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April 21, 2020

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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April 21, 2020

Genome-wide analysis of methyl jasmonate-regulated isoform expression in the medicinal plant Andrographis paniculata

Alternative splicing can increase the complexity of the transcriptome and proteome. The most common mechanism of alternative splicing in plants is intron retention (IR), and the expression levels of IR isoforms can be differentially regulated when facing abiotic stress. The full-length transcriptome of the medicinal plant Andrographis paniculata was sequenced using both Illumina- and SMRT-based RNA-seq and a total of 4846 IR isoforms were identified. The expression levels of 310/296 IR isoforms were up-regulated, and 629/659 IR isoforms were down-regulated at 24?h/48?h after methyl jasmonate (MeJA) treatment, respectively. In the (E,E,E)-geranylgeranyl diphosphate (GGPP) biosynthesis pathway which contributes to the andrographolide biosynthesis, eight genes were alternatively spliced, resulting in a total of 25 isoforms, of which 12 are IR isoforms. After MeJA treatment, four of these IR isoforms showed significant differential expression. RT-PCR and qRT-PCR experiments confirmed the existence of five IR isoforms. This research deepens our understanding of the A. paniculata transcriptome and can assist in the future study of andrographolide biosynthesis.

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April 21, 2020

Potential use of the Pteris vittata arsenic hyperaccumulation-regulation network for phytoremediation.

Arsenic accumulation in soil is a global problem typically addressed using phytoremediation methods. Pteris vittata, a model arsenic hyperaccumulator, has great potential as a genetically engineered plant for phytoremediation. However, the lack of omic information on this species has severely limited the identification and application of its arsenic hyperaccumulation and regulation components. In this study, we used an optimized single-molecular real-time (SMRT) strategy to create a de novo full-length transcriptomic-tonoplast proteomic database for this unsequenced fern and to determine the genetic components underlying its arsenic hyperaccumulation-regulation mechanisms. We established a comprehensive network consisting of six major transporter families, two novel resistance pathways, and a regulatory system by examining alternative splicing (AS) and long non-coding RNA (lncRNA) in different tissues following As(III) and As(V) treatment. The database and network established in this study will deepen our understanding of the unique hyperaccumulation and regulation mechanisms of P. vittata, ultimately providing a valuable resource for futher research on phytoremediation of arsenic-contaminated soil. Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020

Characterization and analysis of the transcriptome in Gymnocypris selincuoensis on the Qinghai-Tibetan Plateau using single-molecule long-read sequencing and RNA-seq.

The lakes on the Qinghai-Tibet Plateau (QTP) are the largest and highest lake group in the world. Gymnocypris selincuoensis is the only cyprinid fish living in lake Selincuo, the largest lake on QTP. However, its genetic resource is still blank, limiting studies on molecular and genetic analysis. In this study, the transcriptome of G. selincuoensis was first generated by using PacBio Iso-Seq and Illumina RNA-seq. A full-length (FL) transcriptome with 75,435 transcripts was obtained by Iso-Seq with N50 length of 3,870 bp. Among all transcripts, 75,016 were annotated to public databases, 64,710 contain complete open reading frames and 2,811 were long non-coding RNAs. Based on all- vs.-all BLAST, 2,069 alternative splicing events were detected, and 80% of them were validated by reverse transcription polymerase chain reaction (RT-PCR). Tissue gene expression atlas showed that the number of detected expressed transcripts ranged from 37,397 in brain to 19,914 in muscle, with 10,488 transcripts detected in all seven tissues. Comparative genomic analysis with other cyprinid fishes identified 77 orthologous genes with potential positive selection (Ka/Ks > 0.3). A total of 56,696 perfect simple sequence repeats were identified from FL transcripts. Our results provide valuable genetic resources for further studies on adaptive evolution, gene expression and population genetics in G. selincuoensis and other congeneric fishes. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020

Full-length transcriptome analysis of Litopenaeus vannamei reveals transcript variants involved in the innate immune system.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

TranscriptClean: variant-aware correction of indels, mismatches and splice junctions in long-read transcripts.

Long-read, single-molecule sequencing platforms hold great potential for isoform discovery and characterization of multi-exon transcripts. However, their high error rates are an obstacle to distinguishing novel transcript isoforms from sequencing artifacts. Therefore, we developed the package TranscriptClean to correct mismatches, microindels and noncanonical splice junctions in mapped transcripts using the reference genome while preserving known variants.Our method corrects nearly all mismatches and indels present in a publically available human PacBio Iso-seq dataset, and rescues 39% of noncanonical splice junctions.All Python and R scripts used in this paper are available at https://github.com/dewyman/TranscriptClean.

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April 21, 2020

The interplay between microRNA and alternative splicing of linear and circular RNAs in eleven plant species.

MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species.In total, we identified 399?401 and 114?574 AS events from linear and circular RNAs, respectively. Among them, there were 64?781 and 41?146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38?913 circRNAs to be overlapping with 45?648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms.The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR.Supplementary data are available at Bioinformatics online. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020

Hybrid sequencing-based personal full-length transcriptomic analysis implicates proteostatic stress in metastatic ovarian cancer.

Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.

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April 21, 2020

The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina- and single-molecule real-time-based RNA-sequencing.

Alternative splicing (AS) plays a critical role in regulating different physiological and developmental processes in eukaryotes, by dramatically increasing the diversity of the transcriptome and the proteome. However, the saturation and complexity of AS remain unclear in lotus due to its limitation of rare obtainment of full-length multiple-splice isoforms. In this study, we apply a hybrid assembly strategy by combining single-molecule real-time sequencing and Illumina RNA-seq to get a comprehensive insight into the lotus transcriptomic landscape. We identified 211,802 high-quality full-length non-chimeric reads, with 192,690 non-redundant isoforms, and updated the lotus reference gene model. Moreover, our analysis identified a total of 104,288 AS events from 16,543 genes, with alternative 3′ splice-site being the predominant model, following by intron retention. By exploring tissue datasets, 370 tissue-specific AS events were identified among 12 tissues. Both the tissue-specific genes and isoforms might play important roles in tissue or organ development, and are suitable for ‘ABCE’ model partly in floral tissues. A large number of AS events and isoform variants identified in our study enhance the understanding of transcriptional diversity in lotus, and provide valuable resource for further functional genomic studies. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_IsoSeq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

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April 21, 2020

NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells.

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.

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April 21, 2020

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.

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April 21, 2020

DNA Methylation at the Schizophrenia and Intelligence GWAS-Implicated MIR137HG Locus May Be Associated with Disease and Cognitive Functions

The largest genome-wide association studies have identified schizophrenia and intelligence associated variants in the MIR137HG locus containing genes encoding microRNA-137 and microRNA-2682. In the present study, we investigated DNA methylation in the MIR137HG intragenic CpG island (CGI) in the peripheral blood of 44 patients with schizophrenia and 50 healthy controls. The CGI included the entire MIR137 gene and the region adjacent to the 5′-end of MIR2682. The aim of the study was to examine the relationship of the CGI methylation with schizophrenia and cognitive functioning. The methylation level of 91 CpG located in the selected region was established for each participant by means of single-molecule real-time bisulfite sequencing. All subjects completed the battery of neuropsychological tests. We found that the CGI was hypomethylated in both groups, except for one site—CpG (chr1: 98?511?049), with significant interindividual variability in methylation. A higher level of methylation of this CpG was seen in male patients and was associated with a decrease in the cognitive index in the combined sample of patients and controls. Our data suggest that further investigation of mechanisms that regulate the MIR137 and MIR2682 genes expression might help to understand the molecular basis of cognitive deficits in schizophrenia.

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April 21, 2020

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

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April 21, 2020

Analysis of transcripts and splice isoforms in Medicago sativa L. by single-molecule long-read sequencing.

The full-length transcriptome of alfalfa was analyzed with PacBio single-molecule long-read sequencing technology. The transcriptome data provided full-length sequences and gene isoforms of transcripts in alfalfa, which will improve genome annotation and enhance our understanding of the gene structure of alfalfa. As an important forage, alfalfa (Medicago sativa L.) is world-wide planted. For its complexity of genome and unfinished whole genome sequencing, the sequences and complete structure of mRNA transcripts remain unclear in alfalfa. In this study, single-molecule long-read sequencing was applied to investigate the alfalfa transcriptome using the Pacific Biosciences platform, and a total of 113,321 transcripts were obtained from young, mature and senescent leaves. We identified 72,606 open reading frames including 46,616 full-length ORFs, 1670 transcription factors from 54 TF families and 44,040 simple sequence repeats from 30,797 sequences. A total of 7568 alternative splicing events was identified and the majority of alternative splicing events in alfalfa was intron retention. In addition, we identified 17,740 long non-coding RNAs. Our results show the feasibility of deep sequencing full-length RNA from alfalfa transcriptome on a single-molecule level.

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