DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

2020 ◽  
Author(s):  
E.V. Korneenko ◽  
◽  
А.E. Samoilov ◽  
I.V. Artyushin ◽  
M.V. Safonova ◽  
...  
Keyword(s):  
High Throughput ◽  
Viral Genome ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Moscow Region ◽  
Fecal Samples ◽  
Genus Level ◽  
Zoonotic Viruses ◽  
Reservoir Hosts ◽  
Blastn Search

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses.

scholarly journals DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

2020 ◽  
Author(s):  
E.V. Korneenko ◽  
◽  
А.E. Samoilov ◽  
I.V. Artyushin ◽  
M.V. Safonova ◽  
...  
Keyword(s):  
High Throughput ◽  
Viral Genome ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Moscow Region ◽  
Fecal Samples ◽  
Genus Level ◽  
Zoonotic Viruses ◽  
Reservoir Hosts ◽  
Blastn Search

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

scholarly journals ATAC-seq with unique molecular identifiers improves quantification and footprinting

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Tao Zhu ◽  
Keyan Liao ◽  
Rongfang Zhou ◽  
Chunjiao Xia ◽  
Weibo Xie
Keyword(s):  
Transcription Factor ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Chromatin Accessibility ◽  
Link Type ◽  
Pcr Duplicates ◽  
Identify Transcription Factor ◽  
Accessible Chromatin ◽  
Accurate Quantification

AbstractATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq.

scholarly journals Monitoring the spread of SARS-CoV-2 variants in Moscow and the Moscow region using targeted high-throughput sequencing

2021 ◽  
Author(s):  
Nadezhda I Borisova ◽  
Ivan A Kotov ◽  
Anton A Kolesnikov ◽  
Valeriia V Kaptelova ◽  
Anna S Speranskaya ◽  
...  
Keyword(s):  
South African ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Biological Properties ◽  
Moscow Region ◽  
Gene Encoding ◽  
Genetic Changes ◽  
Random Samples ◽  
Coronavirus Infection ◽  
Short Time

Since the outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus, the international community has been concerned about the emergence of mutations that alter the biological properties of the pathogen, for example, increasing its infectivity or virulence. In particular, since the end of 2020, several variants of concern have been identified around the world, including variants alpha (B.1.1.7, British), beta (B.1.351, South African), gamma (P.1, Brazilian) and delta (B.1.617.2, Indian). However, the existing mechanism for searching for important mutations and identifying strains may not be effective enough, since only a relatively small fraction of all identified pathogen samples can be examined for genetic changes by whole genome sequencing due to its high cost. In this study, we used the method of targeted high-throughput sequencing of the most significant regions of the gene encoding the S-glycoprotein of the SARS-CoV-2 virus, for which a primer panel was developed. Using this technique, we examined 579 random samples obtained from patients in Moscow and the Moscow region with coronavirus infection from February to June 2021. The study demonstrated the dynamics of the representation in the Moscow region of a number of SARS-CoV-2 strains and its most significant individual mutations in the period from February to June 2021. It was found that the strain B.1.617.2 began to spread rapidly in Moscow and the Moscow region in May, and in June it became dominant, partially displacing other varieties of the virus. The results obtained make it possible to accurately determine the belonging of the samples to the abovementioned and some other strains. The approach can be used to standardize the procedure for searching for new and existing epidemiologically significant mutations in certain regions of the SARS-CoV-2 genome, which allows studying a large number of samples in a short time and to get a more detailed picture of the epidemiological situation in the region.

scholarly journals DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin

2020 ◽  
Author(s):  
Hua-Lin Huang ◽  
Shikui Yin ◽  
Huifang Zhao ◽  
Chao Tian ◽  
Jufang Huang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Magnetic Bead ◽  
Sequencing Data ◽  
Sequencing Library ◽  
Bead Method ◽  
Formalin Fixed ◽  
Better Than

AbstractMawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.

scholarly journals Size-Fractionated Filtration Combined with Molecular Methods Reveals the Size and Diversity of Picophytoplankton

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1280
Author(s):  
Xinze Shuwang ◽  
Jun Sun ◽  
Yuqiu Wei ◽  
Congcong Guo
Keyword(s):  
Flow Cytometry ◽  
Particle Size ◽  
Vertical Distribution ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
Euphotic Zone ◽  
Vertical Distributions ◽  
The Vertical Distribution

In this study, flow cytometry (FCM) and size-fractionated filtration, together with high-throughput molecular sequencing methods (SM), were used to investigate picophytoplankton. A particle separation filter and a higher-throughput sequencing method were used to evaluate the composition of a euphotic zone of picophytoplankton—especially picoeukaryotic phytoplankton—in the Western Pacific, and the results of flow cytometry, which is a classic way to detect picophytoplankton, were used as a standard to evaluate the reliability of the results of the SMs. Within a water column of 200 m, six water depths (5, 25, 50, 113 (DCM), 150, and 200 m) were established. In order to further study the particle size spectra of the picophytoplankton, size-fractionated filtration was used to separate water samples from each water depth into three particle size ranges: 0.2–0.6, 0.6–1.2, and 1.2–2 μm. A total of 36 (6 × 3 × 2) samples were obtained through PCR amplification of the 18S rRNA V4 hypervariable region and 16S rRNA, which were biased toward phytoplankton plastids, and then high-throughput sequencing was performed. The estimation of the picophytoplankton diameter relied on forward scattering (FSC) through FCM. The estimation of the vertical distribution and diameter of the picophytoplankton using the SM was consistent with the results with FCM; thus, we believe that the estimation of picophytoplankton composition with the SM has value as a reference, although the size-fractionated filtration seemed to cause some deviations. In addition to Prochlorococcus and Synechococcus, the SM was used to evaluate the composition of picoeukaryotic phytoplankton, which mainly included Prymnesiophycea (Haptophyta) (38.15%), Cryptophyceae (Cryptophyta) (22.36%), Dictyochophyceae (Chrysophyta) (12.22%), and Mamiellophyceae (Chlorophyta) (3.31%). In addition, the SM also detected Dinophyceae (Dinoflagellata) (11.69%) sequences and a small number of Bacillariophyceae (Diatom) (1.64%) sequences, which are generally considered to have large particle sizes. The results of the SM also showed that the picoeukaryotic phytoplankton were not evenly distributed in the euphotic layer, and the vertical distributions of the different picoeukaryotic phytoplankton were different. An analysis of correlations with environmental factors showed that temperature was the main environmental factor controlling the vertical distribution of picophytoplankton.

scholarly journals Comparison of the Intestinal Microbial Community in Ducks Reared Differently through High-Throughput Sequencing

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Yan Zhao ◽  
Kun Li ◽  
Houqiang Luo ◽  
Longchuan Duan ◽  
Caixia Wei ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
Water Contamination ◽  
High Throughput Sequencing ◽  
Free Access ◽  
Operational Taxonomic Units ◽  
Genus Level ◽  
Significant Difference ◽  
Access To Water ◽  
Poultry Droppings

Birds are an important source of fecal contamination in environment. Many of diseases are spread through water contamination caused by poultry droppings. A study was conducted to compare the intestinal microbial structure of Shaoxing ducks with and without water. Thirty 1-day-old Shaoxing ducks (Qingke No. 3) were randomly divided into two groups; one group had free access to water (CC), while the other one was restricted from water (CT). After 8 months of breeding, caecal samples of 10 birds from each group were obtained on ice for high-throughput sequencing. A total of 1507978 valid sequences were examined and clustered into 1815 operational taxonomic units (OTUs). At phylum level, Firmicutes (41.37%), Bacteroidetes (33.26%), Proteobacteria (13.67%), and Actinobacteria (8.26%) were found to dominate the microbial community in CC birds, while Firmicutes (53.62%), Bacteroidetes (33.06%), and Actinobacteria (11.13%) were uncovered to be the prime phyla in CT ducks. At genus level, Bacteroides (25.02%), Escherichia-Shigella (11.02%), Peptococcus (7.73%) and Parabacteroides (5.86%) were revealed to be the mainly genera in the CC group ducks, while Bacteroides (18.11%), Erysipelatoclostridium (10.94%), Ruminococcaceae_unclassified (10.43%), Lachnospiraceae_unclassified (5.26%), Coriobacteriales_unclassified (5.89%), and Faecalibacterium (4.2%) were detected to staple the microbial flora in the CT birds. One phylum and 13 genera were found to have the significant difference between the two bird groups (p<0.05). At phylum level, Proteobacteria in CT ducks were found to be obviously lower than ducks in CC birds (p<0.05). At genus level, Escherichia-Shigella (p<0.05) and Peptococcus (p<0.05) were found to be notably lower in CT birds, while Erysipelatoclostridium (p<0.05), Ruminococcaceae_unclassified (p<0.01), Coriobacteriales_unclassified (p<0.05), Faecalibacterium (p<0.01), Atopobiaceae_unclassified (p<0.01), Alistipes (p<0.05), Eggerthellaceae_unclassified (p<0.05), Prevotella_7 (<0.05), Rikenellaceae_RC9_gut_group (p<0.05), Prevotellaceae_uncultured (p<0.05), and Shuttleworthia (p<0.05) were observed to be prominently higher in CT ducks. In conclusion, the present study revealed the effects of keeping ducks away from swimming with obvious changes in the microbial community. Though higher microbial richness was found in the ducks without swimming, more pathogenic genera including Eggerthella, Erysipelatoclostridium, Alistipes, Prevotella_7, and Shuttleworthia; zoonotic genera including Eggerthella and Shuttleworthia; inflammatory genus Alistipes; anti-inflammatory Faecalibacterium genus; and tumor genus Rikenellaceae were examined in these ducks. The CT ducks also showed significant changes at genera level regarding the metabolism (Peptococcus, Ruminococcaceae, and Coriobacteriales).

Sign in / Sign up

Export Citation Format

Share Document