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September 22, 2019  |  

Detecting epigenetic motifs in low coverage and metagenomics settings.

It has recently become possible to rapidly and accurately detect epigenetic signatures in bacterial genomes using third generation sequencing data. Monitoring the speed at which a single polymerase inserts a base in the read strand enables one to infer whether a modification is present at that specific site on the template strand. These sites can be challenging to detect in the absence of high coverage and reliable reference genomes.Here we provide a new method for detecting epigenetic motifs in bacteria on datasets with low-coverage, with incomplete references, and with mixed samples (i.e. metagenomic data). Our approach treats motif inference as a kmer comparison problem. First, genomes (or contigs) are deconstructed into kmers. Then, native genome-wide distributions of interpulse durations (IPDs) for kmers are compared with corresponding whole genome amplified (WGA, modification free) IPD distributions using log likelihood ratios. Finally, kmers are ranked and greedily selected by iteratively correcting for sequences within a particular kmer’s neighborhood.Our method can detect multiple types of modifications, even at very low-coverage and in the presence of mixed genomes. Additionally, we are able to predict modified motifs when genomes with “neighbor” modified motifs exist within the sample. Lastly, we show that these motifs can provide an alternative source of information by which to cluster metagenomics contigs and that iterative refinement on these clustered contigs can further improve both sensitivity and specificity of motif detection.https://github.com/alibashir/EMMCKmer.

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September 22, 2019  |  

Searching for convergent pathways in autism spectrum disorders: insights from human brain transcriptome studies.

Autism spectrum disorder (ASD) is one of the most heritable neuropsychiatric conditions. The complex genetic landscape of the disorder includes both common and rare variants at hundreds of genetic loci. This marked heterogeneity has thus far hampered efforts to develop genetic diagnostic panels and targeted pharmacological therapies. Here, we give an overview of the current literature on the genetic basis of ASD, and review recent human brain transcriptome studies and their role in identifying convergent pathways downstream of the heterogeneous genetic variants. We also discuss emerging evidence on the involvement of non-coding genomic regions and non-coding RNAs in ASD.

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September 22, 2019  |  

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

HIV-1 infection of primary CD4(+) T cells regulates the expression of specific HERV-K (HML-2) elements.

Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic.Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1 infected primary human CD4(+) T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read Single Molecule Real-Time sequencing. We show that three HML-2 proviruses, 6q25.1, 8q24.3, and 19q13.42 are up-regulated on average between 3- and 5-fold in HIV-1 infected CD4(+) T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication.In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4(+) T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.Importance Endogenous retroviruses inhabit big portions of our genome. And although they are mainly inert some of the evolutionarily younger members maintain the ability to express both RNA as well as proteins. We have developed an approach using long-read SMRT sequencing that produces long reads, that provides us with ability to obtain detailed and accurate HERV-K HML-2 expression profiles. We have now applied this approach to study HERV-K expression in the presence and absence of productive HIV-1 infection of primary human CD4(+) T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results in this manuscript provide the blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1. Copyright © 2017 American Society for Microbiology.

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September 22, 2019  |  

The state of long non-coding RNA biology.

Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as organizational and guide molecules: most lncRNAs are nuclear-localized and chromatin-associated, with some involved in the formation of specialized subcellular domains. I suggest that a reassessment of the conceptual framework of genetic information and gene expression in the 4-dimensional ontogeny of spatially organized multicellular organisms is required. Together with this and further studies on their biology, the key challenges now are to determine the structure?function relationships of lncRNAs, which may be aided by emerging evidence of their modular structure, the role of RNA editing and modification in enabling epigenetic plasticity, and the role of RNA signaling in transgenerational inheritance of experience.

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September 22, 2019  |  

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019  |  

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

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September 22, 2019  |  

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019  |  

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei

Despite the large interest in the human microbiome in recent years, there are no reports of bacterial DNA methylation in the microbiome. Here metagenomic sequencing using the Pacific Biosciences platform allowed for rapid identification of bacterial GATC methylation status of a bacterial species in human stool samples. For this work, two stool samples were chosen that were dominated by a single species, Bacteroides dorei. Based on 16S rRNA analysis, this species represented over 45% of the bacteria present in these two samples. The B. dorei genome sequence from these samples was determined and the GATC methylation sites mapped. The Bacteroides dorei genome from one subject lacked any GATC methylation and lacked the DNA adenine methyltransferase genes. In contrast, B. dorei from another subject contained 20,551 methylated GATC sites. Of the 4970 open reading frames identified in the GATC methylated B. dorei genome, 3184 genes were methylated as well as 1735 GATC methylations in intergenic regions. These results suggest that DNA methylation patterns are important to consider in multi-omic analyses of microbiome samples seeking to discover the diversity of bacterial functions and may differ between disease states.

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September 22, 2019  |  

The third revolution in sequencing technology.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019  |  

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.

Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis.Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template.Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.

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September 22, 2019  |  

Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S.

The ms3 gene responsible for a male-sterile phenotype in cabbage was mapped to a 187.4-kb genomic fragment. The gene BoTPD1, a homolog of Arabidopsis TPD1, was identified as a strong candidate gene. Cabbage 51S is a spontaneous male-sterile mutant. Phenotypic investigation revealed defects in anther cell differentiation, with failure to form the tapetum layer and complete abortion of microsporocytes before the tetrad stage. Genetic analysis indicated that this male sterility was controlled by a single recessive gene, ms3. Using an F2 population, we mapped ms3 to a 187.4-kb interval. BoTPD1 was identified as a candidate from this interval. Sequence analysis revealed an intronic 182-bp insertion in 51S that interrupted the conserved motif at the 5′ splicing site of the third intron, possibly resulting in a truncated transcript. Analyses of BoTPD1 homologous proteins revealed evolutionarily conserved roles in anther cell fate determination during reproductive development. RT-PCR showed that BoTPD1 was expressed in various tissues, excluding the root, and high expression levels were detected in anthers and buds. A BoTPD1-specific marker based on the 182-bp insertion cosegregated with male sterility and can be used for marker-assisted selection.

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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  |  

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  |  

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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