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Asset Tag: RNA sequencing

September 22, 2019

Long-read based assembly and annotation of a Drosophila simulans genome

Long-read sequencing technologies enable high-quality, contiguous genome assemblies. Here we used SMRT sequencing to assemble the genome of a Drosophila simulans strain originating from Madagascar, the ancestral range of the species. We generated 8 Gb of raw data (~50x coverage) with a mean read length of 6,410 bp, a NR50 of 9,125 bp and the longest subread at 49 kb. We benchmarked six different assemblers and merged the best two assemblies from Canu and Falcon. Our final assembly was 127.41 Mb with a N50 of 5.38 Mb and 305 contigs. We anchored more than 4 Mb of novel sequence to the major chromosome arms, and significantly improved the assembly of peri-centromeric and telomeric regions. Finally, we performed full-length transcript sequencing and used this data in conjunction with short-read RNAseq data to annotate 13,422 genes in the genome, improving the annotation in regions with complex, nested gene structures.

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September 22, 2019

Extensive allele-specific translational regulation in hybrid mice.

Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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September 22, 2019

Genome-wide characterization of human L1 antisense promoter-driven transcripts.

Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active, transposable element in the human genome. L1 sequences comprise approximately 17 % of the human genome, but only the evolutionarily recent, human-specific subfamily is retrotransposition competent. The L1 promoter has a bidirectional orientation containing a sense promoter that drives the transcription of two proteins required for retrotransposition and an antisense promoter. The L1 antisense promoter can drive transcription of chimeric transcripts: 5′ L1 antisense sequences spliced to the exons of neighboring genes.The impact of L1 antisense promoter activity on cellular transcriptomes is poorly understood. To investigate this, we analyzed GenBank ESTs for messenger RNAs that initiate in the L1 antisense promoter. We identified 988 putative L1 antisense chimeric transcripts, 911 of which have not been previously reported. These appear to be alternative genic transcripts, sense-oriented with respect to gene and initiating near, but typically downstream of, the gene transcriptional start site. In multiple cell lines, L1 antisense promoters display enrichment for YY1 transcription factor and histone modifications associated with active promoters. Global run-on sequencing data support the activity of the L1 antisense promoter. We independently detected 124 L1 antisense chimeric transcripts using long read Pacific Biosciences RNA-seq data. Furthermore, we validated four chimeric transcripts by quantitative RT-PCR and Sanger sequencing and demonstrated that they are readily detectable in many normal human tissues.We present a comprehensive characterization of human L1 antisense promoter-driven transcripts and provide substantial evidence that they are transcribed in a variety of human cell-types. Our findings reveal a new wide-reaching aspect of L1 biology by identifying antisense transcripts affecting as many as 4 % of all human genes.

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September 22, 2019

Transcriptome-referenced association study of clove shape traits in garlic.

Genome-wide association studies are a powerful approach for identifying genes related to complex traits in organisms, but are limited by the requirement for a reference genome sequence of the species under study. To circumvent this problem, we propose a transcriptome-referenced association study (TRAS) that utilizes a transcriptome generated by single-molecule long-read sequencing as a reference sequence to score population variation at both transcript sequence and expression levels. Candidate transcripts are identified when both scores are associated with a trait and their potential interactions are ascertained by expression quantitative trait loci analysis. Applying this method to characterize garlic clove shape traits in 102 landraces, we identified 22 candidate transcripts, most of which showed extensive interactions. Eight transcripts were long non-coding RNAs (lncRNAs), and the others were proteins involved mainly in carbohydrate metabolism, protein degradation, etc. TRAS, as an efficient tool for association study independent of a reference genome, extends the applicability of association studies to a broad range of species.

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September 22, 2019

A single-molecule long-read survey of the human transcriptome.

Global RNA studies have become central to understanding biological processes, but methods such as microarrays and short-read sequencing are unable to describe an entire RNA molecule from 5′ to 3′ end. Here we use single-molecule long-read sequencing technology from Pacific Biosciences to sequence the polyadenylated RNA complement of a pooled set of 20 human organs and tissues without the need for fragmentation or amplification. We show that full-length RNA molecules of up to 1.5 kb can readily be monitored with little sequence loss at the 5′ ends. For longer RNA molecules more 5′ nucleotides are missing, but complete intron structures are often preserved. In total, we identify ~14,000 spliced GENCODE genes. High-confidence mappings are consistent with GENCODE annotations, but >10% of the alignments represent intron structures that were not previously annotated. As a group, transcripts mapping to unannotated regions have features of long, noncoding RNAs. Our results show the feasibility of deep sequencing full-length RNA from complex eukaryotic transcriptomes on a single-molecule level.

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September 22, 2019

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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September 22, 2019

High-resolution expression map of the Arabidopsis root reveals alternative splicing and lincRNA regulation.

The extent to which alternative splicing and long intergenic noncoding RNAs (lincRNAs) contribute to the specialized functions of cells within an organ is poorly understood. We generated a comprehensive dataset of gene expression from individual cell types of the Arabidopsis root. Comparisons across cell types revealed that alternative splicing tends to remove parts of coding regions from a longer, major isoform, providing evidence for a progressive mechanism of splicing. Cell-type-specific intron retention suggested a possible origin for this common form of alternative splicing. Coordinated alternative splicing across developmental stages pointed to a role in regulating differentiation. Consistent with this hypothesis, distinct isoforms of a transcription factor were shown to control developmental transitions. lincRNAs were generally lowly expressed at the level of individual cell types, but co-expression clusters provided clues as to their function. Our results highlight insights gained from analysis of expression at the level of individual cell types. Copyright © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019

Full-length isoform sequencing reveals novel transcripts and substantial transcriptional overlaps in a herpesvirus.

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

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September 22, 2019

Evaluation of tools for long read RNA-seq splice-aware alignment.

High-throughput sequencing has transformed the study of gene expression levels through RNA-seq, a technique that is now routinely used by various fields, such as genetic research or diagnostics. The advent of third generation sequencing technologies providing significantly longer reads opens up new possibilities. However, the high error rates common to these technologies set new bioinformatics challenges for the gapped alignment of reads to their genomic origin. In this study, we have explored how currently available RNA-seq splice-aware alignment tools cope with increased read lengths and error rates. All tested tools were initially developed for short NGS reads, but some have claimed support for long Pacific Biosciences (PacBio) or even Oxford Nanopore Technologies (ONT) MinION reads.The tools were tested on synthetic and real datasets from two technologies (PacBio and ONT MinION). Alignment quality and resource usage were compared across different aligners. The effect of error correction of long reads was explored, both using self-correction and correction with an external short reads dataset. A tool was developed for evaluating RNA-seq alignment results. This tool can be used to compare the alignment of simulated reads to their genomic origin, or to compare the alignment of real reads to a set of annotated transcripts. Our tests show that while some RNA-seq aligners were unable to cope with long error-prone reads, others produced overall good results. We further show that alignment accuracy can be improved using error-corrected reads.https://github.com/kkrizanovic/RNAseqEval, https://figshare.com/projects/RNAseq_benchmark/24391.mile.sikic@fer.hr.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

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

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September 22, 2019

A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing.

Alternative splicing (AS) and fusion transcripts produce a vast expansion of transcriptomes and proteomes diversity. However, the reliability of these events and the extend of epigenetic mechanisms have not been adequately addressed due to its limitation of uncertainties about the complete structure of mRNA. Here we combined single-molecule real-time sequencing, Illumina RNA-seq and DNA methylation data to characterize the landscapes of DNA methylation on AS, fusion isoforms formation and lncRNA feature and further to unveil the transcriptome complexity of pig. Our analysis identified an unprecedented scale of high-quality full-length isoforms with over 28,127 novel isoforms from 26,881 novel genes. More than 92,000 novel AS events were detected and intron retention predominated in AS model, followed by exon skipping. Interestingly, we found that DNA methylation played an important role in generating various AS isoforms by regulating splicing sites, promoter regions and first exons. Furthermore, we identified a large of fusion transcripts and novel lncRNAs, and found that DNA methylation of the promoter and gene body could regulate lncRNA expression. Our results significantly improved existed gene models of pig and unveiled that pig AS and epigenetic modify were more complex than previously thought.

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September 22, 2019

Transcriptomic study of Herpes simplex virus type-1 using full-length sequencing techniques

Herpes simplex virus type-1 (HSV-1) is a human pathogenic member of the Alphaherpesvirinae subfamily of herpesviruses. The HSV-1 genome is a large double-stranded DNA specifying about 85 protein coding genes. The latest surveys have demonstrated that the HSV-1 transcriptome is much more complex than it had been thought before. Here, we provide a long-read sequencing dataset, which was generated by using the RSII and Sequel systems from Pacific Biosciences (PacBio), as well as MinION sequencing system from Oxford Nanopore Technologies (ONT). This dataset contains 39,096 reads of inserts (ROIs) mapped to the HSV-1 genome (X14112) in RSII sequencing, while Sequel sequencing yielded 77,851 ROIs. The MinION cDNA sequencing altogether resulted in 158,653 reads, while the direct RNA-seq produced 16,516 reads. This dataset can be utilized for the identification of novel HSV RNAs and transcripts isoforms, as well as for the comparison of the quality and length of the sequencing reads derived from the currently available long- read sequencing platforms. The various library preparation approaches can also be compared with each other.

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September 22, 2019

Transcriptional diversity during lineage commitment of human blood progenitors.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. Copyright © 2014, American Association for the Advancement of Science.

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September 22, 2019

Comprehensive transcriptome analysis of Sarcophaga peregrina, a forensically important fly species.

Sarcophaga peregrina (flesh fly) is a frequently found fly species in Palaearctic, Oriental, and Australasian regions that can be used to estimate minimal postmortem intervals important for forensic investigations. Despite its forensic importance, the genome information of S. peregrina has not been fully described. Therefore, we generated a comprehensive gene expression dataset using RNA sequencing and carried out de novo assembly to characterize the S. peregrina transcriptome. We obtained precise sequence information for RNA transcripts using two different methods. Based on primary sequence information, we identified sets of assembled unigenes and predicted coding sequences. Functional annotation of the aligned unigenes was performed using the UniProt, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. As a result, 26,580,352 and 83,221 raw reads were obtained using the Illumina MiSeq and Pacbio RS II Iso-Seq sequencing applications, respectively. From these reads, 55,730 contigs were successfully annotated. The present study provides the resulting genome information of S. peregrina, which is valuable for forensic applications.

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September 22, 2019

Hybrid sequencing of full-length cDNA transcripts of stems and leaves in Dendrobium officinale.

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem.

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