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September 22, 2019  |  

The genome of an underwater architect, the caddisfly Stenopsyche tienmushanensis Hwang (Insecta: Trichoptera).

Caddisflies (Insecta: Trichoptera) are a highly adapted freshwater group of insects split from a common ancestor with Lepidoptera. They are the most diverse (>16,000 species) of the strictly aquatic insect orders and are widely employed as bio-indicators in water quality assessment and monitoring. Among the numerous adaptations to aquatic habitats, caddisfly larvae use silk and materials from the environment (e.g., stones, sticks, leaf matter) to build composite structures such as fixed retreats and portable cases. Understanding how caddisflies have adapted to aquatic habitats will help explain the evolution and subsequent diversification of the group.We sequenced a retreat-builder caddisfly Stenopsyche tienmushanensis Hwang and assembled a high-quality genome from both Illumina and Pacific Biosciences (PacBio) sequencing. In total, 601.2 M Illumina reads (90.2 Gb) and 16.9 M PacBio subreads (89.0 Gb) were generated. The 451.5 Mb assembled genome has a contig N50 of 1.29 M, has a longest contig of 4.76 Mb, and covers 97.65% of the 1,658 insect single-copy genes as assessed by Benchmarking Universal Single-Copy Orthologs. The genome comprises 36.76% repetitive elements. A total of 14,672 predicted protein-coding genes were identified. The genome revealed gene expansions in specific groups of the cytochrome P450 family and olfactory binding proteins, suggesting potential genomic features associated with pollutant tolerance and mate finding. In addition, the complete gene complex of the highly repetitive H-fibroin, the major protein component of caddisfly larval silk, was assembled.We report the draft genome of Stenopsyche tienmushanensis, the highest-quality caddisfly genome so far. The genome information will be an important resource for the study of caddisflies and may shed light on the evolution of aquatic insects.

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September 22, 2019  |  

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

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September 22, 2019  |  

Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques.

Poxviruses are large DNA viruses that infect humans and animals. Vaccinia virus (VACV) has been applied as a live vaccine for immunization against smallpox, which was eradicated by 1980 as a result of worldwide vaccination. VACV is the prototype of poxviruses in the investigation of the molecular pathogenesis of the virus. Short-read sequencing methods have revolutionized transcriptomics; however, they are not efficient in distinguishing between the RNA isoforms and transcript overlaps. Long-read sequencing (LRS) is much better suited to solve these problems and also allow direct RNA sequencing. Despite the scientific relevance of VACV, no LRS data have been generated for the viral transcriptome to date.For the deep characterization of the VACV RNA profile, various LRS platforms and library preparation approaches were applied. The raw reads were mapped to the VACV reference genome and also to the host (Chlorocebus sabaeus) genome. In this study, we applied the Pacific Biosciences RSII and Sequel platforms, which altogether resulted in 937,531 mapped reads of inserts (1.42 Gb), while we obtained 2,160,348 aligned reads (1.75 Gb) from the different library preparation methods using the MinION device from Oxford Nanopore Technologies.By applying cutting-edge technologies, we were able to generate a large dataset that can serve as a valuable resource for the investigation of the dynamic VACV transcriptome, the virus-host interactions, and RNA base modifications. These data can provide useful information for novel gene annotations in the VACV genome. Our dataset can also be used to analyze the currently available LRS platforms, library preparation methods, and bioinformatics pipelines.

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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  |  

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

Parallel sequencing of a single cell’s genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ~3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

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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  |  

Isoform sequencing provides a more comprehensive view of the Panax ginseng transcriptome.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

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September 22, 2019  |  

Single-Molecule Long-Read Sequencing of Zanthoxylum bungeanum Maxim. Transcriptome: Identification of Aroma-Related Genes

Zanthoxylum bungeanum Maxim. is an economically important tree species that is resistant to drought and infertility, and has potential medicinal and edible value. However, comprehensive genomic data are not yet available for this species, limiting its potential utility for medicinal use, breeding programs, and cultivation. Transcriptome sequencing provides an effective approach to remedying this shortcoming. Herein, single-molecule long-read sequencing and next-generation sequencingapproacheswereusedinparalleltoobtaintranscriptisoformstructureandgenefunctional informationinZ.bungeanum. Intotal, 282,101readsofinserts(ROIs)wereidentified, including134,074 full-length non-chimeric reads, among which 65,711 open reading frames (ORFs), 50,135 simple sequence repeats (SSRs), and 1492 long non-coding RNAs (lncRNAs) were detected. Functional annotation revealed metabolic pathways related to aroma components and color characteristics in Z. bungeanum. Unexpectedly, 30 transcripts were annotated as genes involved in regulating the pathogenesis of breast and colorectal cancers. This work provides a comprehensive transcriptome resource for Z. bungeanum, and lays a foundation for the further investigation and utilization of Zanthoxylum resources.

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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  |  

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  |  

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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September 22, 2019  |  

Computational analysis of alternative splicing in plant genomes.

Computational analyses play crucial roles in characterizing splicing isoforms in plant genomes. In this review, we provide a survey of computational tools used in recently published, genome-scale splicing analyses in plants. We summarize the commonly used software and pipelines for read mapping, isoform reconstruction, isoform quantification, and differential expression analysis. We also discuss methods for analyzing long reads and the strategies to combine long and short reads in identifying splicing isoforms. We review several tools for characterizing local splicing events, splicing graphs, coding potential, and visualizing splicing isoforms. We further discuss the procedures for identifying conserved splicing isoforms across plant species. Finally, we discuss the outlook of integrating other genomic data with splicing analyses to identify regulatory mechanisms of AS on genome-wide scale. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell’s genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

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September 22, 2019  |  

Integrative analysis of three RNA sequencing methods identifies mutually exclusive exons of MADS-box isoforms during early bud development in Picea abies.

Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.

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