phenotypically and genotypically nine C. sedlakii isolates from the feces of horses and then compared them to human-derived isolates using whole genome sequencing and phylogenomic methods. Seven isolates (7/9) were ampicillin-resistant, while at least one isolate was resistant to ceftriaxone, gentamicin, meropenem, and streptomycin. All nine isolates were carriers of the chromosomal-mediated blaSED-1 beta-lactamase gene which confer resistance to ampicillin. One isolate was positive for the mcr-9 gene that confers resistance to colistin, and another isolate had the aac(6')-lid gene that confers resistance to aminoglycosides. Seven isolates (7/9) were carriers of genes that confer metal resistance to copper, silver, and arsenic. Phylogenetically, two horse-derived isolates clustered together with two human-derived isolates from the NDARO database. The results from our study provide insight into the antimicrobial susceptibility of C. sedlakii in horses which was previously lacking, and the specific beta-lactamase gene mediating resistance.

Differential redox sensitivity of tRNA dihydrouridylation.

Various transfer RNA (tRNA) modifications have recently been shown to regulate stress-dependent gene expression by modulating messenger RNA translation. Among these modifications, dihydrouridine stands out for its increase of tRNA structural flexibility. However, whether and how dihydrouridine synthesis reacts to environmental stimuli is largely unknown. In this study, we manipulated the intracellular redox state of Escherichia coli using paraquat, revealing differential sensitivities of the three tRNA-dihydrouridine synthases towards oxidative stress. Using liquid chromatography-mass spectrometryquantification of dihydrouridine in various knockout strains, we validated the use of a specific RNA sequencing method, namely AlkAnilineSeq, for the precise mapping of dihydrouridines throughout E. coli tRNAs. We found DusA showing high activity, followed by DusB and DusC, whose activity was decreased under paraquat treatment. The relative sensitivity is most plausibly explained by a paraquat-dependent drop of NADPH availability. These findings are substantiated by in vitro kinetics, revealing DusA as the most active enzyme, followed by DusB, while DusC showed little activity, likely related to the efficacy of the redox reaction of the flavin coenzyme with NADPH. Overall, our study underscores the intricate interplay between redox dynamics and tRNA modification processes, revealing a new facet of the regulatory mechanisms influencing cellular responses to oxidative stress.

Single Nucleotide Recognition and Mutation Site Sequencing Based on a Barcode Assay and Rolling Circle Amplification

Single nucleotide polymorphisms (SNPs) present significant challenges in microbial detection and treatment, further raising the demands on sequencing technologies. In response to these challenges, we have developed a novel barcode-based approach for highly sensitive single nucleotide recognition. This method leverages a dual-head folded complementary template probe in conjunction with DNA ligase to specifically identify the target base. Upon recognition, the system triggers rolling circle amplification (RCA) followed by the self-assembly of CdSe quantum dots onto polystyrene microspheres, enabling a single-particle fluorescence readout. This approach allows for precise base identification at individual loci, which are then analyzed using a bio-barcode array to screen for base changes across multiple sites. This method was applied to sequence a drug-resistant mutation site in Helicobacter pylori (H. pylori), demonstrating excellent accuracy and stability. Offering high precision, high sensitivity, and single nucleotide resolution, this approach shows great promise as a next-generation sequencing method.

Defining the transcriptome of PIK3CA-altered cells in a human capillary malformation using single cell long-read sequencing

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants—for example how they affect gene expression profiles—remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

Integrated Analysis of Metabolites and Microorganisms Reveals the Anthracnose Resistance Benefits from Cyanidin Mediated by Proteobacteria in Tea Plants

Anthocyanins, key quality components of tea, act as an important bridge between plants and the environment due to their function on protecting plants from biotic and abiotic irritants. This study aimed to assess the interactions between anthocyanins metabolism and the environment. Purple (P) and green (G) leaves with different anthocyanin contents were inoculated with tea plant anthracnose. High-throughput metabolomics and 16S microbial diversity sequencing methods were used to screen the anthocyanin fractions of tea plant leaves responsive to anthracnose. The interconnections between metabolites and the resistance of phyllosphere microorganisms to fungal pathogens were then analyzed. The results showed that leaves with high anthocyanin content (0.14% of diseased area ratio) were less impacted by anthracnose infestation than leaves with low anthocyanin (3.12%). The cyanidin content decreased after infection in purple leaves (PR) and increased in green leaves (GR). The relative abundance of Cyanobacteria was suppressed by the significant enrichment of Proteobacteria after anthracnose infection in green leaves. However, there were no significant differences between these two groups of microorganisms in purple leaves. Collinear network analysis revealed a strong correlation between Cyanobacteria and Dihydrosorbinol and between Proteobacteria and cyanidin metabolites. Among them, OTU456 (Bosea) was identified as the key taxonomic group of bacterial communities in the green-infected leaf network. In summary, the anthracnose resistance benefits from cyanidin mediated by proteobacteria in tea plants. These results deepen our understanding of the regulation of secondary metabolism in tea plants and the formation of plant resistance.

Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell “Flongle.” Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson’s disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r2 > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.

Soil Properties Regulate Soil Microbial Communities During Forest Succession in a Karst Region of Southwest China

Natural vegetation restoration has emerged as an effective and rapid approach for ecological restoration in fragile areas. However, the response of soil microorganisms to natural succession remains unclear. To address this, we utilized high-throughput sequencing methods to assess the dynamics of soil bacterial and fungal communities during forest succession (shrubland, secondary forest, and primary forest) in a karst region of Southwest China. Our study revealed that bacterial α-diversity was significantly higher in secondary forest compared to both shrubland and primary forest. Intriguingly, the soil bacterial community in primary forest exhibited a closer resemblance to that in shrubland yet diverged from the community in secondary forest. Conversely, the soil fungal community underwent notable variations across the different forest stages. Furthermore, analysis of the microbial co-occurrence network revealed that, within these karst forests, the relationships among soil fungi were characterized by fewer but stronger interactions compared to those among bacteria. Additionally, soil properties (including pH, soil organic carbon, total nitrogen, moisture, and available potassium), soil microbial biomass (specifically phosphorus and nitrogen), and plant diversity were the drivers of soil bacterial community dynamics. Notably, soil pH accounted for the majority of the variations observed in the soil fungal community during karst forest succession. Our findings provide valuable insights that can inform the formulation of strategies for ecological restoration and biodiversity conservation in karst regions, particularly from a microbial perspective.

Shotgun Sequencing of 512‐mer Copolyester Allows Random Access to Stored Information

AbstractDigital information encoded in polymers has been exclusively decoded by mass spectrometry. However, the size limit of analytes in mass spectrometry restricts the storage capacity per chain. In addition, sequential decoding hinders random access to the bits of interest without full‐chain sequencing. Here we report the shotgun sequencing of a 512‐mer sequence‐defined polymer whose molecular weight (57.3 kDa) far exceeds the analytical limit of mass spectrometry. A 4‐bit fragmentation code was implemented at aperiodic positions during the synthetic encoding of 512‐bit information without affecting storage capacity per chain. Upon activating the fragmentation code, the polymer chain splits into 18 oligomers, which could be individually decoded by tandem‐mass sequencing. These sequences were computationally reconstructed into a full sequence using an error‐detection method. The proposed sequencing method eliminates the storage limit of a single polymer chain and allows random access to the bits of interest without full‐chain sequencing.

Mitochondrial DNA Alterations in Glioblastoma and Current Therapeutic Targets.

Metabolic reprogramming within tumor cells involves a shift towards either glycolysis or mitochondrial respiration, depending on the stage of tumor progression. Consequently, irreversible dysfunction of the mitochondria is considered a crucial mechanism driving the progression mechanism. While numerous mutations in mitochondrial DNA (mtDNA) have been identified across various tumor types, including glioblastoma, many studies have been limited in the scope, focusing on small segments of mtDNA or utilizing sequencing methods with restricted sensitivity. As a result, several potentially significant mtDNA mutations may have been underestimated, along with their heteroplasmic states, which play a crucial role in determining the phenotypic impact of mtDNA mutation. Although both somatic and germline mtDNA mutations have been observed in different tumor types, research on the mtDNA mutations linked to glioblastoma remains scarce. The mitochondrial genome encodes thirteen protein-coding genes that are essential for the proper functioning of respiratory complex chains. Alterations in mitochondrial function manifest at various levels, including structural and functional changes, impacting mitogenic, hemodynamic, bioenergetic, and apoptotic signaling pathways. These alterations often signify a reduced efficiency of the oxidative phosphorylation system and energy production in tumor cells. As the crucial role of mitochondrial dysfunction in glioma development grows, mitochondria have emerged as promising targets for therapy aimed at overcoming chemoresistance and eliminating cancer cells. This brief review outlines the association between mtDNA alteration and glioblastoma, as well as the current advancements in therapeutic strategies targeting mtDNA alterations.

Real-time and programmable transcriptome sequencing with PROFIT-seq.

The high diversity and complexity of the eukaryotic transcriptome make it difficult to effectively detect specific transcripts of interest. Current targeted RNA sequencing methods often require complex pre-sequencing enrichment steps, which can compromise the comprehensive characterization of the entire transcriptome. Here we describe programmable full-length isoform transcriptome sequencing (PROFIT-seq), a method that enriches target transcripts while maintaining unbiased quantification of the whole transcriptome. PROFIT-seq employs combinatorial reverse transcription to capture polyadenylated, non-polyadenylated and circular RNAs, coupled with a programmable control system that selectively enriches target transcripts during sequencing. This approach achieves over 3-fold increase in effective data yield and reduces the time required for detecting specific pathogens or key mutations by 75%. We applied PROFIT-seq to study colorectal polyp development, revealing the intricate relationship between host immune responses and bacterial infection. PROFIT-seq offers a powerful tool for accurate and efficient sequencing of target transcripts while preserving overall transcriptome quantification, with broad applications in clinical diagnostics and targeted enrichment scenarios.

Generation and analysis of a mouse multitissue genome annotation atlas.

Generating an accurate and complete genome annotation for an organism is complex because the cells within each tissue can express a unique set of transcript isoforms from a unique set of genes. A comprehensive genome annotation should contain information on what tissues express what transcript isoforms at what level. This tissue-level isoform information can then inform a wide range of research questions as well as experiment designs. Long-read sequencing technology combined with advanced full-length cDNA library preparation methods has now achieved throughput and accuracy where generating these types of annotations is achievable. Here, we show this by generating a genome annotation of the mouse (Mus musculus). We used the nanopore-based R2C2 long-read sequencing method to generate 64 million highly accurate full-length cDNA consensus reads-averaging 5.4 million reads per tissue for a dozen tissues. Using the Mandalorion tool, we processed these reads to generate the Tissue-level Atlas of Mouse Isoforms which is available as a trackhub for the UCSC Genome Browser and contains at least one full-length isoform for the vast majority of expressed genes in each tissue.

Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization

The species of the genus Deschampsia are difficult for identification, and the genus is difficult for taxonomic treatment. The regions of 35S rRNA genes were studied for the species of the genus Deschampsia of different geographical origin with a method of sequencing by Sanger (ITS1–5.8S rRNA gene–ITS2, 14 species) and with a method of a locus-specific next-generation sequencing (NGS) on the Illumina platform (ITS1–5.8S rRNA, 7 species). All species of Deschampsia formed one clade; the species, referred by some authors on the basis of morphological characters to the species D. cespitosa s.l., entered one subclade. Subantarctic species formed a separate subclade and their ribotypes formed their own subnetwork. Avenella flexuosa, earlier referred to Deschampsia, entered the other clade, though this species contains some ribotypes common with some Deschampsia species. Deschampsia pamirica and related mountain species have their own specific ribotype groups. On the network of the ribotypes, one can see that D. cespitosa from Great Britain forms a network with some species, but D. cespitosa from the USA forms its own network. Ribotype analysis of each sample revealed traces of introgression with Deyeuxia/Calamagrostis in D. cespitosa and with A. flexuosa and probable introgression of Northern and subantarctic species.