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September 22, 2019

Differential increases of specific FMR1 mRNA isoforms in premutation carriers.

Over 40% of male and ~16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology.To address this question, we have applied a long-read sequencing approach using single-molecule real-time (SMRT) sequencing and qRT-PCR. Our SMRT sequencing analysis performed on peripheral blood mononuclear cells, fibroblasts and brain tissue samples derived from premutation carriers and controls revealed the existence of 16 isoforms of 24 predicted variants. Although the relative abundance of all mRNA isoforms was significantly increased in the premutation group, as expected based on the bulk increase in mRNA levels, there was a disproportionate (fourfold to sixfold) increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14, specifically Iso10 and Iso10b, containing the complete exon 15 and differing only in splicing in exon 17.These findings suggest that RNA toxicity may arise from a relative increase of all FMR1 mRNA isoforms. Interestingly, the Iso10 and Iso10b mRNA isoforms, lacking the C-terminal functional sites for fragile X mental retardation protein function, are the most increased in premutation carriers relative to normal, suggesting a functional relevance in the pathology of FMR1-associated disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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September 22, 2019

Alternative polyadenylation: methods, findings, and impacts.

Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

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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

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

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September 22, 2019

L_RNA_scaffolder: scaffolding genomes with transcripts.

Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments.We describe L_RNA_scaffolder, a novel genome scaffolding method that uses long transcriptome reads to order, orient and combine genomic fragments into larger sequences. To demonstrate the accuracy of the method, the zebrafish genome was scaffolded. With expanded human transcriptome data, the N50 of human genome was doubled and L_RNA_scaffolder out-performed most scaffolding results by existing scaffolders which employ mate-pair libraries. In these two examples, the transcript coverage was almost complete, especially for long transcripts. We applied L_RNA_scaffolder to the highly polymorphic pearl oyster draft genome and the gene model length significantly increased.The simplicity and high-throughput of RNA-seq data makes this approach suitable for genome scaffolding. L_RNA_scaffolder is available at http://www.fishbrowser.org/software/L_RNA_scaffolder.

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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 demonstrate superior precision and recall for simulations compared to state-of-the-art isoform reconstruction methods. BIISQ shows the most gains for low abundance isoforms, with 36% more isoforms correctly inferred at low coverage versus a multi-sample method and 170% more versus single-sample methods. We estimate isoforms in the GEUVADIS RNA-seq data and validate inferred isoforms by associating genetic variants with isoform ratios.

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September 22, 2019

Transcriptome profiling using single-molecule direct RNA sequencing approach for in-depth understanding of genes in secondary metabolism pathways of Camellia sinensis.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL) splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.

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September 22, 2019

PacBio full-length transcriptome profiling of insect mitochondrial gene expression.

In this study, we sequenced the first full-length insect transcriptome using the Erthesina fullo Thunberg based on the PacBio platform. We constructed the first quantitative transcription map of animal mitochondrial genomes and built a straightforward and concise methodology to investigate mitochondrial gene transcription, RNA processing, mRNA maturation and several other related topics. Most of the results were consistent with the previous studies, while to the best of our knowledge some findings were reported for the first time in this study. The new findings included the high levels of mitochondrial gene expression, the 3′ polyadenylation and possible 5′ m(7)G caps of rRNAs, the isoform diversity of 12S rRNA, the polycistronic transcripts and natural antisense transcripts of mitochondrial genes et al. These findings could challenge and enrich fundamental concepts of mitochondrial gene transcription and RNA processing, particularly of the rRNA primary (sequence) structure. The methodology constructed in this study can also be used to study gene expression or RNA processing of nuclear genomes.

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September 22, 2019

Computational identification of novel genes: current and future perspectives.

While it has long been thought that all genomic novelties are derived from the existing material, many genes lacking homology to known genes were found in recent genome projects. Some of these novel genes were proposed to have evolved de novo, ie, out of noncoding sequences, whereas some have been shown to follow a duplication and divergence process. Their discovery called for an extension of the historical hypotheses about gene origination. Besides the theoretical breakthrough, increasing evidence accumulated that novel genes play important roles in evolutionary processes, including adaptation and speciation events. Different techniques are available to identify genes and classify them as novel. Their classification as novel is usually based on their similarity to known genes, or lack thereof, detected by comparative genomics or against databases. Computational approaches are further prime methods that can be based on existing models or leveraging biological evidences from experiments. Identification of novel genes remains however a challenging task. With the constant software and technologies updates, no gold standard, and no available benchmark, evaluation and characterization of genomic novelty is a vibrant field. In this review, the classical and state-of-the-art tools for gene prediction are introduced. The current methods for novel gene detection are presented; the methodological strategies and their limits are discussed along with perspective approaches for further studies.

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September 22, 2019

High-confidence coding and noncoding transcriptome maps.

The advent of high-throughput RNA sequencing (RNA-seq) has led to the discovery of unprecedentedly immense transcriptomes encoded by eukaryotic genomes. However, the transcriptome maps are still incomplete partly because they were mostly reconstructed based on RNA-seq reads that lack their orientations (known as unstranded reads) and certain boundary information. Methods to expand the usability of unstranded RNA-seq data by predetermining the orientation of the reads and precisely determining the boundaries of assembled transcripts could significantly benefit the quality of the resulting transcriptome maps. Here, we present a high-performing transcriptome assembly pipeline, called CAFE, that significantly improves the original assemblies, respectively assembled with stranded and/or unstranded RNA-seq data, by orienting unstranded reads using the maximum likelihood estimation and by integrating information about transcription start sites and cleavage and polyadenylation sites. Applying large-scale transcriptomic data comprising 230 billion RNA-seq reads from the ENCODE, Human BodyMap 2.0, The Cancer Genome Atlas, and GTEx projects, CAFE enabled us to predict the directions of about 220 billion unstranded reads, which led to the construction of more accurate transcriptome maps, comparable to the manually curated map, and a comprehensive lncRNA catalog that includes thousands of novel lncRNAs. Our pipeline should not only help to build comprehensive, precise transcriptome maps from complex genomes but also to expand the universe of noncoding genomes.© 2017 You et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ~360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ~25-35 Mya and CFHR1 and CFHR3 ~7-13 Mya). Remarkably, all evolutionary breakpoints share a common ~4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

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September 22, 2019

SparseIso: a novel Bayesian approach to identify alternatively spliced isoforms from RNA-seq data.

Recent advances in high-throughput RNA sequencing (RNA-seq) technologies have made it possible to reconstruct the full transcriptome of various types of cells. It is important to accurately assemble transcripts or identify isoforms for an improved understanding of molecular mechanisms in biological systems.We have developed a novel Bayesian method, SparseIso, to reliably identify spliced isoforms from RNA-seq data. A spike-and-slab prior is incorporated into the Bayesian model to enforce the sparsity for isoform identification, effectively alleviating the problem of overfitting. A Gibbs sampling procedure is further developed to simultaneously identify and quantify transcripts from RNA-seq data. With the sampling approach, SparseIso estimates the joint distribution of all candidate transcripts, resulting in a significantly improved performance in detecting lowly expressed transcripts and multiple expressed isoforms of genes. Both simulation study and real data analysis have demonstrated that the proposed SparseIso method significantly outperforms existing methods for improved transcript assembly and isoform identification.The SparseIso package is available at http://github.com/henryxushi/SparseIso.xuan@vt.edu.Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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September 22, 2019

cDNA library enrichment of full length transcripts for SMRT long read sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

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September 22, 2019

Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.

Danshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

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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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