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Sunday, September 22, 2019

Full-length transcriptome sequencing and modular organization analysis of naringin/neoeriocitrin related gene expression pattern in Drynaria roosii.

Drynaria roosii (Nakaike) is a traditional Chinese medicinal fern, known as ‘GuSuiBu’. The effective components, naringin and neoeriocitrin, share a highly similar chemical structure and medicinal function. Our HPLC-tandem mass spectrometry (MS/MS) results showed that the accumulation of naringin/neoeriocitrin depended on specific tissues or ages. However, little was known about the expression patterns of naringin/neoeriocitrin-related genes involved in their regulatory pathways. Due to a lack of basic genetic information, we applied a combination of single molecule real-time (SMRT) sequencing and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of D. roosii. According to the SGS data, the differentially…

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Sunday, September 22, 2019

Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human.

Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5′-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences.We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5′ cap selection was performed on the embryo library to provide methodological comparison.…

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Sunday, September 22, 2019

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to…

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Sunday, September 22, 2019

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress.

Arabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing.Correction of SMRT long reads by SGS short reads…

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Sunday, September 22, 2019

Comparative transcriptome analysis of genes involved in Na+ transport in the leaves of halophyte Halogeton glomeratus.

Compartmentalization of Na+ into vacuoles is considered to be the most critical aspect of salt tolerance in H. glomeratus, an annual, succulent halophyte. Previous analysis of transcriptome involved in the H. glomeratus salt stress response relied on next-generation sequencing technologies that limit the capture of accurately spliced, full-length isoforms. To gain deeper insights into its salt stress response, we used the H. glomeratus Iso-Seq transcriptome database as a reference, and subsequent next-generation sequencing was subjected to various NaCl concentrations of leaves from plants revealed 115 upregulated and 87 downregulated differentially expressed isoforms (core DEIs). The majority of the core DEIs…

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Sunday, September 22, 2019

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their…

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Sunday, September 22, 2019

The state of play in higher eukaryote gene annotation.

A genome sequence is worthless if it cannot be deciphered; therefore, efforts to describe – or ‘annotate’ – genes began as soon as DNA sequences became available. Whereas early work focused on individual protein-coding genes, the modern genomic ocean is a complex maelstrom of alternative splicing, non-coding transcription and pseudogenes. Scientists – from clinicians to evolutionary biologists – need to navigate these waters, and this has led to the design of high-throughput, computationally driven annotation projects. The catalogues that are being produced are key resources for genome exploration, especially as they become integrated with expression, epigenomic and variation data sets.…

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Sunday, September 22, 2019

Full-length transcriptome of Misgurnus anguillicaudatus provides insights into evolution of genus Misgurnus.

Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497?bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905…

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Sunday, September 22, 2019

Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina-and SMRT-based RNA-seq datasets

The genome of the wild diploid strawberry species Fragaria vesca, an ideal model system of cultivated strawberry (Fragaria × ananassa, octoploid) and other Rosaceae family crops, was first published in 2011 and followed by a new assembly (Fvb). However, the annotation for Fvb mainly relied on ab initio predictions and included only predicted coding sequences, therefore an improved annotation is highly desirable. Here, a new annotation version named v2.0.a2 was created for the Fvb genome by a pipeline utilizing one PacBio library, 90 Illumina RNA-seq libraries, and 9 small RNA-seq libraries. Altogether, 18,641 genes (55.6% out of 33,538 genes) were…

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Sunday, September 22, 2019

Circular RNA architecture and differentiation during leaf bud to young leaf development in tea (Camellia sinensis).

Circular RNA (circRNA) discovery, expression patterns and experimental validation in developing tea leaves indicates its correlation with circRNA-parental genes and potential roles in ceRNA interaction network. Circular RNAs (circRNAs) have recently emerged as a novel class of abundant endogenous stable RNAs produced by circularization with regulatory potential. However, identification of circRNAs in plants, especially in non-model plants with large genomes, is challenging. In this study, we undertook a systematic identification of circRNAs from different stage tissues of tea plant (Camellia sinensis) leaf development using rRNA-depleted circular RNA-seq. By combining two state-of-the-art detecting tools, we characterized 3174 circRNAs, of which 342…

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Sunday, September 22, 2019

Transcriptome comparative analysis of salt stress responsiveness in chrysanthemum (Dendranthema grandiflorum) roots by Illumina- and Single-Molecule Real-Time-based RNA sequencing.

Salt response has long been considered a polygenic-controlled character in plants. Under salt stress conditions, plants respond by activating a great amount of proteins and enzymes. To develop a better understanding of the molecular mechanism and screen salt responsive genes in chrysanthemum under salt stress, we performed the RNA sequencing (RNA-seq) on both salt-processed chrysanthemum seedling roots and the control group, and gathered six cDNA databases eventually. Moreover, to overcome the Illumina HiSeq technology’s limitation on sufficient length of reads and improve the quality and accuracy of the result, we combined Illumina HiSeq with single-molecule real-time sequencing (SMRT-seq) to decode…

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Sunday, September 22, 2019

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These “phasiRNAs” are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic…

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Sunday, September 22, 2019

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1a, Nrxn1ß, Nrxn2ß, Nrxn3a, and Nrxn3ß mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs,…

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Sunday, September 22, 2019

High-quality reference transcript datasets hold the key to transcript-specific RNA-sequencing analysis in plants.

Re-programming of the transcriptome involves both transcription and alternative splicing (AS). Some genes are regulated only at the AS level with no change in expression at the gene level. AS data must be incorporated as an essential aspect of the regulation of gene expression. RNA-sequencing (RNA-seq) can deliver both transcriptional and AS information, but accurate methods to analyse the added complexity in RNA-seq data are needed. The construction of a comprehensive reference transcript dataset (RTD) for a specific plant species, variety or accession, from all available sequence data, will immediately allow more robust analysis of RNA-seq data. RTDs will continually…

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