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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

Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession.

Although we understand the ecological processes eliciting changes in plant community composition during secondary succession, we do not understand whether co-occurring changes in plant detritus shape saprotrophic microbial communities in soil. In this study, we investigated soil microbial composition and function across an old-field chronosequence ranging from 16 to 86 years following agricultural abandonment, as well as three forests representing potential late-successional ecosystems. Fungal and bacterial community composition was quantified from ribosomal DNA, and insight into the functional potential of the microbial community to decay plant litter was gained from shotgun metagenomics and extracellular enzyme assays. Accumulation of soil organic matter across the chronosequence exerted a positive and significant effect on fungal phylogenetic ß-diversity and the activity of extracellular enzymes with lignocellulolytic activity. In addition, the increasing abundance of lignin-rich C4 grasses was positively related to the composition of fungal genes with lignocellulolytic function, thereby linking plant community composition, litter biochemistry, and microbial community function. However, edaphic properties were the primary agent shaping bacterial communities, as bacterial ß-diversity and variation in functional gene composition displayed a significant and positive relationship to soil pH across the chronosequence. The late-successional forests were compositionally distinct from the oldest old fields, indicating that substantial changes occur in soil microbial communities as old fields give way to forests. Taken together, our observations demonstrate that plants govern the turnover of soil fungal communities and functional characteristics during secondary succession, due to the continual input of detritus and differences in litter biochemistry among plant species.

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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

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 insert length (for paired-end reads), sequencing coverage, read alignment and k-mer frequency. ALE pinpoints synthetic errors in both single and metagenomic assemblies, including single-base errors, insertions/deletions, genome rearrangements and chimeric assemblies presented in metagenomes. At the genome level with real-world data, ALE identifies three large misassemblies from the Spirochaeta smaragdinae finished genome, which were all independently validated by Pacific Biosciences sequencing. At the single-base level with Illumina data, ALE recovers 215 of 222 (97%) single nucleotide variants in a training set from a GC-rich Rhodobacter sphaeroides genome. Using real Pacific Biosciences data, ALE identifies 12 of 12 synthetic errors in a Lambda Phage genome, surpassing even Pacific Biosciences’ own variant caller, EviCons. In summary, the ALE framework provides a comprehensive, reference-independent and statistically rigorous measure of single genome and metagenome assembly accuracy, which can be used to identify misassemblies or to optimize the assembly process.ALE is released as open source software under the UoI/NCSA license at http://www.alescore.org. It is implemented in C and Python.

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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

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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September 22, 2019

Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

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September 22, 2019

The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of ‘jumping genes’ as a source of major phenotypic novelty.

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September 22, 2019

Identification of microbial profile of Koji using Single Molecule, Real-Time Sequencing technology.

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples.© 2017 Institute of Food Technologists®.

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September 22, 2019

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line.

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.© 2018 Nattestad et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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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 the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.Cappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

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September 22, 2019

The new world of isoform sequencing

Not too long ago, the life sciences community was still debating whether sequencers would ever overtake microarrays as the preferred means of measuring gene expression. Today, not only have sequencers become the standard workhorse for gene expression studies, but newer sequencing technology has delivered the ability to generate novel expression data even in the most well-characterized cells or organisms. Truly, it is a remarkable time for comprehensive studies of which genes are being transcribed, with the goal of providing functional insight into various biological processes. The key advantage sequencing holds over microarrays is its ability to deeply survey an entire transcriptome, while microarrays are limited to interrogating known genes using probes designed from a reference genome assembly. As next-generation sequencing became more affordable, scientists were eager to switch to this approach, which became known as RNA sequencing or simply RNA-seq. © Mary Ann Liebert, Inc.

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