Sequencing Of Fragments Research Articles

Community Structure and Diversity of Endophytic Bacteria in Melon (Cucumis melo L.) Seeds

Endophytic bacteria play a crucial role in the life cycle of Spermatophyta, and exploring the beneficial interactions between seeds and endophytic bacteria is an effective strategy for sustainable agricultural development and improved crop productivity. In this study, we employed Illumina Hiseq high-throughput sequencing technology to analyze the amplified fragments of microbial 16S rRNA sequences from melon seeds of MR-1, HH and their F1 generation, which displayed varying levels of resistance to bacterial fruit blotch disease. Initially, we extracted endophytes from eight melon samples and performed sequencing, obtaining an average of 84,758 effective tags per sample. At the 97% similarity level, the number of operational taxonomic units (OTUs) derived from melon seed endophytes ranged from 447 to 492. After filtering out low-level OTUs, we obtained an average of 65,866 valid tags from the eight samples, representing approximately 160 genera and 167 species across eight phyla. It was found that the composition of the endophyte communities in the eight samples was basically similar, and their abundance showed some variability. Furthermore, we utilized the KO and COG databases to functionally predict the genes of endophytes in melon seeds. The analysis revealed a significant enrichment of genes associated with pathways related to protein synthesis, carbohydrate metabolism, substance transport, and metabolite synthesis. These findings greatly contribute to our understanding of endophytic bacteria in melon seeds and serve as a vital reference for the selection and breeding of high-quality melon seeds, as well as the prevention of seed diseases.

Construction of a high-density genetic map and mapping of a spike length locus for rye.

Genetic maps provide the foundation for QTL mapping of important traits of crops. As a valuable food and forage crop, rye (Secale cereale L., RR) is also one of the tertiary gene sources of wheat, especially wild rye, Secale cereale subsp. segetale, possessing remarkable stress tolerance, tillering capacity and numerous valuable traits. In this study, based on the technique of specific-locus amplified fragment sequencing (SLAF-seq), a high-density single nucleotide polymorphism (SNP) linkage map of the cross-pollinated (CP) hybrid population crossed by S. cereale L (female parent) and S. cereale subsp. segetale (male parent) was successfully constructed. Following preprocessing, the number of 1035.11 M reads were collected and 2425800 SNP were obtained, of which 409134 SNP were polymorphic. According to the screening process, 9811 SNP markers suitable for constructing linkage groups (LGs) were selected. Subsequently, all of the markers with MLOD values lower than 3 were filtered out. Finally, an integrated map was constructed with 4443 markers, including 1931 female mapping markers and 3006 male mapping markers. A major quantitative trait locus (QTL) linked with spike length (SL) was discovered at 73.882 cM on LG4, which explained 25.29% of phenotypic variation. Meanwhile two candidate genes for SL, ScWN4R01G329300 and ScWN4R01G329600, were detected. This research presents the first high-quality genetic map of rye, providing a substantial number of SNP marker loci that can be applied to marker-assisted breeding. Additionally, the finding could help to use SLAF marker mapping to identify certain QTL contributing to important agronomic traits. The QTL and the candidate genes identified through the high-density genetic map above may provide diverse potential gene resources for the genetic improvement of rye.

Microclimate shapes the phylosymbiosis of rodent gut microbiota in Jordan's Great Rift Valley.

Host phylogeny and the environment play vital roles in shaping animal microbiomes. However, the effects of these variables on the diversity and richness of the gut microbiome in different bioclimatic zones remain underexplored. In this study, we investigated the effects of host phylogeny and bioclimatic zone on the diversity and composition of the gut microbiota of two heterospecific rodent species, the spiny mouse Acomys cahirinus and the house mouse Mus musculus, in three bioclimatic zones of the African Great Rift Valley (GRV). We confirmed host phylogeny using the D-loop sequencing method and analyzed the influence of host phylogeny and bioclimatic zone parameters on the rodent gut microbiome using high-throughput amplicon sequencing of 16S rRNA gene fragments. Phylogenetic analysis supported the morphological identification of the rodents and revealed a marked genetic difference between the two heterospecific species. We found that bioclimatic zone had a significant effect on the gut microbiota composition while host phylogeny did not. Microbial alpha diversity of heterospecific hosts was highest in the Mediterranean forest bioclimatic zone, followed by the Irano-Turanian shrubland, and was lowest in the Sudanian savanna tropical zone. The beta diversity of the two rodent species showed significant differences across the Mediterranean, Irano-Turanian, and Sudanian regions. The phyla Firmicutes and Bacteroidetes were highly abundant, and Deferribacterota, Cyanobacteria and Proteobacteria were also prominent. Amplicon sequence variants (ASVs) were identified that were unique to the Sudanian bioclimatic zone. The core microbiota families recovered in this study were consistent among heterospecific hosts. However, diversity decreased in conspecific host populations found at lower altitudes in Sudanian bioclimatic zone. The composition of the gut microbiota is linked to the adaptation of the host to its environment, and this study underscores the importance of incorporating climatic factors such as elevation and ambient temperature, in empirical microbiome research and is the first to describe the rodent gut microbiome from the GRV.

Fragmented imaginary-time evolution for early-stage quantum signal processors

Simulating quantum imaginary-time evolution (QITE) is a significant promise of quantum computation. However, the known algorithms are either probabilistic (repeat until success) with unpractically small success probabilities or coherent (quantum amplitude amplification) with circuit depths and ancillary-qubit numbers unrealistically large in the mid-term. Our main contribution is a new generation of deterministic, high-precision QITE algorithms that are significantly more amenable experimentally. A surprisingly simple idea is behind them: partitioning the evolution into a sequence of fragments that are run probabilistically. It causes a considerable reduction in wasted circuit depth every time a run fails. Remarkably, the resulting overall runtime is asymptotically better than in coherent approaches, and the hardware requirements are even milder than in probabilistic ones. Our findings are especially relevant for the early fault-tolerance stages of quantum hardware.

Potential zoonotic agents in sea turtles: Chlamydia species and Listeria monocytogenes

Abstract Background Sea turtles are well recognized sentinels of marine environment as well as carrier of pathogens and contaminants. Chlamydial species have been described in reptiles, but scarce data regarding sea turtles are available in literature (Pace et al., 2022). Hitherto, only one case of listeriosis has been described in marine reptiles (Di Renzo et al., 2022). This study reports the detection of Chlamydia species and Listeria monocytogenes in loggerhead sea turtles (Caretta caretta), suggesting the role of these animals as carriers of potential zoonotic agents. Methods In 2022, a total of 11 sea turtles found stranded along Emilia-Romagna coast, Northwestern Adriatic Sea, were subjected to post mortem investigation. Cloacal swabs were collected from 10 of the investigated animals and screened for Chlamydiaceae family by qPCR targeting 23S rRNA gene, followed by confirmation via 16S rRNA fragment sequencing. For positive samples, Chlamydia species identification was performed by species-specific qPCR for C. abortus, C. pneumoniae, C. pecorum, C. psittaci. One subject showed internal organs disseminated with multiple nodular lesions that were sampled for histopathology analyses, alongside microbiological examination. The cultured isolates were identified by MALDI-TOF (MALDI Biotyper; Bruker Daltonics Inc). Results In six out of 10 cloacal swabs Chlamydiaceae DNA was demonstrated by qPCR and confirmed by sequencing of 16S rRNA, but species identification was not achieved. In the turtle with nodular lesions, histopathology revealed the presence of granulomas: the Ziehl-Neelsen stain was negative for acid-fast organisms. L. monocytogenes was isolated from heart and liver and listeriosis was diagnosed. Conclusions The results highlight how sea turtles can be carriers, symptomatic or not, of pathogens relevant for public health. Given the zoonotic potential of these microorganisms, particular precautions are recommended especially for professionals handling these animals. Key messages • Sea turtles can play an active role as carriers of possibly pathogenetic microorganisms. • Further studies of molecular identification of Chlamydia spp. from sea turtles are needed in atypical strains.

Identification and introgression of a novel leaf rust resistance gene from Thinopyrum intermedium chromosome 7Js into wheat.

A novel leaf rust resistance locus located on a terminal segment (0-69.29 Mb) of Thinopyrum intermedium chromosome arm 7JsS has been introduced into wheat genome for disease resistance breeding. Xiaoyan 78829, a wheat-Thinopyrum intermedium partial amphiploid, exhibits excellent resistance to fungal diseases in wheat. To transfer its disease resistance to common wheat (Triticum aestivum), we previously developed a translocation line WTT26 using chromosome engineering. Disease evaluation showed that WTT26 was nearly immune to 14 common races of leaf rust pathogen (Puccinia triticina) and highly resistant to Ug99 race PTKST of stem rust pathogen (P. graminis f. sp. tritici) at the seedling stage. It also displayed high adult plant resistance to powdery mildew (caused by Blumeria graminis f. sp. tritici). Cytogenetic and molecular marker analysis revealed that WTT26 carried a T4BS·7JsS chromosome translocation. Once transferred into the susceptible wheat genetic background, chromosome 7JsS exhibited its resistance to leaf rust, indicating that the resistance locus was located on this alien chromosome. To enhance the usefulness of this locus in wheat breeding, we further developed several new translocation lines with small Th. intermedium segments using irradiation and developed 124 specific markers using specific-locus amplified fragment sequencing, which increased the marker density of chromosome 7JsS. Furthermore, a refined physical map of chromosome 7JsS was constructed with 74 specific markers, and six bins were thus arranged according to the co-occurrence of markers and alien chromosome segments. Combining data from specific marker amplification and resistance evaluation, we mapped a new leaf rust resistance locus in the 0-69.29Mb region on chromosome 7JsS. The translocation lines carrying the new leaf rust resistance locus and its linked markers will contribute to wheat disease-resistance breeding.

Genome-wide SNP-based diversity analysis and phylogeographic inference in the ark shell (Anadara broughtonii)

The ark shell (Anadara broughtonii) is a marine bivalve inhabiting the West Pacific coasts. Its natural resources have declined considerably in recent years due to overexploitation. Understanding of the genetic diversity and population structure of A. broughtonii is important for conservation and species-specific management. The Specific-Locus Amplified Fragment Sequencing (SLAF-seq) method was used to screen the SNPs for exploring the genetic diversity and phylogeographic characterization in 40 Anadara broughtonii individuals, 10 each from 4 sample locations: China, Japan, Korea, and Russia. We generated 394,768 SLAFs and developed 452,144 polymorphic SNPs. The observed and expected heterozygosity (Ho and He) ranged from 0.1805 to 0.3216 and from 0.1504 to 0.2156, respectively. Relatively strong differentiation was detected between populations from China/Russia and those from Japan/Korea, possibly related to the barrier effects from the Shandong Peninsula and the Korean Peninsula. Phylogenetic and Discriminant analysis of principal components (DAPC) supported the 40 individuals were mainly classified into four genetically distinct groups according to their sampling locations. Overall, Japanese and Korean populations owned higher genetic diversity. However, the two native populations may probably be affected by human activities such as artificial culture and commercial transportation. In order to avoid the further admixture of the native population of A. broughtonii, more attention should be paid to the germplasm resources protection of this species. Our results demonstrated the capability and high resolution of SNPs derived from SLAF-seq to measure the population genetic diversity and phylogeographic structure of A. broughtonii.

Development and Evaluation of Real-Time Polymerase Chain Reaction (PCR) to Authenticate Sablefish (Anoplopoma fimbria) in Commercial Seafood Products

ABSTRACT By comparing the cytochrome b fragment sequences of sablefish and 16 varieties susceptible to adulteration, a real-time polymerase chain reaction (PCR) method was established to detect sablefish-derived components. The PCR method was used to analyze commercial products. The method developed was highly specific without any cross-reaction in the other 16 species and with a limit of detection for deoxyribonucleic acid (DNA) of 0.002 ng µL−1 for sablefish. In addition, the mixed muscle tissues were detected at as low as 0.01 g kg−1. The applicability of the PCR method to 50 commercial prepackaged products revealed the accuracy of this method.

Phylogenetic analysis of Aciagrion Selys (Odonata: Coenagrionidae)

ABSTRACT Odonates are useful biological indicators of environmental health because of their pollution-free habitat requirements and amphibiotic life cycle. Due to habitat destruction by industrial development, there is a need to name these odonates and obtain their genetic data to be used in understanding their evolutionary history for biodiversity conservation. Aciagrion is a poorly known and taxonomically difficult genus of oriental damselflies. Species of this genus cannot be distinguished on the basis of morphological characters alone as they are morphologically similar to the damselflies of other genera (Ischnura and Amphiallagma) and may also be referred to as cryptic species. Here, therefore, molecular studies are carried out to distinguish the species of this genus. The phylogenetic relationships of five species of genus Aciagrion have been explored using molecular (based on concatenated sequences of mitochondrial genes- COI, ND1 and 16S rRNA genes) as well as external morphological characters. Single-linkage cluster analysis of morphological data sets did not explain the relationships appropriately among the species of this genus as outgroup species were also retrieved within ingroup species. Interspecific genetic divergence, conserved, variable, parsimony-informative sites, nucleotide base composition and transition/transversion bias based on concatenated gene sequences have been calculated for these species. It is found that a multigene concatenation approach interprets phylogeny more appropriately than the morphological data alone. Presently, a COI gene fragment sequence for one species (the endemic Aciagrion hisopa), 16S rRNA gene fragments for four species and ND1 gene fragments for five species have been submitted for the first time to GenBank, and phylogenetic relationships based on three genes have also been determined for the first time. Aciagrion migratum is a new record from India.

Searching for sequencing signal anomalies associated with genome structural variations

Genomic structural variations (SVs) are one of the main sources of genetic diversity. Structural variants as mutagens may have a significant impact on human health and lead to hereditary diseases and cancers. Existing methods of finding structural variants are based on analysis of high-throughput sequencing data and despite significant progress in the development of the detection methods, there is still a need for improving the identification of structural variations with accuracy appropriate for use in a diagnostic procedure. Analysis of the signal of sequencing coverage (i.e., the number of sequencing fragments that aligned to every point of a genome) holds new potential for the design of approaches for structural variations discovery, and can be used as time-series analysis. Here, we present an approach for identification of patterns in the coverage signal. The method has been developed based on algorithms used for analysis of time series data, namely KNN (K-nearest neighbour) search algorithm and the SAX (Symbolic Aggregation Approximation) method. Using the rich dataset encompassing full genomes of 911 individuals with different ethnic backgrounds generated by the Human Genome Diversity Project initiative, we constructed generalized patterns of signal coverage in the vicinity of breakpoints corresponding to various structural variant types. Also, with the benefit of the SAX models of the motifs we developed a software package for fast detection of anomalies in the coverage signal.

Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction

ObjectiveTo establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes. MethodsA database of capsular polysaccharide (cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping. ResultsA total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains. ConclusionA total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a "Booster" for SARS-CoV-2 Point-of-Care Testing.

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a "booster" by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

Label free optofluidic DNA hybridization detection based on suspended core fiber whispering gallery mode resonator

An optofluidic DNA biosensor based on a suspended core fiber (SCF) has been proposed for the detection of specific DNA sequence fragments. The sensor is formed by coupling a micro/nano tapered optical fiber to the cladding of an SCF, producing a whispering gallery mode (WGM) effect. The WGM resonance is sensitive to samples within the channels of the SCF. The diameter of the SCF is reduced by etching to increase refractive index sensitivity, up to 274 nm/RIU. Biomarkers are captured on the inner surface of the microfluidic channel leading to a change in the local refractive index. The experimental results show that the sensor can quantitatively detect complementary DNA (cDNA) by immobilizing probe DNA (pDNA) on the surface of the microfluidic channel with a limit of detection of 1.5 nM. The proposed DNA biosensor provides a label-free method for DNA detection, which has great potential for multiplexed detection and realizing disease diagnostics.

Mosquito Transposon-Mediated Transgenesis.

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as "enhancer traps" to identify endogenous genes with specific expression characteristics.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody–antigen interactions. The method, which we named ‘deep screening’, involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument’s flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

HycDemux: a hybrid unsupervised approach for accurate barcoded sample demultiplexing in nanopore sequencing

DNA barcodes enable Oxford Nanopore sequencing to sequence multiple barcoded DNA samples on a single flow cell. DNA sequences with the same barcode need to be grouped together through demultiplexing. As the number of samples increases, accurate demultiplexing becomes difficult. We introduce HycDemux, which incorporates a GPU-parallelized hybrid clustering algorithm that uses nanopore signals and DNA sequences for accurate data clustering, alongside a voting-based module to finalize the demultiplexing results. Comprehensive experiments demonstrate that our approach outperforms unsupervised tools in short sequence fragment clustering and performs more robustly than current state-of-the-art demultiplexing tools for complex multi-sample sequencing data.

Molecular Detection of Rickettsia hoogstraalii in Hyalomma anatolicum and Haemaphysalis sulcata: Updated Knowledge on the Epidemiology of Tick-Borne Rickettsia hoogstraalii.

Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99-100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts.

Sequencing of a fragment of the leptin gene in adolescents with different weight status

Background. Obesity is a significant social problem among the population of the world. The leptin gene (LEP) is currently considered as a potential candidate gene influencing metabolic disorders associated with predisposition to overweight and obesity. Leptin plays an important role in body weight homeostasis by influencing food intake and energy expenditure and maintaining constant energy stores. A defect in the leptin gene may be one of the causes of obesity and, as a result, of various obesity-associated pathologies. The aim of the study. To search for single-nucleotide polymorphisms (SNP) of the leptin gene in adolescents with different weight status. Methods. The study involved 20 adolescents aged 11–17 years with normal body weight and overweight/obesity. Research methods: assessment of clinical status with anthropometry; Sanger sequencing of the leptin gene fragment localized in the intron of this gene – (5’-AGCCTTGTTTTCATCATCTGGA, 3’-TGGGAGGAATCGCTCTCAGA). We also carried out bioinformatic processing of sequencing results. Results. As a result of the study, the optimal conditions for amplification of the 891 bps leptin gene region were selected for the above mentioned primer pair of the LEP gene (s16_L891, s16_R891). Based on the results of sequencing, 45 single nucleotide substitutions of the LEP gene were identified, including 23 single nucleotide substitutions which were not previously registered in GenBank. In the group of adolescents with overweight and obesity, 14 unregistered single nucleotide substitutions of the LEP gene and 13 registered SNPs were identified in the GenBank database. In the group of adolescents with normal body weight, these SNPs were not found.

Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells.

Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.