Ambiguous Sequences Research Articles

Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline.

Infecting millions of people, the SARS‐CoV‐2 is evolving at an unprecedented rate, demanding advanced and specified analytic pipeline to capture the mutational spectra. In order to explore mutations and deletions in the spike (S) protein — the most‐discussed protein of SARS‐CoV‐2 — we comprehensively analyzed 35,750 complete S protein‐coding sequences through a custom Python‐based pipeline. This GISAID‐collected dataset of until 24 June 2020 covered six continents and five major climate zones. We identified 27,801 (77.77% sequences) mutated strains compared to reference Wuhan‐Hu‐1 wherein 84.40% of these strains mutated by only a single amino acid (aa). An outlier strain (EPI_ISL_463893) from Bosnia and Herzegovina possessed six aa substitutions. We also identified 11 residues with high aa mutation frequency, and each contains four types of aa variations. The infamous D614G variant has spread worldwide with ever‐rising dominance and across regions with different climatic conditions alongside L5F and D936Y mutants, which have been documented throughout all regions and climate zones, respectively. We also found 988 unique aa substitutions spanned across 660 residues, which differed significantly among different continents (p = .003) and climatic zones (p = .021) as inferred with the Kruskal–Wallis test. Besides, 17 in‐frame deletions at four sites adjacent to receptor‐binding‐domain were determined that may have a possible impact on attenuation. This study provides a fast and accurate pipeline for identifying mutations and deletions from the large dataset for coding and also non‐coding sequences as evidenced by the representative analysis on existing S protein data. By using separate multi‐sequence alignment, removing ambiguous sequences and in‐frame stop codons, and utilizing pairwise alignment, this method can derive both synonymous and non‐synonymous mutations (strain_ID reference aa:mutation position:strain aa). We suggest that the pipeline will aid in the evolutionary surveillance of any SARS‐CoV‐2 encoded proteins and will prove to be crucial in tracking the ever‐increasing variation of many other divergent RNA viruses in the future. The code is available at https://github.com/SShaminur/Mutation-Analysis.

Molecular identification of Bactrocera passiflorae (Diptera: Tephritidae): Challenge and solution for DNA barcoding

AbstractFruit flies cause significant damage to crop and fruit production worldwide. Therefore, it is essential to identify these organisms to species level; however, immature stages are often impossible to be identified morphologically; thus, the application of DNA barcoding has greatly assisted in species identification. Nuclear, mitochondrial pseudo‐COI (NUMT) can be co‐amplified with mitochondrial DNA when using generic primers and therefore impair the efficacy of DNA barcoding. This study detected two types of NUMTs from Bactrocera passiflorae, one of them is novel. Therefore, the new finding will assist future species identification by avoiding misidentification using ambiguous NUMT sequences. In addition, this study has developed primers to target the COI gene of B. passiflorae, not the NUMT copies. The newly designed primers have demonstrated its efficiency in amplifying the Mt‐COI of B. passiflorae and can be used in routine diagnostics.

GAD: A Python Script for Dividing Genome Annotation Files into Feature-Based Files.

Nowadays, the manipulation and analysis of genomic data stored in publicly accessible repositories have become a daily task in genomics and bioinformatics laboratories. Due to the enormous advancement in the field of genome sequencing and the emergence of many projects, bioinformaticians have pushed for the creation of a variety of programs and pipelines that will automatically analyze such big data, in particular the pipelines of gene annotation. Dealing with annotation files using easy and simple programs is very important, particularly for non-developers, enhancing the genomic data analysis acceleration. One of the first tasks required to work with genomic annotation files is to extract different features. In this regard, we have developed GAD ( https://github.com/bio-projects/GAD ) using Python to be a fast, easy, and controlled script that has a high ability to handle annotation files such as GFF3 and GTF. GAD is a cross-platform graphical interface tool used to extract genome features such as intergenic regions, upstream, and downstream genes. Besides, GAD finds all names of ambiguous sequence ontology, and either extracts them or considers them as genes or transcripts. The results are produced in a variety of file formats, such as BED, GTF, GFF3, and FASTA, supported by other bioinformatics programs. The GAD can handle large sizes of different genomes and an infinite number of files with minimal user effort. Therefore, our script could be integrated into various pipelines in all genomic laboratories to accelerate data analysis.

Difficulties in DNA barcoding-based authentication of snapper products due to ambiguous nucleotide sequences in public databases

Fishery products are traded worldwide, have long and branched supply chains and are among the most counterfeit foods. Food control authorities are responsible of controlling food labelling in order to ensure the safety and quality of products. This is particularly important for tropical and subtropical fish species, such as snappers (Lutjanidae), because certain species are known to be frequently contaminated with ciguatoxins that cause harmful Ciguatera Fish Poisoning outbreaks. The analytical method of choice for the identification of a sample at species level is ‘DNA barcoding’, the sequencing of specific genetic markers and the comparison of the sequences with international nucleotide databases, such as GenBank and BOLD. However, the results of these analyses can be severely impaired by an insufficient data basis and especially by database entries with incorrect species annotations. In this work, the available nucleotide sequences for common genetic markers (COI, cytb, 16S rDNA, 12S rDNA, rag1 and ITS) of snapper species in GenBank were subjected to careful examination with regard to their unambiguousness and clarity. Phylogenetic neighbour joining trees were prepared and checked for ambiguous and contradictory placement of species' sequences with special emphasis on the Malabar blood snapper (Lutjanus malabaricus), the two-spot red snapper (L. bohar) and the crimson snapper (L. erythropterus). The results indicate that ambiguous and contradictory database nucleotide sequences impede the DNA barcoding-based authentication of snapper products at species level. A species assignment for the Malabar snapper and the crimson snapper based solely on database queries seems questionable.

A Conceptual Framework for Abundance Estimation of Genomic Targets in the Presence of Ambiguous Short Sequencing Reads

RNA sequencing (RNA-seq) is widely used to study gene-, transcript-, or exon expression. To quantify the expression level, millions of short sequenced reads need to be mapped back to a reference genome or transcriptome. Read mapping makes it possible to find a location to which a read is identical or similar. Based upon this alignment, expression summaries, that is, read counts are generated. However, reads may be matched to multiple locations. Such ambiguously mapped reads are often ignored in the analysis, which is a potential loss of information and may cause bias in expression estimation. We present the general principles underlying multiread allocation and unbiased estimation of the expression level of genes, exons, or transcripts in the presence of multiple mapped reads. The underlying principles are derived from a theoretical concept that identifies important sources of information such as the number of uniquely mapped reads, the total target length, and the length of the shared target regions. We show with simulation studies that methods incorporating some or all of the aforementioned sources of information estimate the expression levels of genes, exons, and/or transcripts with a higher precision and accuracy than methods that do not use this information. We identify important sources of information that should be taken into account by methods that estimate the abundance of genes, exons, and/or transcripts to achieve good precision and accuracy.

Word form shapes are selected to be morphotactically indicative

Abstract This paper explores the hypothesis that morphotactically ambiguous segment sequences should be dispreferred and selected against in the evolution of languages. We define morphotactically ambiguous sequences as sequences that can occur both within morphemes and across boundaries, such as final /nd/ or /mz/ in ModE, which occur in simple forms like wind or alms and in complex ones like sinned or seems. We test the hypothesis in two diachronic corpus studies of Middle and Early Modern English word forms ending in clusters of sonorants followed by /d/ or /t/ and /s/ or /z/. These clusters became highly frequent after the loss of unstressed vowels in final syllables and were highly ambiguous when they emerged. Our data show that the ambiguity of these final clusters was indeed reduced so that the distribution of the final clusters became increasingly skewed: clusters ending in voiceless coronals became significantly clearly indicative of simple forms, while clusters ending in voiced ones came to signal inflectional complexity more reliably.

An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii.

Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast, and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.

The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi

The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent sea-water habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37 °C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for β-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.

PhylotaR: An Automated Pipeline for Retrieving Orthologous DNA Sequences from GenBank in R.

The exceptional increase in molecular DNA sequence data in open repositories is mirrored by an ever-growing interest among evolutionary biologists to harvest and use those data for phylogenetic inference. Many quality issues, however, are known and the sheer amount and complexity of data available can pose considerable barriers to their usefulness. A key issue in this domain is the high frequency of sequence mislabeling encountered when searching for suitable sequences for phylogenetic analysis. These issues include, among others, the incorrect identification of sequenced species, non-standardized and ambiguous sequence annotation, and the inadvertent addition of paralogous sequences by users. Taken together, these issues likely add considerable noise, error or bias to phylogenetic inference, a risk that is likely to increase with the size of phylogenies or the molecular datasets used to generate them. Here we present a software package, phylotaR that bypasses the above issues by using instead an alignment search tool to identify orthologous sequences. Our package builds on the framework of its predecessor, PhyLoTa, by providing a modular pipeline for identifying overlapping sequence clusters using up-to-date GenBank data and providing new features, improvements and tools. We demonstrate and test our pipeline’s effectiveness by presenting trees generated from phylotaR clusters for two large taxonomic clades: Palms and primates. Given the versatility of this package, we hope that it will become a standard tool for any research aiming to use GenBank data for phylogenetic analysis.

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other ‘biversal’ analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine ‘universal base’ and ‘mixed sequence’ probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

Codiagnosability Analysis of Bounded Petri Nets

In this paper, we propose a novel approach to perform codiagnosability analysis of labeled bounded Petri nets. A set of sites observe the system evolution, each one with its own observation mask. Sites do not exchange information with each other but communicate with a coordinator. The coordinator is able to detect a fault if and only if at least one site is able to do that. In a previous work by some of us, it has been proven that a necessary and sufficient condition for codiagnosability under such a framework is the absence of sequences that are “ambiguous” with respect to all sites and whose length may grow indefinitely after the occurrence of some fault. The novelties of the approach consist in using the notion of basis markings to avoid exhaustive enumeration of the set of reachable markings, and in the construction of an automaton, called Verifier, that allows one to detect the presence of ambiguous sequences. Finally, we introduce the notion of K-codiagnosability: a system is K-codiagnosable if and only if faults can be detected in the above framework within at most K observations after their occurrence. An algorithm is provided to compute the smallest value of K such that the system is K-codiagnosable.

Hepatitis B, C and D virus genotypes detected in HBsAg- or anti-HCV-positive people from the Republic of Moldova.

In the Republic of Moldova, little is known about hepatitis B, C and D virus (HBV, HCV, HDV) genotypes, although the genetic variant may influence the course and outcome of disease. For HBV genotyping, 301 hepatitis B surface antigen (HBsAg)-positive sera collected in 2010 and 2011 from drug users, prison inmates, commercial sex workers, and the general population in different geographical regions were investigated. The 31 HBsAg-positive sera collected in 2011 were also tested for HDV. Eighty-eight anti-HCV-positive sera collected between 2010 and 2011 from the general population and health care workers were used for HCV genotyping. Phylogenetic analysis of 84 HBV sequences showed that most of the viruses belonged to genotype D (n=82, 97.6%), predominantly to the subgenotype D1/D2 cluster (n=75/82, 91.5%). One sequence (74110) clustered as an outlier to this cluster, and six sequences belonged to subgenotype D3. Only two subgenotype A2 sequences were found. Cloning of six samples with ambiguous sequence chromatogram signals showed no mixed infections. Phylogenetic analysis of HCV sequences from 66 patients showed a predominance of subtype 1b (n=63, 95.5%). Two sequences belonged to subtype 3a, and one to subtype 2a. HDV RNA belonging to genotype 1 was found in two sera (2/31, 6.5%). Thus, genotypes prevalent in Europe were detected for all three hepatitis viruses. For both HBV and HCV, one genotype was dominant, while occasional other variants seem to be restricted to certain cohorts and/or transmission routes.

Back from the future: Volitional postdiction of perceived apparent motion direction

Among physical events, it is impossible that an event could alter its own past for the simple reason that past events precede future events, and not vice versa. Moreover, to do so would invoke impossible self-causation. However, mental events are constructed by physical neuronal processes that take a finite duration to execute. Given this fact, it is conceivable that later brain events could alter the ongoing interpretation of previous brain events if they arrive within this finite duration of interpretive processing, before a commitment is made to what happened. In the current study, we show that humans can volitionally influence how they perceive an ambiguous apparent motion sequence, as long as the top-down command occurs up to 300ms after the occurrence of the actual motion event in the world. This finding supports the view that there is a temporal integration period over which perception is constructed on the basis of both bottom-up and top-down inputs.

Integrative modeling techniques applied to resolving ambiguous sequence assignment in DNAPKcs

Integrative modeling techniques applied to resolving ambiguous sequence assignment in DNAPKcs

Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing.

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.

P162 Probing neural mechanisms of auditory streaming in humans by transcranial direct current stimulation

Introduction One hypothesis concerning the neural underpinnings of auditory streaming states that frequency tuning of tonotopically organized neurons in primary auditory fields in combination with physiological forward suppression leads to separable representations of high-frequency A and low-frequency B tones. The extent of spatial overlap between the tonotopic activations of A and B tones is thought to underlie the perceptual organization of streaming sequences into one coherent or two separate streams. The present study attempted to interfere with these mechanisms by transcranial direct current stimulation (tDCS) and to probe behavioral outcomes reflecting the perception of ABAB streaming sequences. Objectives We hypothesized that depending on the polarity, tDCS modulates the frequency selectivity and the degree of forward suppression in the neural populations activated by tones A and B. With depolarizing anodal stimulation, tonotopic activation should be broadened. Hyperpolarizing cathodal stimulation should narrow the spatial activation in auditory cortex (AC). As a perceptual effect, tDCS should therefore modulate the proportion of one-stream and two-stream perception whit anodal tDCS reducing two-stream and enhancing one-stream perception, whereas cathodal tDCS enhancing two-stream and reducing one-stream perception. Material and methods Twenty-two subjects were presented with ambiguous ABAB sequences of three different frequency separations and had to decide on their current percept. Before this psychophysical task, the subjects received sham, anodal, or cathodal tDCS over the left AC for 15 min. Each subject performed three consecutive sessions at 1-week intervals to avoid interference effects. In two out of three sessions, subjects were stimulated with anodal and cathodal direct current, respectively, and in one session they underwent a sham condition. Results We could confirm our hypothesis at the most ambiguous ΔF condition of 6 semitones. For anodal compared to sham and cathodal stimulation, we found a significant decrease in the proportion of two-stream and an increase in the proportion of one-stream perception. Conclusion The results demonstrate the feasibility of using tDCS to probe mechanisms underlying auditory streaming through the use of behavioral measures.

Using the ITS2 sequence-structure as a DNA mini-barcode: A case study in authenticating the traditional medicine“Fang Feng”

An ideal DNA barcode should be short enough to be easily amplified. However, barcodes that are too short may lack discriminatory efficacy, and how additional phylogenetic information can be obtained and used without the addition of nucleotides is a major concern. In this study, we selected 135 individuals that represented 97 species from 21 genera to explore the possibility of employing the short ITS2 fragment as a barcode to identify the traditional medicine “Fang Feng” using sequence and structure information. The results revealed that all of the ITS2 sequences had a common four-helix folding structure and some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from highly divergent taxa. The sequence-structure data yielded a maximum parsimony (MP) tree in which the species referred to as “Fang Feng” was successfully identified. Additionally, we compared the species-level discriminatory powers of the sequence-structure ITS2 and the full-length ITS (ITS1, 5.8S, ITS2), and found that their efficacies were very similar. These findings support the possibility of incorporating the ITS2 secondary structure into DNA barcode analysis. Therefore, we propose that ITS2 sequence-structure information could be used as an alternative mini-barcode when a full-length ITS is not present.

Sensitivity and Bias in the Resolution of Stream-Bounce Stimuli.

The audiovisual stream-bounce effect refers to the resolution of ambiguous motion sequences as streaming or bouncing depending on the presence or absence of a sound. We used a novel experimental design and signal detection theory (SDT) to determine its sensory or decisional origins. To account for issues raised by Witt etal. on the interpretation of SDT results, we devised a pure signal detection (as opposed to signal discrimination) paradigm and measured participants' sensitivity and criterion when detecting a weak tone concurrent with objectively streaming or bouncing visual displays. We observed no change in sensitivity but a significant change in criterion with participants' criterion more liberal with bouncing targets than for streaming targets with. In a second experiment, we tasked participants with detecting a weak tone in noise while viewing an ambiguous motion sequence. They also indicated whether the targets appeared to stream or bounce. Participants' reported equivalent, mostly bouncing responses for hit and false alarm trials, and equivalent, mostly streaming responses for correct rejection and miss trials. Further, differences in participants' sensitivity and criterion measures for detecting tones in subjectively streaming compared to subjectively bouncing targets were inconsistent with sensory factors. These results support a decisional account of the sound-induced switch from mostly streaming to mostly bouncing responses in audiovisual stream-bounce displays.

Probing neural mechanisms underlying auditory stream segregation in humans by transcranial direct current stimulation (tDCS)

One hypothesis concerning the neural underpinnings of auditory streaming states that frequency tuning of tonotopically organized neurons in primary auditory fields in combination with physiological forward suppression is necessary for the separation of representations of high-frequency A and low-frequency B tones. The extent of spatial overlap between the tonotopic activations of A and B tones is thought to underlie the perceptual organization of streaming sequences into one coherent or two separate streams. The present study attempts to interfere with these mechanisms by transcranial direct current stimulation (tDCS) and to probe behavioral outcomes reflecting the perception of ABAB streaming sequences. We hypothesized that tDCS by modulating cortical excitability causes a change in the separateness of the representations of A and B tones, which leads to a change in the proportions of one-stream and two-stream percepts. To test this, 22 subjects were presented with ambiguous ABAB sequences of three different frequency separations (∆F) and had to decide on their current percept after receiving sham, anodal, or cathodal tDCS over the left auditory cortex. We could confirm our hypothesis at the most ambiguous ∆F condition of 6 semitones. For anodal compared with sham and cathodal stimulation, we found a significant decrease in the proportion of two-stream perception and an increase in the proportion of one-stream perception. The results demonstrate the feasibility of using tDCS to probe mechanisms underlying auditory streaming through the use of various behavioral measures. Moreover, this approach allows one to probe the functions of auditory regions and their interactions with other processing stages.

Challenges in the analysis of viral metagenomes.

Genome sequencing technologies continue to develop with remarkable pace, yet analytical approaches for reconstructing and classifying viral genomes from mixed samples remain limited in their performance and usability. Existing solutions generally target expert users and often have unclear scope, making it challenging to critically evaluate their performance. There is a growing need for intuitive analytical tooling for researchers lacking specialist computing expertise and that is applicable in diverse experimental circumstances. Notable technical challenges have impeded progress; for example, fragments of viral genomes are typically orders of magnitude less abundant than those of host, bacteria, and/or other organisms in clinical and environmental metagenomes; observed viral genomes often deviate considerably from reference genomes demanding use of exhaustive alignment approaches; high intrapopulation viral diversity can lead to ambiguous sequence reconstruction; and finally, the relatively few documented viral reference genomes compared to the estimated number of distinct viral taxa renders classification problematic. Various software tools have been developed to accommodate the unique challenges and use cases associated with characterizing viral sequences; however, the quality of these tools varies, and their use often necessitates computing expertise or access to powerful computers, thus limiting their usefulness to many researchers. In this review, we consider the general and application-specific challenges posed by viral sequencing and analysis, outline the landscape of available tools and methodologies, and propose ways of overcoming the current barriers to effective analysis.