Nucleic Acid Sequence Analysis Research Articles

Molecular epidemiology of foot-and-mouth disease (review)

Molecular epidemiological studies are an important tool for regional and global surveillance of foot-and-mouth disease (FMD). These tests are based on constantly progressing technologies of nucleic acid sequencing and phylogenetic analysis. The use of these technologies made it possible to assess the genetic diversity of the FMD virus, to analyze the evolution of the virus in the enzootic regions, and track the pathways of FMD epizootic and panzootic spread. Molecular epidemiological studies have shown that within the long-known seven serotypes of the FMD virus, there are numerous topotypes (geographical types), genetic lineages and sublineages. Usually, the foot-and-mouth disease virus of a certain topotype and genetic lineage evolves within a certain area, periodically causing regional epizootics. However, over the past 30 years, two FMD panzootics have occurred, invloving several continents. The first panzootic occurred in the late 1990s – early 2000s and was caused by O/ME-SA/PanAsia FMDV, and the second, caused by O/ME-SA/Ind-2001 virus, began in 2013 and continues to the present. The emergence of FMD panzootics is probably a consequence of the economic globalization. FMD is not enzootic in Russia, but sporadic outbreaks of this disease are periodically reported. Molecular epidemiological studies have shown that these outbreaks are caused by the infection introduction from neighboring Asian countries, mainly from China. The FMD virus, which has come to the Russian Federation from other countries, is characterized by great genetic diversity and belongs to three serotypes, five topotypes and eight genetic lineages: O/Cathay, O/ME-SA/PanAsia, O/SEA/Mya-98, O/ME-SA/Ind-2001, O/ME-SA/unnamed, A/Asia/Iran-05, A/Asia/Sea-97, Asia1/V. The results of molecular epidemiological studies are taken into account when vaccine strains are to be selected for preventive vaccination of livestock in FMD high-risk areas. The review is based on the analysis of 68 literature sources.

Reassortment and genomic analysis of a G9P[8]-E2 rotavirus isolated in China

ObjectiveTo isolate a prevalent G9P[8] group A rotavirus (RVA) (N4006) in China and investigate its genomic and evolutionary characteristics, with the goal of facilitating the development of a new rotavirus vaccine.MethodsThe RVA G9P[8] genotype from a diarrhea sample was passaged in MA104 cells. The virus was evaluated by TEM, polyacrylamide gel electrophoresis, and indirect immunofluorescence assay. The complete genome of virus was obtained by RT-PCR and sequencing. The genomic and evolutionary characteristics of the virus were evaluated by nucleic acid sequence analysis with MEGA ver. 5.0.5 and DNASTAR software. The neutralizing epitopes of VP7 and VP4 (VP5* and VP8*) were analyzed using BioEdit ver. 7.0.9.0 and PyMOL ver. 2.5.2.ResultsThe RVA N4006 (G9P[8] genotype) was adapted in MA104 cells with a high titer (105.5 PFU/mL). Whole-genome sequence analysis showed N4006 to be a reassortant rotavirus of Wa-like G9P[8] RVA and the NSP4 gene of DS-1-like G2P[4] RVA, with the genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). Phylogenetic analysis indicated that N4006 had a common ancestor with Japanese G9P[8]-E2 rotavirus. Neutralizing epitope analysis showed that VP7, VP5*, and VP8* of N4006 had low homology with vaccine viruses of the same genotype and marked differences with vaccine viruses of other genotypes.ConclusionThe RVA G9P[8] genotype with the G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2) constellation predominates in China and may originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] rotaviruses. The antigenic variation of N4006 with the vaccine virus necessitates an evaluation of the effect of the rotavirus vaccine on G9P[8]-E2 genotype rotavirus.

Metrological Support of Nucleic Acid Sequence Analysis

Metrological support for the process of molecular genetic identification is proposed. An establishment of a nucleotide sequence is the main objective of the DNA/RNA analysis. Such nucleotide sequence provides key information about the role and function of nucleic acids in living systems, as well as the origin of biological objects during molecular genetic identification. The molecular genetic identification is based on the modern methods of studying DNA sequences, which are inherited by the living organisms from generation to generation. In some cases, comparison of DNA sequences is required to establish the authenticity of biological objects, as well as determine biological affiliation or kinship with other biological objects. The problems of metrological support of molecular genetic identification by Sanger DNA sequencing using fluorescent-labeled reaction terminators and capillary electrophoresis are considered. Methods and means of metrological support of the DNA/RNA analysis were developed, which include the determination of a nucleotide as a nucleic acid monomer, the use of a nucleotide sequence of nucleic acids used in certified methods and procedures, the use of a genetic analyzer as a measurement tool along with related auxiliary equipment, as well as the use of a standard sample of a fragment of human mitochondrial DNA has been developed and approved. A Nanofor 05 domestic genome analyzer was tested. The following two procedures were developed and certified: for measuring the nucleotide sequence of the section of the control region of mitochondrial DNA of fish (sturgeon and paddlefish families) by Sanger sequencing, and for measuring the nucleotide sequence of the 5' region of the COI gene of the mitochondrial DNA of aquatic biological resources by Sanger sequencing. Both measurement procedures are based on the use of fluorescent-labeled reaction terminators and capillary electrophoresis. The results are important for use in the DNA studies in the field of biological sciences, food industry, biotechnology, and DNA forensics.

Metrological support of sequence analysis of nucleic acids

Metrological support for the process of molecular genetic identification is provided. An establishment of a nucleotide sequence is the general aim in nucleic acids analysis. A nucleotide sequence of DNA/RNA is the key information which gives an idea of the role and functioning of nucleic acids in living systems, as well as it allows establishing the origin of biological objects during molecular genetic identification. The molecular genetic identification is based on modern methods of research in the sequencing of DNA, which is inherited from generation to next generation of living organisms. DNA sequence comparison in some cases is mandatory to establish the authenticity of biological objects and to determine biological affiliation or kinship with other biological objects. The problems of metrological support of molecular genetic identification by Sanger DNA sequencing using fluorescently labeled reaction terminators and the use of capillary electrophoresis on the example of fish identification are considered. Metrological support of the process includes the use of such definitions as a nucleotide (the nucleic acid monomer), a sequence of nucleotides of nucleic acids. Also, it involves the procedures used in certified methods, a standard sample of a known DNA sequence of an approved type, and a genetic analyzer.Standard Reference Materials of a fragment of human mitochondrial DNA was developed, approved, entered into the Federal registry. The tests of the domestic genetic analyzer Nanophore 05 were carried out. Two measurement procedures “The measurement methodology the partial nucleotide sequence of the control region of mitochondrial DNA of fish (families Acipenseridae and Polyodontidae) by Sanger sequencing with fluorescently-labeled terminators and using capillary electrophoresis” and “The measurement methodology the nucleotide sequence of the 5’ region of the mitochondrial COI gene of water biological resources by Sanger sequencing with fluorescently-labeled terminators and using capillary electrophoresis” were tested and certified. The results are relevant to research in biological sciences, the food industry, biotechnology, and DNA forensics.

Identification of three new ‘Candidatus Liberibacter solanacearum’ haplotypes in four psyllid species (Hemiptera: Psylloidea)

Eleven haplotypes of the bacterium, ‘Candidatus Liberibacter solanacearum’, have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for ‘Ca. L. solanacearum’ by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the ‘Ca. L. solanacearum’ 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of ‘Ca. L. solanacearum’ haplotype F was not detected in this study.

Филогенетическое разнообразие микроорганизмов источника Абаканский Аржан – потенциальных продуцентов микробной энергии

Microbial energy is a promising area of innovative development in bio- and nanotechnology. Recent studies have revealed that microbial communities of thermal springs have excellent implementation prospects in this area. The present article introduces the microbial diversity of the Abakan Arzhan thermal spring and their isolates that are potentially applicable in microbial electricity synthesis. 
 The research featured microbial isolates obtained from a microbiota analysis of water and slit samples from the Abakan Arzhan thermal spring. The study involved a metagenomic analysis of the microbial community, as well as such molecular biology methods as nucleic acid extraction, PCR, sequencing, phylogenetic, and bioinformatic analysis. The Silva library was used to compare 16S RNA sequences
 Firmicutes, Bacteroides, and Proteobacteria proved to be the dominant phylotypes for water samples, while Firmicutes, Thermomonas, Gammaproteobacteria, and Proteobacteria were the dominant phylotypes for slit samples. The analysis of minor phylotypes confirmed the presence of Geobacter and Shewanella in the samples. The total number of obtained enrichment cultures was nine. Two types of resistant colonies were discovered during the isolation of extremophilic iron-reducing isolates. The samples were grown on a medium containing iron (III) acetate and iron (III) nitrate, and the isolates appeared to be in the process of Fe(III) reduction. The isolates showed an intense iron recovery of 409 and 407 µg/mL after 72 h of cultivation.
 The study confirmed the ability of the acquired isolates to reduce iron, making them a priority for future microbial energy research. The isolates belonged to the Shewanella algae and Geobacter sulfurreducens species, as determined by 16S RNA morphology and phylogenetic analyses.

Postgenomic Bioinformatic Analysis of Nucleic Acid Sequences Expressed in the Salivary Gland Epithelial Cells of Primary Sjögren's Syndrome Patients in Search of Microorganisms and Endogenous Retroviruses.

Several previous studies from our laboratory have indicated that the salivary gland epithelia of primary Sjögren's syndrome (SS) patients are not only the target of autoimmune immune responses, but also key instigators of the chronic salivary gland inflammatory infiltrates of patients. In particular, the comparative analysis of salivary gland tissue specimens and of in-vitro cultured non-neoplastic salivary gland epithelial cell lines (SGEC, of ductal type) from SS-patients and non-SS disease-controls, have unequivocally highlighted the presence of intrinsic activation in the ductal epithelia of SS-patients and of aberrant expression of inflammagenic molecules thereof, that correlate with the severity of local histopathologic changes, as well as of systemic manifestations of the disease. In the same context, we have recently shown that the ductal epithelia of SS-patients manifest cell-autonomous activation of the AIM2 inflammasome owing to the presence of aberrant cytoplasmic accumulations of damaged DNA. These findings not only provide a mechanistic explanation for the intrinsic activation and inflammatory status of SS ductal epithelia, but may also point towards the putative instigating role of an exogenous or endogenous agent (i.e., a micro-organism or an endogenous retrovirus, respectively). On this basis and to further explore the nature of epithelial cell-intrinsic activation in SS, the present proposal aims to investigate the expression of endogenous retroviral and/or non-human nucleic acid sequences of microbial origin in the ductal salivary gland epithelia of SS-patients, using metagenomic analysis of high throughput DNA and RNA genome sequencing data, which will be obtained from SGEC lines derived from SS-patients and disease-controls.

Nonclimacteric ‘Jen-Ju Bar’ guava ripening behavior is caused by Copia LTR retrotransposon insertion in the promoter region of PgACS1, a System-2 ACC synthase gene

‘Jen-Ju Bar’ (‘JJB’) guava (Psidium guajava L.), a nonclimacteric guava variety featuring delayed peel coloration and pulp softening, exhibits very low ethylene production during the fruit ripening stage and long storability due to the barely detectable activity of 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid (ACC) content in the pulp. PgACS1, a System-2 ACS gene, has been demonstrated to be a major, if not the only, responsive ACS associated with massive ethylene production during ripening in climacteric guava, such as ‘Li-Tzy Bar’ (‘LTB’). After alignment of the nucleotide sequences of PgACS1 genomic clones between ‘JJB’ and ‘LTB’, the promoter region of ‘JJB’ PgACS1 was found to be interrupted by a retrotransposon. We cloned and characterized the full-length retrotransposon in the promoter region of ‘JJB’ PgACS1. Nucleic acid sequence analysis indicated that the 5487 bp insertion was a Copia long terminal repeat (LTR) retrotransposon, which is oriented in the antisense transcriptional direction of PgACS1 at − 6 upstream of the putative transcription start site (TSS). These results imply that retrotransposon insertion in the promoter region of PgACS1 renders a massive frameshift of the ethylene response element (ERE) cis-element; therefore, the gene was almost silent at the ripening stage. Consequently, ‘JJB’ behaved in a nonclimacteric ripening manner and is feasible for distant marketing. Specific PCR primers targeting the PgACS1 promoter were designed and successfully discriminated the ripening behavior among ten guava cultivars. The primers were suggested for application as molecular markers to assist in early ripening-behavior screening among progenies of a guava breeding project.

Fatal Sarcocystis calchasi-associated meningoencephalitis in 2 captive vulturine guineafowl.

Two captive vulturine guineafowl (Acryllium vulturinum) were presented with lethargy, hyporexia, weight loss, and progressive neurologic signs. One of the guineafowl was seropositive for Sarcocystis falcatula (1:50 dilution). Both guineafowl died within 5 d of presentation. Histologic examination revealed nonsuppurative meningoencephalitis with gliosis, associated with occasional schizonts in the neuropil. Using fresh-frozen brain tissue, PCR was performed to amplify the ITS1 RNA region and portions of the 18S ribosomal RNA gene (18S gene) and the 28S ribosomal RNA gene (28S gene). Analysis of nucleic acid sequences from the resulting amplicons indicated that Sarcocystis calchasi was the likely cause of disease. To our knowledge, S. calchasi-associated disease has not been reported previously in the order Galliformes.

Detection and molecular characterisation of Mycoplasma spp. from respiratory tract infections in pigs

Achieving food security involves the maintenance of intensive production systems and large animal populations where infectious diseases are the most important challenge that need to be addressed. Respiratory infections in pigs are a formidable threat to swine farmers worldwide. It is multifactorial and is caused by the interplay between bacterial pathogens, viral agents and environmental factors. The present study aimed to identify mycoplasmal agents causing respiratory infections in pigs. A total of 43 samples, comprising of nasal swabs, and necropsy samples with pneumonic lesions were collected. The DNA extracted from the samples were subjected to Mycoplasma genus-specific, followed by species-specific PCR. Twenty five samples were positive for Mycoplasma spp., but further molecular detection performed through species-specific primers, revealed that 12 samples were positive for M. hyorhinis. None of the samples were positive for M. hyopneumoniae. The identity of the amplicons were confirmed by nucleic acid sequencing and BLAST analysis. The evolutionary relationship between the detected organisms was also studied by phylogenetic analysis. This study hints at the significance of M. hyorhinis in causing respiratory infections in swine and also suggests that M. hyopneumoniae may not be a significant health hazard for swine populations of north Kerala.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa (P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

Molecular characterization of Anaplasma marginale based on the msp1a and msp1b genes

Anaplasma marginale is an intracellular rickettsial bacterium causing anaplasmosis in ruminants. A. marginale is transmitted biologically by ticks and mechanically by blood-sucking vectors. Anaplasmosis occurs in tropical and subtropical areas of the world. This disease causes huge economic losses due to decreasing meat yield and milk production. The aims of this study were to determine the genetic diversity and antigenicity of A. marginale based on the msp1a and msp1b genes in cattle in Thailand. The A. marginale msp1a and msp1b genes were amplified by the polymerase chain reaction (PCR). There have been four copies of MSP1a tandem repeats among A. marginale Thailand strain, and thirteen different MSP1a tandem repeats were found including repeats B, 25, 27, M, 3, S, C, H, β, 80, 4, TH1 and TH2. Notably, this study showed two copies of the novel conserved tandem sequences namely Thailand Type 1 (TH1) and Type 2 (TH2). The phylogenetic analysis revealed that A. marginale msp1a and msp1b genes were genetically diverse and showed 9 and 5 clades with similarity ranging from 98 to 100% and 79.5 to 100%, respectively, when compared within the isolates of this study. The results of diversity analysis showed 18 and 16 haplotypes of the msp1a and msp1b genes, respectively. The entropy analyses of msp1a and msp1b nucleic acid sequences showed 39 and 900 high entropy peaks with values ranging from 0.35 to 0.85 and from 0.41 to 1.48, respectively, while those of MSP1a and MSP1b amino acid sequences exhibited 75 and 72 high entropy peaks with values ranging from 0.35 to 1.06 and from 0.41 to 1.55, respectively. In addition, B-cell and T-cell epitopes have also been investigated in this study. Hence, our results could be employed to improve the insight input of molecular phylogenetics, genetic diversity and antigenicity of A. marginale Thailand strain.

Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand

Leucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi. The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant (p < 0.05). Hence, our results could be used to improve the immunodiagnostic methods and to find appropriate preventive control strategies or vaccination programs against leucocytozoonosis in order to mitigate or eliminate the harmful impact of this infection on chicken industry.

A Method of Chemical-Mechanical Polishing of a Thick Silver Layer on Patterned Silicon Wafer

Microfluidics-based biochips for biochemical analysis are receiving much attention nowadays. These composite microsystems, also known as lab-on-a-chip or bio-MEMS, offer a number of advantages over conventional laboratory procedures. They automate highly repetitive laboratory tasks by replacing cumbersome equipment with miniaturized and integrated systems, and they enable the handling of small amounts, e.g., micro- and nano-liters, of fluids. Thus they are able to provide ultra-sensitive detection at significantly lower costs per assay than traditional methods, and in a significantly smaller amount of laboratory space. Advances in microfluidics technology offer exciting possibilities in the realm of enzymatic analysis (e.g., glucose and lactate assays), DNA analysis (e.g., PCR and nucleic acid sequence analysis), proteomic analysis involving proteins and peptides, immuno-assays, and toxicity monitoring.Biochips can be used to detect molecules and/or sequence nucleic acid molecules with aid of an array of electrochemical detector cells containing one or more electrodes per cell where each cell being electrically isolated at a wafer level. For many applications it is beneficial to form a non-planar working electrode using a metal which will corrode during CMP steps.We’ve developed a novel Ag and Cu integration process which a) solves problems of metal corrosion over 3D topography during CMP step and b) prevents metal surface contamination with CMP residual slurry and polishing byproducts not usually removable by brush or megasonic cleaning.Electrochemical studies show corrosion behavior of thick Ag layers in different CMP slurries used in this process. SEM images of cross-sectioned metal lined electrochemical cells (vias) clearly demonstrate advantages of new process (right) without traces of corrosion vs conventional CMP process (left).This process can be used also for polishing of other metals and dielectrics over topography where aggressive cleaning post-CMP steps are not desirable. Figure 1

A de novo mutation of the SOX10 gene associated with inner ear malformation in a Guangxi family with Waardenburg syndrome type II

ObjectiveWaardenburg syndrome type 2 (WS2) is a rare neural-crest disorder, characterized by heterochromic irides or blue eyes and sensorineural hearing loss. The aim of this study was to analyze the clinical features and investigate the genetic cause of WS2 in a small family from Guangxi Zhuang Autonomous region. MethodsWhole-exome sequencing and mutational analysis were used to identify disease-causing genes in this family. ResultsA de novo missense mutation, C.355C > T (p. Arg119Cys), in exon 2 of SOX10 was related to inner ear malformation in the proband and identified by whole exon sequencing, but this mutation was absent in normal controls and any public databases. According to nucleic acid sequence and protein bioinformatic analysis, this mutation is considered the cause of WS2 without neurologic involvement in the proband. ConclusionsOur findings provide an accurate genetic diagnosis, counseling, and rehabilitation for family members and may contribute to further genotype-phenotype correlation studies of the SOX10 gene.

ILearnPlus: a comprehensive and automated machine-learning platform for nucleic acid and protein sequence analysis, prediction and visualization.

Sequence-based analysis and prediction are fundamental bioinformatic tasks that facilitate understanding of the sequence(-structure)-function paradigm for DNAs, RNAs and proteins. Rapid accumulation of sequences requires equally pervasive development of new predictive models, which depends on the availability of effective tools that support these efforts. We introduce iLearnPlus, the first machine-learning platform with graphical- and web-based interfaces for the construction of machine-learning pipelines for analysis and predictions using nucleic acid and protein sequences. iLearnPlus provides a comprehensive set of algorithms and automates sequence-based feature extraction and analysis, construction and deployment of models, assessment of predictive performance, statistical analysis, and data visualization; all without programming. iLearnPlus includes a wide range of feature sets which encode information from the input sequences and over twenty machine-learning algorithms that cover several deep-learning approaches, outnumbering the current solutions by a wide margin. Our solution caters to experienced bioinformaticians, given the broad range of options, and biologists with no programming background, given the point-and-click interface and easy-to-follow design process. We showcase iLearnPlus with two case studies concerning prediction of long noncoding RNAs (lncRNAs) from RNA transcripts and prediction of crotonylation sites in protein chains. iLearnPlus is an open-source platform available at https://github.com/Superzchen/iLearnPlus/ with the webserver at http://ilearnplus.erc.monash.edu/.

Comparison of methods for preservation of activated sludge samples for high-throughput nucleic acid sequencing and bacterial diversity analysis

Preservation of environmental samples is an important step in maintaining the original microbial community until the nucleic acid extraction in the laboratory. Here, we collected activated sludge samples to both immediately extract nucleic acids (control) and submit to different storage/preservation methods for 48 h before nucleic acids extraction: room temperature storage, storage on ice, direct storage at −20 °C, rapid freezing in liquid nitrogen followed by storage at −20 °C, and preservation with TRIzol®, RNAlater®, and Nucleic Acid Preservation (NAP) buffers. Bacterial community was compared by high-throughput 16S rRNA gene sequencing from total DNA and RNA. Among the evaluated methods, TRIzol® and rapid freezing with liquid nitrogen were the least indicated, since they led to significant changes in the microbial community structure and abundance of functional groups involved in ammonia metabolism. Compared to control, storage on ice and NAP preserved more than 80% and 75% of genera at the DNA and RNA level respectively, indicating that these methods are the most suitable for storage/preservation of activated sludge samples intended for nucleic acids sequencing.

A Unified Dynamic Programming Framework for the Analysis of Interacting Nucleic Acid Strands: Enhanced Models, Scalability, and Speed.

Dynamic programming algorithms within the NUPACK software suite enable analysis of nucleic acid sequences over complex and test tube ensembles containing arbitrary numbers of interacting strand species, serving the needs of researchers in molecular programming, nucleic acid nanotechnology, synthetic biology, and across the life sciences. Here, to enhance the underlying physical model, ensure scalability for large calculations, and achieve dramatic speedups when calculating diverse physical quantities over complex and test tube ensembles, we introduce a unified dynamic programming framework that combines three ingredients: (1) recursions that specify the dependencies between subproblems and incorporate the details of the structural ensemble and the free energy model, (2) evaluation algebras that define the mathematical form of each subproblem, (3) operation orders that specify the computational trajectory through the dependency graph of subproblems. The physical model is enhanced using new recursions that operate over the complex ensemble including coaxial and dangle stacking subensembles. The recursions are coded generically and then compiled with a quantity-specific evaluation algebra and operation order to generate an executable for each physical quantity: partition function, equilibrium base-pairing probabilities, MFE energy and proxy structure, suboptimal proxy structures, and Boltzmann sampled structures. For large complexes (e.g., 30 000 nt), scalability is achieved for partition function calculations using an overflow-safe evaluation algebra, and for equilibrium base-pairing probabilities using a backtrack-free operation order. A new blockwise operation order that treats subcomplex blocks for the complex species in a test tube ensemble enables dramatic speedups (e.g., 20-120× ) using vectorization and caching. With these performance enhancements, equilibrium analysis of substantial test tube ensembles can be performed in ≤ 1 min on a single computational core (e.g., partition function and equilibrium concentration for all complex species of up to six strands formed from two strand species of 300 nt each, or for all complex species of up to two strands formed from 80 strand species of 100 nt each). A new sampling algorithm simultaneously samples multiple structures from the complex ensemble to yield speedups of an order of magnitude or more as the number of structures increases above ≈103. These advances are available within the NUPACK 4.0 code base (www.nupack.org) which can be flexibly scripted using the all-new NUPACK Python module.

Genomic Evidence for a Case of Reinfection with SARS-CoV-2

The degree of protective immunity conferred by infection with SARS-CoV-2 is currently unknown. As such, the possibility of reinfection with this virus is not well understood. Herein, we describe the data from an investigation of two instances of SARS-CoV-2 infection in the same individual. Through nucleic acid sequence analysis, the viruses associated with each instance of infection were found to possess a degree of genetic discordance that cannot be explained reasonably through short-term in vivo evolution. We conclude that it is possible for humans to become infected multiple times by SARS-CoV-2, but the generalizability of this finding is not known.

NASQAR: a web-based platform for high-throughput sequencing data analysis and visualization

BackgroundAs high-throughput sequencing applications continue to evolve, the rapid growth in quantity and variety of sequence-based data calls for the development of new software libraries and tools for data analysis and visualization. Often, effective use of these tools requires computational skills beyond those of many researchers. To ease this computational barrier, we have created a dynamic web-based platform, NASQAR (Nucleic Acid SeQuence Analysis Resource).ResultsNASQAR offers a collection of custom and publicly available open-source web applications that make extensive use of a variety of R packages to provide interactive data analysis and visualization. The platform is publicly accessible at http://nasqar.abudhabi.nyu.edu/. Open-source code is on GitHub at https://github.com/nasqar/NASQAR, and the system is also available as a Docker image at https://hub.docker.com/r/aymanm/nasqarall. NASQAR is a collaboration between the core bioinformatics teams of the NYU Abu Dhabi and NYU New York Centers for Genomics and Systems Biology.ConclusionsNASQAR empowers non-programming experts with a versatile and intuitive toolbox to easily and efficiently explore, analyze, and visualize their Transcriptomics data interactively. Popular tools for a variety of applications are currently available, including Transcriptome Data Preprocessing, RNA-seq Analysis (including Single-cell RNA-seq), Metagenomics, and Gene Enrichment.