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Wednesday, October 23, 2019

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly…

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Wednesday, October 23, 2019

Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity.

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal…

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Wednesday, October 23, 2019

Improved production of propionic acid using genome shuffling.

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by-products gene-knockout strategies as a logical solution to increase yield. However, removing by-product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand…

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Wednesday, October 23, 2019

Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector…

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Sunday, September 22, 2019

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (?123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3’UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ?123…

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Sunday, September 22, 2019

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have…

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Sunday, September 22, 2019

Global dissection of alternative splicing uncovers transcriptional diversity in tissues and associates with the flavonoid pathway in tea plant (Camellia sinensis).

Alternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea.In our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~?20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc.…

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Sunday, September 22, 2019

Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques.

Poxviruses are large DNA viruses that infect humans and animals. Vaccinia virus (VACV) has been applied as a live vaccine for immunization against smallpox, which was eradicated by 1980 as a result of worldwide vaccination. VACV is the prototype of poxviruses in the investigation of the molecular pathogenesis of the virus. Short-read sequencing methods have revolutionized transcriptomics; however, they are not efficient in distinguishing between the RNA isoforms and transcript overlaps. Long-read sequencing (LRS) is much better suited to solve these problems and also allow direct RNA sequencing. Despite the scientific relevance of VACV, no LRS data have been generated…

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Sunday, September 22, 2019

Molecular genetic diversity and characterization of conjugation genes in the fish parasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is the etiologic agent of “white spot”, a commercially important disease of freshwater fish. As a parasitic ciliate, I. multifiliis infects numerous host species across a broad geographic range. Although Ichthyophthirius outbreaks are difficult to control, recent sequencing of the I. multifiliis genome has revealed a number of potential metabolic pathways for therapeutic intervention, along with likely vaccine targets for disease prevention. Nonetheless, major gaps exist in our understanding of both the life cycle and population structure of I. multifiliis in the wild. For example, conjugation has never been described in this species, and it is unclear whether…

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Sunday, September 22, 2019

Single-molecule long-read transcriptome profiling of Platysternon megacephalum mitochondrial genome with gene rearrangement and control region duplication.

Platysternon megacephalum is the sole living representative of the poorly studied turtle lineage Platysternidae. Their mitochondrial genome has been subject to gene rearrangement and control region duplication, resulting in a unique mitochondrial gene order in vertebrates. In this study, we sequenced the first full-length turtle (P. megacephalum) liver transcriptome using single-molecule real-time sequencing to study the transcriptional mechanisms of its mitochondrial genome. ND5 and ND6 anti-sense (ND6AS) forms a single transcript with the same expression in the human mitochondrial genome, but here we demonstrated differential expression of the rearranged ND5 and ND6AS genes in P. megacephalum. And some polycistronic transcripts…

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Sunday, September 22, 2019

A high-quality annotated transcriptome of swine peripheral blood.

High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes and/or transcriptomes. However, neither the reference genome nor the peripheral blood transcriptome of the pig have been sufficiently assembled and annotated to support such profiling assays in this emerging biomedical model organism. We aimed to assemble published and novel RNA-seq data to provide a comprehensive, well-annotated blood transcriptome for pigs by integrating a de novo assembly with a genome-guided assembly.A de novo…

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Sunday, September 22, 2019

ALE: a generic assembly likelihood evaluation framework for assessing the accuracy of genome and metagenome assemblies.

Researchers need general purpose methods for objectively evaluating the accuracy of single and metagenome assemblies and for automatically detecting any errors they may contain. Current methods do not fully meet this need because they require a reference, only consider one of the many aspects of assembly quality or lack statistical justification, and none are designed to evaluate metagenome assemblies.In this article, we present an Assembly Likelihood Evaluation (ALE) framework that overcomes these limitations, systematically evaluating the accuracy of an assembly in a reference-independent manner using rigorous statistical methods. This framework is comprehensive, and integrates read quality, mate pair orientation and…

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Sunday, September 22, 2019

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

The initiating nucleotide found at the 5′ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5’end of primary transcripts, but rather attempt to deplete non-targeted RNA.We developed a method, Cappable-seq, for directly enriching for the 5′ end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying…

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Sunday, September 22, 2019

A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing.

Alternative splicing (AS) and fusion transcripts produce a vast expansion of transcriptomes and proteomes diversity. However, the reliability of these events and the extend of epigenetic mechanisms have not been adequately addressed due to its limitation of uncertainties about the complete structure of mRNA. Here we combined single-molecule real-time sequencing, Illumina RNA-seq and DNA methylation data to characterize the landscapes of DNA methylation on AS, fusion isoforms formation and lncRNA feature and further to unveil the transcriptome complexity of pig. Our analysis identified an unprecedented scale of high-quality full-length isoforms with over 28,127 novel isoforms from 26,881 novel genes. More…

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Sunday, September 22, 2019

Transcriptomic study of Herpes simplex virus type-1 using full-length sequencing techniques

Herpes simplex virus type-1 (HSV-1) is a human pathogenic member of the Alphaherpesvirinae subfamily of herpesviruses. The HSV-1 genome is a large double-stranded DNA specifying about 85 protein coding genes. The latest surveys have demonstrated that the HSV-1 transcriptome is much more complex than it had been thought before. Here, we provide a long-read sequencing dataset, which was generated by using the RSII and Sequel systems from Pacific Biosciences (PacBio), as well as MinION sequencing system from Oxford Nanopore Technologies (ONT). This dataset contains 39,096 reads of inserts (ROIs) mapped to the HSV-1 genome (X14112) in RSII sequencing, while Sequel…

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