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Asset Tag: RNA sequencing

September 22, 2019

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

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September 22, 2019

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.

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September 22, 2019

PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme.

High-throughput transcriptome sequencing (RNA-seq) technology promises to discover novel protein-coding and non-coding transcripts, particularly the identification of long non-coding RNAs (lncRNAs) from de novo sequencing data. This requires tools that are not restricted by prior gene annotations, genomic sequences and high-quality sequencing.We present an alignment-free tool called PLEK (predictor of long non-coding RNAs and messenger RNAs based on an improved k-mer scheme), which uses a computational pipeline based on an improved k-mer scheme and a support vector machine (SVM) algorithm to distinguish lncRNAs from messenger RNAs (mRNAs), in the absence of genomic sequences or annotations. The performance of PLEK was evaluated on well-annotated mRNA and lncRNA transcripts. 10-fold cross-validation tests on human RefSeq mRNAs and GENCODE lncRNAs indicated that our tool could achieve accuracy of up to 95.6%. We demonstrated the utility of PLEK on transcripts from other vertebrates using the model built from human datasets. PLEK attained >90% accuracy on most of these datasets. PLEK also performed well using a simulated dataset and two real de novo assembled transcriptome datasets (sequenced by PacBio and 454 platforms) with relatively high indel sequencing errors. In addition, PLEK is approximately eightfold faster than a newly developed alignment-free tool, named Coding-Non-Coding Index (CNCI), and 244 times faster than the most popular alignment-based tool, Coding Potential Calculator (CPC), in a single-threading running manner.PLEK is an efficient alignment-free computational tool to distinguish lncRNAs from mRNAs in RNA-seq transcriptomes of species lacking reference genomes. PLEK is especially suitable for PacBio or 454 sequencing data and large-scale transcriptome data. Its open-source software can be freely downloaded from https://sourceforge.net/projects/plek/files/.

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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 LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens – as it does in mammals – despite an apparent absence of GPIHBP1.

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September 22, 2019

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

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September 22, 2019

Single Molecule Sequencing: new outlooks for solving genome assembly and transcripts identification challenges

In this review, we introduce a novel sequencing technology, named Single Molecule Real Time sequencing. Also called Single Molecule Sequencing, as it do not requires any amplification, this new technology is able to pro- duce much longer reads than previous NGS technologies such as Illumina. This read size improvements, which can reach 150 fold, will solve many challenges caused by the actual NGS technologies. Short NGS reads, reach- ing a maximum size of 300 bp, make it hard to reconstitute a whole genome and are always leading to fragmented genome assembly. It is also difficult to correctly infer transcript quantification and identification when there is a high isoforms diversity. Despite their higher error rate, long reads have shown very promising result concerning these actual issues. We show that longer reads can produce less fragmented assembly, with a better quality, but also sequence from start to end mRNA, making it much more easier to infer correct transcript quantification, and even allow new intron structure and so new isoforms discovery.

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September 22, 2019

Global identification of alternative splicing via comparative analysis of SMRT- and Illumina-based RNA-seq in strawberry.

Alternative splicing (AS) is a key post-transcriptional regulatory mechanism, yet little information is known about its roles in fruit crops. Here, AS was globally analyzed in the wild strawberry Fragaria vesca genome with RNA-seq data derived from different stages of fruit development. The AS landscape was characterized and compared between the single-molecule, real-time (SMRT) and Illumina RNA-seq platform. While SMRT has a lower sequencing depth, it identifies more genes undergoing AS (57.67% of detected multiexon genes) when it is compared with Illumina (33.48%), illustrating the efficacy of SMRT in AS identification. We investigated different modes of AS in the context of fruit development; the percentage of intron retention (IR) is markedly reduced whereas that of alternative acceptor sites (AA) is significantly increased post-fertilization when compared with pre-fertilization. When all the identified transcripts were combined, a total of 66.43% detected multiexon genes in strawberry undergo AS, some of which lead to a gain or loss of conserved domains in the gene products. The work demonstrates that SMRT sequencing is highly powerful in AS discovery and provides a rich data resource for later functional studies of different isoforms. Further, shifting AS modes may contribute to rapid changes of gene expression during fruit set.© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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September 22, 2019

Cataloguing over-expressed genes in Epstein Barr Virus immortalized lymphoblastoid cell lines through consensus analysis of PacBio transcriptomes corroborates hypomethylation of chromosome 1

The ability of Epstein Barr Virus (EBV) to transform resting cell B-cells into immortalized lymphoblastoid cell lines (LCL) provides a continuous source of peripheral blood lymphocytes that are used to model conditions in which these lymphocytes play a key role. Here, the PacBio generated transcriptome of three LCLs from a parent-daughter trio (SRAid:SRP036136) provided by a previous study [1] were analyzed using a kmer-based version of YeATS (KEATS). The set of over-expressed genes in these cell lines were determined based on a comparison with the PacBio transcriptome of twenty tissues pro- vided by another study (hOPTRS) [2]. MIR155 long non-coding RNA (MIR155HG), Fc fragment of IgE receptor II (FCER2), T-cell leukemia/lymphoma 1A (TCL1A), and germinal center associated signaling and motility (GCSAM) were genes having the highest expression counts in the three LCLs with no expression in hOPTRS. Other over-expressed genes, having low expression in hOPTRS, were membrane spanning 4-domains A1 (MS4A1) and ribosomal protein S2 pseudogene 55 (RPS2P55). While some of these genes are known to be over-expressed in LCLs, this study provides a comprehensive cataloguing of such genes. A recent work involving a patient with EBV-positive large B-cell lymphoma was “unusually lacking various B-cell markers”, but over-expressing CD30 [3] – a gene ranked 79 among uniquely expressed genes here. Hypomethylation of chromosome 1 observed in EBV immortalized LCLs [4, 5] is also corroborated here by mapping the genes to chromosomes. Extending previous work identifying un-annotated genes [6], 80 genes were identified which are expressed in the three LCLs, not in hOPTRS, and missing in the GENCODE, RefSeq and RefSeqGene databases. KEATS introduces a method of determining expression counts based on a partitioning of the known annotated genes, has runtimes of a few hours on a personal workstation and provides detailed reports enabling proper debugging.

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September 22, 2019

Characterization of the Rosellinia necatrix transcriptome and genes related to pathogenesis by single-molecule mRNA sequencing.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world’s most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

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September 22, 2019

Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.

We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score = 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (~2%; 4/216) and TRAF7 (~2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

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September 22, 2019

Capturing a long look at our genetic library.

Long-read sequencing, coupled to cDNA capture, provides an unrivaled view of the transcriptome of chromosome 21, revealing surprises about the splicing of long noncoding RNAs. Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019

Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure.

The United States is in the midst of an opiate epidemic, with abuse of prescription and illegal opioids increasing steadily over the past decade. While it is clear that there is a genetic component to opioid addiction, there is a significant portion of heritability that cannot be explained by genetics alone. The current study was designed to test the hypothesis that maternal exposure to opioids prior to pregnancy alters abuse liability in subsequent generations. Female adolescent Sprague Dawley rats were administered morphine at increasing doses (5-25 mg/kg, s.c.) or saline for 10 days (P30-39). During adulthood, animals were bred with drug-naïve colony males. Male and female adult offspring (F1 animals) were tested for morphine self-administration acquisition, progressive ratio, extinction, and reinstatement at three doses of morphine (0.25, 0.75, 1.25 mg/kg/infusion). Grand offspring (F2 animals, from the maternal line) were also examined. Additionally, gene expression changes within the nucleus accumbens were examined with RNA deep sequencing (PacBio) and qPCR. There were dose- and sex-dependent effects on all phases of the self-administration paradigm that indicate decreased morphine reinforcement and attenuated relapse-like behavior. Additionally, genes related to synaptic plasticity, as well as myelin basic protein (MBP), were dysregulated. Some, but not all, effects persisted into the subsequent (F2) generation. The results demonstrate that even limited opioid exposure during adolescence can have lasting effects across multiple generations, which has implications for mechanisms of the transmission of drug abuse liability in humans. Copyright © 2016 Elsevier Ltd. All rights reserved.

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September 22, 2019

Introduction to isoform sequencing using Pacific Biosciences technology (Iso-Seq)

Alternative RNA splicing is a known phenomenon, but we still do not have a complete catalog of isoforms that explain variability in the human transcriptome. We have made significant progress in developing methods to study variability of the transcriptome, but we are far away of having a complete picture of the transcriptome. The initial methods to study gene expression were based on cloning of cDNAs and Sanger sequencing. The strategy was labor-intensive and expensive. With the development of microarrays, different methods based on exon arrays and tiling arrays provided valuable information about RNA expression. However, the microarray presented significant limitations. Most of the limitations became apparent by 2005, but it was not until 2008 that an alternative method to study the transcriptome was developed. RNA Sequencing using next-generation sequencing (RNA-Seq) quickly became the technology of choice for gene expression profiling. Recently, the precision and sensitivity of RNA-Seq have come into question, especially for transcriptome reconstruction. This chapter will describe a relatively new method, “Isoform Sequencing (Iso-Seq). Iso-Seq was developed by Pacific Biosciences (PacBio), and it is capable of identifying new isoforms with extraordinary precision due to its long-read technology. The technique to create libraries is straightforward, and the PacBio RS II instrument generates the information in hours. The bioinformatics analysis is performed using the freely available SMRT® Portal software. The SMRT Portal is easy to use and capable of performing all the steps necessary to analyze the raw data and to generate high-quality full-length isoforms. For the universal acceptance of the Iso-Seq method, the capacity of the SMRT Cells needs to improve at least 10- to 100-fold to make the system affordable and attractive to users.

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September 22, 2019

JAFFA: High sensitivity transcriptome-focused fusion gene detection.

Genomic instability is a hallmark of cancer and, as such, structural alterations and fusion genes are common events in the cancer landscape. RNA sequencing (RNA-Seq) is a powerful method for profiling cancers, but current methods for identifying fusion genes are optimised for short reads. JAFFA (https://github.com/Oshlack/JAFFA/wiki) is a sensitive fusion detection method that outperforms other methods with reads of 100 bp or greater. JAFFA compares a cancer transcriptome to the reference transcriptome, rather than the genome, where the cancer transcriptome is inferred using long reads directly or by de novo assembling short reads.

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September 22, 2019

Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes.

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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