Sequencing Of Fragments Research Articles

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.

Филогенетические связи представителей рода Mesocestoides Vaillant, 1863 от микромаммалий востока России и Аляски

The article presents the data on structures of the nucleotide sequence fragments of the 12S ribosomal RNA gene (12S rRNA) for 19 tetrathyridia specimens of the genus Mesocestoides Vaillant, 1863 from representatives of 10 insectivore and rodent species from different regions of RussiaТs East and Alaska (USA). For the first time, the participation of three shrew species in the life cycle of Mesocestoides has been established. Nucleotide sequence fragments of the 12S rRNA gene have been analyzed phylogenetically. The researched tetrathyridia do not assumingly belong to any of the confirmed species of the genus; however, the identity of five of the 12S rRNA sequence fragments from micromammalian tetrathyridia and two nucleotide sequences of 12S rRNA from carnivorous mammals of Mongolia, placed in GenBank, has been established.

Advanced file carving: ontology, models and methods

File carving techniques are important in the field of digital forensics. At the same time, the rapid growth in the amount and types of data requires the development of file carving methods in terms of capabilities, accuracy, and computational efficiency. However, most of the methods are developed to solve specific tasks and are based on a certain set of assumptions and a priori knowledge about the files to be recovered. There is a lack of research that systematizes methods and structures approaches to identify gaps and determine perspective directions for development, considering the latest advances in information technology and artificial intelligence. The subject matter of this article is the structure, factors, efficiency criteria, methods, and tools of file carving, as well as the current state and tendencies of development of file carving methods. The goal of this study is to systematize knowledge about advanced file carving methods and identify perspective directions for their development. The tasks to be solved are as follows: to identify the main stages of file carving and analyze approaches to their implementation; to build an ontological scheme of file carving; and to identify perspective directions for the development of carving methods. The methods used were literature review, systematization, and summarization. The obtained results are as follows. An ontological scheme for the file carving concept is constructed. The scheme includes the principles, properties, phases, techniques, evaluation criteria, tools used, and factors influencing file carving. The features, limitations, and fields of application of the data recovery methods are provided. It was established that the most widespread approach to file reconstruction is still a manually detailed analysis of the internal structure of files and/or their contents, identifying specific patterns that allow reassembling the sequence of data fragments in the correct order. However, most of the methods do not provide one hundred percent guaranteed results. This article analyzes the current state and prospects of using artificial intelligence methods in the field of digital forensics, particularly for identifying data blocks, clustering, and reconstructing files, as well as restoring the contents of media files with damaged or lost headers. The necessity of having priori information about the file structure or content for successfully carving fragmented data is determined. Conclusions. The scientific novelty of the obtained results is as follows: for the first time, advanced file carving methods are systematized and analyzed by directions of development and the perspectives of using artificial intelligence for identifying data blocks, clustering, and file content restoration; for the first time, an ontological scheme of file carving is constructed, which can be used as a roadmap for developing new advanced systems in the digital forensics field.

Metagenomic Analysis of the Composition of Microbial Consortia Involved in Spruce Degradation over Time in Białowieża Natural Forest.

Deadwood plays an important role in forest ecology; its degradation and, therefore, carbon assimilation is carried out by fungi and bacteria. To quantify the abundance and distribution of microbial taxa inhabiting dead spruce logs fallen over a span of 50 years and the soil beneath, we used taxonomic profiling with NGS sequencing of hypervariable DNA fragments of ITS1 and 16S V3-V4, respectively. The analysis of sequencing data revealed a high level of diversity in microbial communities participating in the degradation of spruce logs. Differences in the relative abundance of microbial taxa between the samples of the wood that died in 1974 and 2014, and of the soil in its immediate vicinity, were visible, especially at the genus level. Based on the Lefse analysis significantly higher numbers of classified bacterial taxa were observed in the wood and soil samples from 2014 (wood: 1974-18 and 2014-28 taxa; soil: 1974-8 and 2014-41 taxa) while the number of classified fungal taxa was significantly higher in the wood and soil samples from 1974 (wood: 1974-17 and 2014-9 taxa; soil: 1974-57 and 2014-28 taxa). Most of the bacterial and fungal amplicon sequence variants (ASVs) unique to wood were found in the samples from 1974, while those unique to soil were detected in the samples from 2014. The ATR-FTIR method supported by CHN analysis revealed physicochemical changes in deadwood induced by the activity of fungal and bacterial organisms.

Molecular characterization of juvenile fish from the Amazon estuary using DNA barcoding approach.

The efficiency of the DNA barcoding relies on sequencing fragment of the Cytochrome C Subunit I (COI) gene, which has been claimed as a tool to biodiversity identification from distinct groups. Accordingly, the goal of this study was to identify juvenile fish species along an estuary of Caeté River in the Brazilian Blue Amazon based on. For this purpose, we applied the DNA barcoding and discuss this approach as a tool for discrimination of species in early ontogenetic stages. A 500-bp fragment was obtained from 74 individuals, belonging to 23 species, 20 genera, 13 families and seven orders. About 70% of the 46 haplotypes revealed congruence between morphological and molecular species identification, while 8% of them failed in identification of taxa and 22% demonstrated morphological misidentification. These results proved that COI fragments were effective to diagnose fish species at early life stages, allowing identifying all samples to a species-specific status, except for some taxa whose COI sequences remain unavailable in public databases. Therefore, we recommend the incorporation of DNA barcoding to provide additional support to traditional identification, especially in morphologically controversial groups. In addition, periodic updates and comparative analyses in public COI datasets are encouraged.

DPP-IV Inhibitory Peptides from Coix Seed Prolamins: Release, Identification, and Analysis of the Interaction between Key Residues and Enzyme Domains.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides can regulate type 2 diabetes by inhibiting the cleavage of glucagon-like peptide-1 and prolonging its half-life. The development of DPP-IV inhibitory peptides is still a hot topic. The primary structure of coix seed prolamins contains peptide sequence fragments that potentially inhibit DPP-IV; however, limited information is available regarding the extraction of peptides from coix seeds and the analysis of their conformational relationships. In this study, novel coix seed prolamin-derived peptides were obtained through single hydrolysis and double-enzyme stepwise hydrolysis. The inhibitory activity of these peptides against DPP-IV was evaluated to explore new functional properties of coix seeds. The results evidenced that the step-by-step enzymolysis (papain and alcalase) compared to single enzymolysis promoted the secondary structure disruption of the hydrolysates, enhanced the β-turn structure, significantly increased the content of peptides below 1 kDa, and exhibited a substantial increase in DPP-IV inhibitory activity (97% inhibition). Three nontoxic DPP-IV inhibitory peptides, namely, LPFYPN, TFFPQ, and ATFFPQ (IC50 = 70.24, 176.87, 268.31 μM), were isolated and identified. All three peptides exhibited strong interactions with DPP-IV (all KA values >103). LPFYPN exhibited competitive inhibition, while TFFPQ and ATFFPQ demonstrated mixed competitive-noncompetitive inhibition. Hydrogen bonding and hydrophobic interactions were the main contributors to the coix seed prolamin peptides binding to DPP-IV. The central residue was a key amino acid in the parent peptide sequence, forming a more stable π-π stacking with residues in the active pocket, which may facilitate peptide activity. This study provides theoretical support for the development of coix seed-derived hypoglycemic peptides.

Chicken nuclear DNA in chicken egg whites

Poultry eggs contain high-quality proteins, fats, vitamins and minerals, which makes them an indispensable product in human nutrition. Egg whites have exceptional foaming, emulsifying, gelling, and heat setting properties, making them popular for use in baked goods. The knowledge accumulated so far about egg whites has now settled on a stable opinion that there are no cells, and no DNA, in them. However, this study demonstrates the possibility of extracting chicken genomic DNA from the outer thin whites of chicken eggs. For DNA extraction, foremost, protein degradation of the samples by trypsin was used. The amount of isolated DNA ranged from 0.3880 ± 0.0348 to 0.6380 ± 0.0545 μg/ml of the white sample. A PCR test based on 18S ribosomal DNA showed that the DNA isolated from the outer thin white contained avian genomic DNA. Furthermore, cloning and sequencing of the bird-specific PCR fragment showed that this DNA belongs to chickens (Gallus gallus). In addition, staining with Hoechst 33342 showed the presence of cell nuclei in the samples. Therefore, this study demonstrates that chicken egg whites contain chicken genomic (nuclear) DNA and cell nuclei (i. e., and cells).

A new species of Rhyncholagena Lang, 1944 (Copepoda, Harpacticoida, Miraciidae) from Palau.

A new species of Miraciidae Dana, 1846, Rhyncholagenacuspissp. nov., was described from Palau. Morphological descriptions and gene fragment sequence barcoding were performed on the 11th species of Rhyncholagena Lang, 1944 collected from sandy sediment samples in the subtidal zone of the Philippine Sea, Palau. Morphological characteristics were compared and an updated identification key was provided. A new species, Rhyncholagenacuspissp. nov., was found to be morphologically similar to Rhyncholagenalittoralis Por, 1967 and R.bermudensis Malt, 1990. This is the first record of the genus Rhyncholagena in Palau. The study provides basic data for future studies and highlights the need for continued exploration of marine biodiversity in Palau and other regions.