scholarly journals Altered Nucleoprotein Binding to Influenza Virus RNA Impacts Packaging Efficiency and Replication

2019 ◽  
Author(s):  
Valerie Le Sage ◽  
Jack P. Kanarek ◽  
Eric Nturibi ◽  
Adalena V. Nanni ◽  
Dan J. Snyder ◽  
...  
Keyword(s):  
Influenza A ◽  
High Throughput Sequencing ◽  
Particle Production ◽  
De Novo ◽  
Sequence Similarity ◽  
Independent Manner ◽  
Binding Profile ◽  
Genome Wide ◽  
Packaging Efficiency ◽  
Replication Fitness

AbstractThe genome of Influenza A viruses consists of eight negative-sense RNA segments that are bound by viral nucleoprotein (NP). We recently showed that NP binding is not uniform along the segments but exhibits regions of enrichment as well as depletion. Furthermore, genome-wide NP binding profiles are distinct even in strains with high sequence similarity, such as the two H1N1 strains A/WSN/1933 and A/California/07/2009. Here, we performed interstrain segment swapping experiments with segments of either high or low congruency in NP binding, which suggested that a segment with a similar overall NP binding profile preserved replication fitness of the resulting virus. Further sub-segmental swapping experiments demonstrated that NP binding is affected by changes to the underlying nucleotide sequence, as NP peaks can either become lost or appear de novo at mutated regions. Unexpectedly, these local nucleotide changes in one segment not only affect NP binding in cis, but also impact the genome-wide NP binding profile on other segments in a vRNA sequence-independent manner, suggesting that primary sequence alone is not the sole determinant for NP association to vRNA. Moreover, we observed that sub-segmental mutations that affect NP binding profiles can result in reduced replication fitness, which is caused by defects in vRNA packaging efficiency and an increase in semi-infectious particle production. Taken together, our results indicate that the pattern of NP binding to vRNA is important for efficient virus replication.Author SummaryEach viral RNA (vRNA) segment is bound by the polymerase complex at the 5′ and 3′ ends, while the remainder of the vRNA is coated non-uniformly and non-randomly by nucleoprotein (NP). To explore the constraints of NP binding to vRNA, we used high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of mutant H1N1 strains with exchanged vRNA sequences and observed that NP binding can be changed based on vRNA sequence. The most striking observation of our study is that nucleotide changes in one segment can have genome-wide effects on the NP binding profile of other segments. We refer to this phenomenon as the ‘butterfly effect’ of influenza packaging. Our results provide an important context in which to consider future studies regarding influenza packaging and assembly.

scholarly journals Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 749 ◽  
Author(s):  
Melanie Hiltbrunner ◽  
Gerald Heckel
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Phylogenetic Analyses ◽  
Common Vole ◽  
Natural Host ◽  
Sequence Information ◽  
Coding Region ◽  
Sequence Capture ◽  
Genome Wide ◽  
The Impact

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

scholarly journals Analysis of the Genomic Basis of Functional Diversity in Dinoflagellates using a Transcriptome-Based Sequence Similarity Network

2017 ◽  
Author(s):  
Arnaud Meng ◽  
Erwan Corre ◽  
Ian Probert ◽  
Andres Gutierrez-Rodriguez ◽  
Raffaele Siano ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Sequence Similarity ◽  
Connected Components ◽  
Similarity Network ◽  
Network Analyses ◽  
Comprehensive Picture ◽  
Core Proteome ◽  
Functional Features ◽  
Genomic Basis

ABSTRACTDinoflagellates are one of the most abundant and functionally diverse groups of eukaryotes. Despite an overall scarcity of genomic information for dinoflagellates, constantly emerging high-throughput sequencing resources can be used to characterize and compare these organisms. We assembled de novo and processed 46 dinoflagellate transcriptomes and used a sequence similarity network (SSN) to compare the underlying genomic basis of functional features within the group. This approach constitutes the most comprehensive picture to date of the genomic potential of dinoflagellates. A core proteome composed of 252 connected components (CCs) of putative conserved protein domains (pCDs) was identified. Of these, 206 were novel and 16 lacked any functional annotation in public databases. Integration of functional information in our network analyses allowed investigation of pCDs specifically associated to functional traits. With respect to toxicity, sequences homologous to those of proteins involved in toxin biosynthesis pathways (e.g. sxtA1-4 and sxtG) were not specific to known toxin-producing species. Although not fully specific to symbiosis, the most represented functions associated with proteins involved in the symbiotic trait were related to membrane processes and ion transport. Overall, our SSN approach led to identification of 45,207 and 90,794 specific and constitutive pCDs of respectively the toxic and symbiotic species represented in our analyses. Of these, 56% and 57% respectively (i.e. 25,393 and 52,193 pCDs) completely lacked annotation in public databases. This stresses the extent of our lack of knowledge, while emphasizing the potential of SSNs to identify candidate pCDs for further functional genomic characterization.

scholarly journals Phylogenetic relationships of endornaviruses in common bean from the western highlands of Kenya and global sequences

2018 ◽  
Author(s):  
James M Wainaina ◽  
Elijah Ateka ◽  
Timothy Makori ◽  
Monica A Kehoe ◽  
Laura M Boykin
Keyword(s):  
Phaseolus Vulgaris ◽  
Complete Genome ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Current Knowledge ◽  
Sequence Similarity ◽  
Evolutionary Relationship ◽  
Sub Saharan Africa ◽  
Complete Genomes ◽  
Sub Saharan

Background: Endornaviruses are non-pathogenic viruses infecting multiple agricultural important crops including legumes, with global distribution. However, there is an absence on the complete genome of endornaviruses from legumes in particular with the sub-Saharan region. In this study, we report the first complete genomes of PvEV1 and PvEV2, and the evolutionary relationship of these genomes. Methods: Viral symptomatic common beans (Phaseolus vulgaris) showing Bean common mosaic necrosis virus (BCMNV) symptoms from Vihiga county, in the western highlands of Kenya were collected during field survey’s in the region. High throughput sequencing (RNA-Seq) was carried out on total RNA isolated from symptomatic leaf samples. Subsequently, de novo assembly and reference mapping was carried out to obtain the complete genomes of PvEV-1 and PvEV-2. Results: We identified the complete genome of Phaseolus vulgaris endornavirus 1 and 2 (PvEV-1 and PvEV-2) from sub-Saharan Africa (SSA). The average genome size of PvEV-1 was ~13,890 nucleotides (nt) while PvEV-2 was ~14,698 nt, encoding a single open reading frame (ORF). Single ORFs ranged from 4,632 to 4,633 aa in PvEV-1 and from 4,899 – to 4,954 aa in PvEV-2. Both ORFs encoded for the RNA-dependent RNA polymerase (RdRP) gene. The percentage sequence similarity between PvEV-1, PvEV-2 from this study GenBanks sequences was 29 % to 99 %. Bayesian phylogenetic analysis resolved in two well-supported monophyletic clades, with isolates from this study clustering with those from Brazil sequences. Discussion: This study provides the first insights into the evolutionary relationships of PvEV from SSA diverse and contributes towards filling the current knowledge gaps on endornaviruses

scholarly journals Phylogenetic relationships of endornaviruses in common bean from the western highlands of Kenya and global sequences

2018 ◽  
Author(s):  
James M Wainaina ◽  
Elijah Ateka ◽  
Timothy Makori ◽  
Monica A Kehoe ◽  
Laura M Boykin
Keyword(s):  
Phaseolus Vulgaris ◽  
Complete Genome ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Current Knowledge ◽  
Sequence Similarity ◽  
Evolutionary Relationship ◽  
Sub Saharan Africa ◽  
Complete Genomes ◽  
Sub Saharan

Background: Endornaviruses are non-pathogenic viruses infecting multiple agricultural important crops including legumes, with global distribution. However, there is an absence on the complete genome of endornaviruses from legumes in particular with the sub-Saharan region. In this study, we report the first complete genomes of PvEV1 and PvEV2, and the evolutionary relationship of these genomes. Methods: Viral symptomatic common beans (Phaseolus vulgaris) showing Bean common mosaic necrosis virus (BCMNV) symptoms from Vihiga county, in the western highlands of Kenya were collected during field survey’s in the region. High throughput sequencing (RNA-Seq) was carried out on total RNA isolated from symptomatic leaf samples. Subsequently, de novo assembly and reference mapping was carried out to obtain the complete genomes of PvEV-1 and PvEV-2. Results: We identified the complete genome of Phaseolus vulgaris endornavirus 1 and 2 (PvEV-1 and PvEV-2) from sub-Saharan Africa (SSA). The average genome size of PvEV-1 was ~13,890 nucleotides (nt) while PvEV-2 was ~14,698 nt, encoding a single open reading frame (ORF). Single ORFs ranged from 4,632 to 4,633 aa in PvEV-1 and from 4,899 – to 4,954 aa in PvEV-2. Both ORFs encoded for the RNA-dependent RNA polymerase (RdRP) gene. The percentage sequence similarity between PvEV-1, PvEV-2 from this study GenBanks sequences was 29 % to 99 %. Bayesian phylogenetic analysis resolved in two well-supported monophyletic clades, with isolates from this study clustering with those from Brazil sequences. Discussion: This study provides the first insights into the evolutionary relationships of PvEV from SSA diverse and contributes towards filling the current knowledge gaps on endornaviruses

scholarly journals Genome-Wide Analysis of Estrogen Receptor α DNA Binding and Tethering Mechanisms Identifies Runx1 as a Novel Tethering Factor in Receptor-Mediated Transcriptional Activation

2010 ◽  
Vol 30 (16) ◽  
pp. 3943-3955 ◽  
Author(s):  
Joshua D. Stender ◽  
Kyuri Kim ◽  
Tze Howe Charn ◽  
Barry Komm ◽  
Ken C. N. Chang ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Estrogen Receptor Alpha ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Motif Analysis ◽  
Phenotypic Properties ◽  
Genome Wide

ABSTRACT Nuclear receptor estrogen receptor alpha (ERα) controls the expression of hundreds of genes responsible for target cell phenotypic properties, but the relative importance of direct versus tethering mechanisms of DNA binding has not been established. In this first report, we examine the genome-wide chromatin localization of an altered-specificity mutant ER with a DNA binding domain deficient in binding to estrogen response element (ERE)-containing DNA (DBDmut ER) versus wild-type ERα. Using high-throughput sequencing of ER chromatin immunoprecipitations (ChIP-Seq) and mRNA transcriptional profiling, we show that direct ERE binding is required for most of (75%) estrogen-dependent gene regulation and 90% of hormone-dependent recruitment of ER to genomic binding sites. De novo motif analysis of the chromatin binding regions in MDA-MB-231 human breast cancer cells defined unique transcription factor profiles responsible for genes regulated through tethering versus direct ERE binding, with Runx motifs enriched in ER-tethered sites. We confirmed a role for Runx1 in mediating ERα genomic recruitment and regulation of tethering genes. Our findings delineate the contributions of direct receptor ERE binding versus binding through response elements for other transcription factors in chromatin localization and ER-dependent gene regulation, paradigms likely to underlie the gene regulatory actions of other nuclear receptors as well.

scholarly journals Genome-wide profiling of heritable and de novo STR variations

2016 ◽  
Author(s):  
Thomas Willems ◽  
Dina Zielinski ◽  
Assaf Gordon ◽  
Melissa Gymrek ◽  
Yaniv Erlich
Keyword(s):  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Genetic Diseases ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
High Throughput Sequencing Data ◽  
Genome Wide ◽  
A Genome ◽  
Short Tandem

AbstractShort tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases, population genetics applications, and forensic casework. However, STRs have proven problematic to genotype from high-throughput sequencing data. Here, we describe HipSTR, a novel haplotype-based method for robustly genotyping, haplotyping, and phasing STRs from whole genome sequencing data and report a genome-wide analysis and validation of de novo STR mutations.

scholarly journals Comparison of three clustering approaches for detecting novel environmental microbial diversity

2015 ◽  
Author(s):  
Dominik Forster ◽  
Micah Dunthorn ◽  
Thorsten Stoeck ◽  
Frédéric Mahé
Keyword(s):  
Graph Theory ◽  
Microbial Ecology ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Sequence Similarity ◽  
Pairwise Alignment ◽  
Reference Database ◽  
Clustering Methods ◽  
Underlying Network ◽  
Network Topologies

Discovery of novel diversity in high-throughput sequencing (HTS) studies is a central task in environmental microbial ecology. To evaluate the effects that amplicon clustering methods have on novel diversity discovery, we clustered an environmental marine protist HTS dataset of protist reads together with accessions from the taxonomically curated PR2 reference database using three de novo approaches: sequence similarity networks, USEARCH, and Swarm. The novel diversity uncovered by each clustering approach differed drastically in the number of operational taxonomic units (OTUs) and the number of environmental amplicons in these novel diversity OTUs. Global pairwise alignment comparisons revealed that numerous amplicons classified as novel by USEARCH and Swarm were actually highly similar to reference accessions. Using graph theory we found additional novel diversity within OTUs that would have gone unnoticed without further using their underlying network topologies. Our results suggest that novel diversity inferred from clustering approaches requires further validation, whereas graph theory provides a powerful tool for microbial ecology and the analyses of environmental HTS datasets.

scholarly journals Comparison of three clustering approaches for detecting novel environmental microbial diversity

2015 ◽  
Author(s):  
Dominik Forster ◽  
Micah Dunthorn ◽  
Thorsten Stoeck ◽  
Frédéric Mahé
Keyword(s):  
Graph Theory ◽  
Microbial Ecology ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Sequence Similarity ◽  
Pairwise Alignment ◽  
Reference Database ◽  
Clustering Methods ◽  
Underlying Network ◽  
Network Topologies

Discovery of novel diversity in high-throughput sequencing (HTS) studies is a central task in environmental microbial ecology. To evaluate the effects that amplicon clustering methods have on novel diversity discovery, we clustered an environmental marine protist HTS dataset of protist reads together with accessions from the taxonomically curated PR2 reference database using three de novo approaches: sequence similarity networks, USEARCH, and Swarm. The novel diversity uncovered by each clustering approach differed drastically in the number of operational taxonomic units (OTUs) and the number of environmental amplicons in these novel diversity OTUs. Global pairwise alignment comparisons revealed that numerous amplicons classified as novel by USEARCH and Swarm were actually highly similar to reference accessions. Using graph theory we found additional novel diversity within OTUs that would have gone unnoticed without further using their underlying network topologies. Our results suggest that novel diversity inferred from clustering approaches requires further validation, whereas graph theory provides a powerful tool for microbial ecology and the analyses of environmental HTS datasets.

scholarly journals Prediction of mitochondrial genome-wide variation through sequencing of mitochondrion-enriched extracts

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kelsey E. Fisher ◽  
Steven P. Bradbury ◽  
Brad S. Coates
Keyword(s):  
Mitochondrial Genome ◽  
Nadh Dehydrogenase ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Short Read ◽  
Deletion Mutations ◽  
Haplotype Variation ◽  
Cell Extracts ◽  
Genome Wide ◽  
Mtdna Haplotype

Abstract Although mitochondrial DNA (mtDNA) haplotype variation is often applied for estimating population dynamics and phylogenetic relationships, economical and generalized methods for entire mtDNA genome enrichment prior to high-throughput sequencing are not readily available. This study demonstrates the utility of differential centrifugation to enrich for mitochondrion within cell extracts prior to DNA extraction, short-read sequencing, and assembly using exemplars from eight maternal lineages of the insect species, Ostrinia nubilalis. Compared to controls, enriched extracts showed a significant mean increase of 48.2- and 86.1-fold in mtDNA based on quantitative PCR, and proportion of subsequent short sequence reads that aligned to the O. nubilalis reference mitochondrial genome, respectively. Compared to the reference genome, our de novo assembled O. nubilalis mitochondrial genomes contained 82 intraspecific substitution and insertion/deletion mutations, and provided evidence for correction of mis-annotated 28 C-terminal residues within the NADH dehydrogenase subunit 4. Comparison to a more recent O. nubilalis mtDNA assembly from unenriched short-read data analogously showed 77 variant sites. Twenty-eight variant positions, and a triplet ATT codon (Ile) insertion within ATP synthase subunit 8, were unique within our assemblies. This study provides a generalizable pipeline for whole mitochondrial genome sequence acquisition adaptable to applications across a range of taxa.

scholarly journals Hybrid decay: a transgenerational epigenetic decline in vigor and viability triggered in backcross populations of teosinte with maize

2019 ◽  
Author(s):  
Wei Xue ◽  
Sarah N. Anderson ◽  
Xufeng Wang ◽  
Liyan Yang ◽  
Peter A. Crisp ◽  
...  
Keyword(s):  
Transposable Elements ◽  
De Novo ◽  
Genome Instability ◽  
Sequence Similarity ◽  
Hybrid Incompatibility ◽  
Advanced Backcross ◽  
Genome Wide ◽  
Normal Maize ◽  
Plant Phenotype ◽  
Backcross Populations

ABSTRACTIn the course of generating populations of maize with teosinte chromosomal introgressions, an unusual sickly plant phenotype was noted in individuals from crosses with two teosinte accessions collected near Valle de Bravo, Mexico. The plants of these Bravo teosinte accessions appear phenotypically normal themselves and the F1plants appear similar to typical maize x teosinte F1s. However, upon backcrossing to maize, the BC1and subsequent generations display a number of detrimental characteristics including shorter stature, reduced seed set and abnormal floral structures. This phenomenon is observed in all BC individuals and there is no chromosomal segment linked to the sickly plant phenotype in advanced backcross generations. Once the sickly phenotype appears in a lineage, normal plants are never again recovered by continued backcrossing to the normal maize parent. Whole-genome shotgun sequencing reveals a small number of genomic sequences, some with homology to transposable elements, that have increased in copy number in the backcross populations. Transcriptome analysis of seedlings, which do not have striking phenotypic abnormalities, identified segments of 18 maize genes that exhibit increased expression in sickly plants. Ade novoassembly of transcripts present in plants exhibiting the sickly phenotype identified a set of 59 up-regulated novel transcripts. These transcripts include some examples with sequence similarity to transposable elements and other sequences present in the recurrent maize parent (W22) genome as well as novel sequences not present in the W22 genome. Genome-wide profiles of gene expression, DNA methylation and sRNAs are similar between sickly plants and normal controls, although a few up-regulated transcripts and transposable elements are associated with altered sRNA or methylation profiles. This study documents hybrid incompatibility and genome instability triggered by the backcrossing of Bravo teosinte with maize. We name this phenomenon “hybrid decay” and present ideas on the mechanism that may underlie it.

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