Comparative analysis of Illumina and Ion Torrent high-throughput sequencing platforms for identification of plant components in herbal teas

© 2018 Elsevier Ltd The rapid development of high-throughput sequencing (HTS) methods offers new opportunities for food quality control and identification of food components using the DNA barcoding approach (metabarcoding in cases of complex mixes). However, the protocols of DNA barcoding applied to food analysis are not yet fully established; testing and optimization are required to achieve the highest accuracy and cost efficiency. We report here a comparative study of the two most widely used sequencing platforms - Illumina and Ion Torrent - for composition analysis of herbal teas, and show that both technologies yield congruent results, both qualitatively and quantitatively. They have revealed the substitution of fireweed (Epilobium angustifolium L.) by Lythrum sp. in one of the samples. It was confirmed by classic methods of botanical analysis (anatomy and palynology). In most samples, undeclared components have been detected, such as bindweed (Convolvulus) and ragweed (Ambrosia), which are known toxic and allergy-causing plants

Development and Testing of the Method for the Detection of Lassa virus RNA, Based on real-Time Polymerase Chain reaction with reverse Transcription

Abstract. Objective of the study was the development of a method for the detection and quantitative analysis (realtime RT-PCR) to identify genetic markers of Lassa virus - LASV-Fl. Materials and methods. We utilized all the available in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) Lassa virus sequences that have been aligned to identify conservative sites applying the BioEdit 7.2.5 software package (IbisBiosciences, USA). To test the developed PCR kit, the control panel of Lassa virus RNA and pseudo-viral particles, 27 viral strains belonging to different fami­lies, as well as 37 serum samples from patients with feverish diseases selected in medical institutions of the Republic of Guinea in 2016-2018 and 55 samples of organ suspensions from multi-spiked mice were used. Results and discussion. The analytical sensitivity of the method varied from 103 copies/ml to 105 copies/ml and had 96.4 % diagnostic sensitivity, while the analytical and diagnostic specificity was 100 %. It is shown that the developed technique can be successfully introduced into practice for the detection of Lassa virus in the Republic of Guinea, using various types of material from small mammals, including whole blood and organ suspensions of M. natalensis, as well as samples of human blood sera collected 3-7 days after the onset of the disease. It is also suggested that this method can be used for strains of Lassa virus, common not only in Guinea but also in other endemic areas, but this fact must be confirmed in further studies

Comparative analysis of Illumina and Ion Torrent high-throughput sequencing platforms for identification of plant components in herbal teas

© 2018 Elsevier Ltd The rapid development of high-throughput sequencing (HTS) methods offers new opportunities for food quality control and identification of food components using the DNA barcoding approach (metabarcoding in cases of complex mixes). However, the protocols of DNA barcoding applied to food analysis are not yet fully established; testing and optimization are required to achieve the highest accuracy and cost efficiency. We report here a comparative study of the two most widely used sequencing platforms - Illumina and Ion Torrent - for composition analysis of herbal teas, and show that both technologies yield congruent results, both qualitatively and quantitatively. They have revealed the substitution of fireweed (Epilobium angustifolium L.) by Lythrum sp. in one of the samples. It was confirmed by classic methods of botanical analysis (anatomy and palynology). In most samples, undeclared components have been detected, such as bindweed (Convolvulus) and ragweed (Ambrosia), which are known toxic and allergy-causing plants

Small-molecule inhibitors of hepatitis C virus (HCV) non-structural protein 5A (NS5A): a patent review (2010-2015)

<p><b>Introduction</b>: Non-structural 5A (NS5A) protein has achieved a considerable attention as an attractive target for the treatment of hepatitis C (HCV). A number of novel NS5A inhibitors have been reported to date. Several drugs having favorable ADME properties and mild side effects were launched into the pharmaceutical market. For instance, daclatasvir was launched in 2014, elbasvir is currently undergoing registration, ledipasvir was launched in 2014 as a fixed-dose combination with sofosbuvir (NS5B inhibitor).</p> <p><b>Areas covered</b>: Thomson integrity database and SciFinder database were used as a valuable source to collect the patents on small-molecule NS5A inhibitors. All the structures were ranked by the date of priority. Patent holder and antiviral activity for each scaffold claimed were summarized and presented in a convenient manner. A particular focus was placed on the best-in-class bis-pyrrolidine-containing NS5A inhibitors.</p> <p><b>Expert opinion</b>: Several first generation NS5A inhibitors have recently progressed into advanced clinical trials and showed superior efficacy in reducing viral load in infected subjects. Therapy schemes of using these agents in combination with other established antiviral drugs with complementary mechanisms of action can address the emergence of resistance and poor therapeutic outcome frequently attributed to antiviral drugs.</p

Genomic Variability of SARS-CoV-2 Omicron Variant Circulating in the Russian Federation during Early December 2021 and Late January 2022

Analysis of genomic variability of pathogens associated with heightened public health concerns is an opportunity to track transmission routes of the disease and helps to develop more effective vaccines and specific diagnostic tests. We present the findings of a detailed genomic analysis of the genomic variability of the SARS-CoV-2 Omicron variant that spread in Russia between 8 December 2021 and 30 January 2022. We performed phylogenetic analysis of Omicron viral isolates collected in Moscow (n = 589) and downloaded from GISAID (n = 397), and identified that the BA.1 lineage was predominant in Russia during this period. The BA.2 lineage was also identified early in December 2021. We identified three cases of BA.1/BA.2 coinfections and one case of Delta/Omicron coinfection. A comparative genomic analysis of SARS-CoV-2 viral variants that spread in other countries allowed us to identify possible cases of transmission. We also found that some mutations that are quite rare in the Global Omicron dataset have a higher incidence rate, and identified genetic markers that could be associated with ways of Omicron transmission in Russia. We give the genomic variability of single nucleotide variations across the genome and give a characteristic of haplotype variability of Omicron strains in both Russia and around the world, and we also identify them

Synthesis and Biological Evaluation of Novel <i>Dispiro</i>-Indolinones with Anticancer Activity

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCTwt, and HCT(−/−). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC50 = 1.2–3.5 µM) and a reasonable selectivity index (SI = 3–10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index—estimated as LD50/ED50—for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method&#8217;s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: (1) the DNA extraction method greatly influences amplification success; (2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and (3) the &#8220;non-amplifiable&#8222; samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components

SARS-CoV-2 Recombination and Coinfection Events Identified in Clinical Samples in Russia

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line