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Sunday, September 22, 2019

TACO produces robust multisample transcriptome assemblies from RNA-seq.

Accurate transcript structure and abundance inference from RNA sequencing (RNA-seq) data is foundational for molecular discovery. Here we present TACO, a computational method to reconstruct a consensus transcriptome from multiple RNA-seq data sets. TACO employs novel change-point detection to demarcate transcript start and end sites, leading to improved reconstruction accuracy compared with other tools in its class. The tool is available at http://tacorna.github.io and can be readily incorporated into RNA-seq analysis workflows.

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Sunday, September 22, 2019

Evaluating approaches to find exon chains based on long reads.

Transcript prediction can be modeled as a graph problem where exons are modeled as nodes and reads spanning two or more exons are modeled as exon chains. Pacific Biosciences third-generation sequencing technology produces significantly longer reads than earlier second-generation sequencing technologies, which gives valuable information about longer exon chains in a graph. However, with the high error rates of third-generation sequencing, aligning long reads correctly around the splice sites is a challenging task. Incorrect alignments lead to spurious nodes and arcs in the graph, which in turn lead to incorrect transcript predictions. We survey several approaches to find the exon…

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Sunday, September 22, 2019

neoantigenR: An annotation based pipeline for tumor neoantigen identification from sequencing data

Studies indicate that more than 90% of human genes are alternatively spliced, suggesting the complexity of the transcriptome assembly and analysis. The splicing process is often disrupted, resulting in both functional and non-functional end-products (Sveen et al. 2016) in many cancers. Harnessing the immune system to fight against malignant cancers carrying aberrantly mutated or spliced products is becoming a promising approach to cancer therapy. Advances in immune checkpoint blockade have elicited adaptive immune responses with promising clinical responses to treatments against human malignancies (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Emerging data suggest that recognition of patient-specific mutation-associated cancer antigens…

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Sunday, September 22, 2019

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the…

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Sunday, September 22, 2019

Interactive analysis of Long-read RNA isoforms with Iso-Seq Browser

Background: Long-read RNA sequencing, such as Pacific Biosciences Iso-Seq method, enables generation of sequencing reads that are 10 kilobases or even longer. These reads are ideal for discovering splice junctions and resolving full-length gene transcripts without time-consuming and error-prone techniques such as transcript assembly and junction inference. Results: Iso-Seq Browser is a Web-based visual analytics tool for long-read RNA sequencing data produced by Pacific Biosciences isoform sequencing (Iso-Seq) techniques. Key features of the Iso-Seq Browser are: 1) an exon-only web-based interface with zooming and exon highlighting for exploring reference gene transcripts and novel gene isoforms, 2) automated grouping of transcripts…

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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

Androgen receptor variant AR-V9 is co-expressed with AR-V7 in prostate cancer metastases and predicts abiraterone resistance.

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies.  Accordingly, efforts are underway to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC).  The purpose of this study was to understand whether other AR variants may be co-expressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design:  We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models.  Co-expression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. …

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Sunday, September 22, 2019

Bayesian nonparametric discovery of isoforms and individual specific quantification.

Most human protein-coding genes can be transcribed into multiple distinct mRNA isoforms. These alternative splicing patterns encourage molecular diversity, and dysregulation of isoform expression plays an important role in disease etiology. However, isoforms are difficult to characterize from short-read RNA-seq data because they share identical subsequences and occur in different frequencies across tissues and samples. Here, we develop BIISQ, a Bayesian nonparametric model for isoform discovery and individual specific quantification from short-read RNA-seq data. BIISQ does not require isoform reference sequences but instead estimates an isoform catalog shared across samples. We use stochastic variational inference for efficient posterior estimates and…

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Sunday, September 22, 2019

wtf genes are prolific dual poison-antidote meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote…

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Sunday, September 22, 2019

Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis.

RNA-sequencing (RNA-seq) is an essential technique for transcriptome studies, hundreds of analysis tools have been developed since it was debuted. Although recent efforts have attempted to assess the latest available tools, they have not evaluated the analysis workflows comprehensively to unleash the power within RNA-seq. Here we conduct an extensive study analysing a broad spectrum of RNA-seq workflows. Surpassing the expression analysis scope, our work also includes assessment of RNA variant-calling, RNA editing and RNA fusion detection techniques. Specifically, we examine both short- and long-read RNA-seq technologies, 39 analysis tools resulting in ~120 combinations, and ~490 analyses involving 15 samples…

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Sunday, September 22, 2019

Long-read sequencing revealed an extensive transcript complexity in herpesviruses.

Long-read sequencing (LRS) techniques are very recent advancements, but they have already been used for transcriptome research in all of the three subfamilies of herpesviruses. These techniques have multiplied the number of known transcripts in each of the examined viruses. Meanwhile, they have revealed a so far hidden complexity of the herpesvirus transcriptome with the discovery of a large number of novel RNA molecules, including coding and non-coding RNAs, as well as transcript isoforms, and polycistronic RNAs. Additionally, LRS techniques have uncovered an intricate meshwork of transcriptional overlaps between adjacent and distally located genes. Here, we review the contribution of…

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Sunday, September 22, 2019

Lipoprotein lipase reaches the capillary lumen in chickens despite an apparent absence of GPIHBP1.

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken…

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Sunday, September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT…

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Sunday, September 22, 2019

Dual platform long-read RNA-sequencing dataset of the human Cytomegalovirus Lytic transcriptome

RNA-sequencing has revolutionized transcriptomics and the way we measure gene expression (Wang et al., 2009). As of today, short-read RNA sequencing is more widely used, and due to its low price and high throughput, is the preferred tool for the quantitative analysis of gene expression. However, the annotation of transcript isoforms is rather difficult using only short-read sequencing data, because the reads are shorter than most transcripts (Steijger et al., 2013). Long-read sequencing, on the other hand, can provide full contig information about transcripts, including exon-connectivity, and its merits in transcriptome profiling are being increasingly acknowledged (Sharon et al., 2013;…

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Sunday, September 22, 2019

Widespread polycistronic transcripts in fungi revealed by single-molecule mRNA sequencing.

Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of…

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