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April 21, 2020

Plant ISOform sequencing database (PISO): a comprehensive repertory of full-length transcripts in plants.

In higher eukaryotes, alternative splicing (AS) and alternative polyadenylation (APA) events can produce multiple transcript isoforms in the majority of genes, which significantly increase the protein- coding potential of a genome (Pan et al., 2008; Anvar et al., 2018). Different transcript isoforms might encode proteins with different functions or affect the mRNA stability and translational capacity, in some sense AS and APA events can dramatically increase the complexity and flexibility of the entire transcriptome and proteome (Yang et al., 2016; Feng et al., 2015; Li et al., 2017a; Wang et al., 2017a). Many databases contained AS events and transcripts in animals are available in some public resources such as ASTD and MAASE (Zheng et al., 2005), whereas there is no database containing full-length transcripts and AS events in plants up to now. Next-generation sequencing (NGS) technology has limitation for identifying AS and APA events due to short reads and low accuracy. In recent years, isoform sequencing (Iso-Seq) using Pacbio single molecule real-time sequencing (SMRT) platform can generate full-length sequences and provide accurate information about AS and transcriptional start sites (Li et al., 2017a). In this study, we collected the plant Iso-Seq data sequenced by Pacbio platform from NCBI database up to the end of 2017, and employed unified pipelines to process all the full-length transcripts in different species. Based on these data, we constructed Plant ISOform sequencing database (PISO, http://cbi.hzau.edu.cn/piso/).

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April 21, 2020

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

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April 21, 2020

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.

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April 21, 2020

Transcriptome analysis reveals multiple signal network contributing to the Verticillium wilt resistance in eggplant

Verticillium wilt is a devastating disease in eggplants. In order to understand the molecular mechanism of disease resistance in eggplants, transcriptomes of Verticillium wilt infected eggplants were detected. A total of 480, 518, 887 and 1 046 Verticillium wilt related differentially expressed genes were identified at 6 (V6), 12 (V12), 24 (V24) and 48?h (V48), respectively. COG function classification revealed that most of DEGs functioned in “Amino acid transport and metabolism”, “Cytoskeleton” and “Cell motility”. In addition, compared the control plants (V0) to infected eggplants (V6-V48), a total of 111 common DEGs were identified. Except for “General function prediction only”, most of the DEGs enriched in “Signal transduction”. DEGs associated to different hormone signals, including GID1B, ROPGAP1, OPT3 and CDPK, were identified throughout the whole infection process. Cross-talk among defense signal pathways plays major roles in the Verticillium wilt disease resistance in eggplants.

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April 21, 2020

5’UTR-mediated regulation of Ataxin-1 expression.

Expression of mutant Ataxin-1 with an abnormally expanded polyglutamine domain is necessary for the onset and progression of spinocerebellar ataxia type 1 (SCA1). Understanding how Ataxin-1 expression is regulated in the human brain could inspire novel molecular therapies for this fatal, dominantly inherited neurodegenerative disease. Previous studies have shown that the ATXN1 3’UTR plays a key role in regulating the Ataxin-1 cellular pool via diverse post-transcriptional mechanisms. Here we show that elements within the ATXN1 5’UTR also participate in the regulation of Ataxin-1 expression. PCR and PacBio sequencing analysis of cDNA obtained from control and SCA1 human brain samples revealed the presence of three major, alternatively spliced ATXN1 5’UTR variants. In cell-based assays, fusion of these variants upstream of an EGFP reporter construct revealed significant and differential impacts on total EGFP protein output, uncovering a type of genetic rheostat-like function of the ATXN1 5’UTR. We identified ribosomal scanning of upstream AUG codons and increased transcript instability as potential mechanisms of regulation. Importantly, transcript-based analyses revealed significant differences in the expression pattern of ATXN1 5’UTR variants between control and SCA1 cerebellum. Together, the data presented here shed light into a previously unknown role for the ATXN1 5’UTR in the regulation of Ataxin-1 and provide new opportunities for the development of SCA1 therapeutics. Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

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April 21, 2020

The Single-molecule long-read sequencing of Scylla paramamosain.

Scylla paramamosain is an important aquaculture crab, which has great economical and nutritional value. To the best of our knowledge, few full-length crab transcriptomes are available. In this study, a library composed of 12 different tissues including gill, hepatopancreas, muscle, cerebral ganglion, eyestalk, thoracic ganglia, intestine, heart, testis, ovary, sperm reservoir, and hemocyte was constructed and sequenced using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. A total of 284803 full-length non-chimeric reads were obtained, from which 79005 high-quality unique transcripts were obtained after error correction and sequence clustering and redundant. Additionally, a total of 52544 transcripts were annotated against protein database (NCBI nonredundant, Swiss-Prot, KOG, and KEGG database). A total of 23644 long non-coding RNAs (lncRNAs) and 131561 simple sequence repeats (SSRs) were identified. Meanwhile, the isoforms of many genes were also identified in this study. Our study provides a rich set of full-length cDNA sequences for S. paramamosain, which will greatly facilitate S. paramamosain research.

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April 21, 2020

Genetic basis of functional variability in adhesion G protein-coupled receptors.

The enormous sizes of adhesion G protein-coupled receptors (aGPCRs) go along with complex genomic exon-intron architectures giving rise to multiple mRNA variants. There is a need for a comprehensive catalog of aGPCR variants for proper evaluation of the complex functions of aGPCRs found in structural, in vitro and animal model studies. We used an established bioinformatics pipeline to extract, quantify and visualize mRNA variants of aGPCRs from deeply sequenced transcriptomes. Data analysis showed that aGPCRs have multiple transcription start sites even within introns and that tissue-specific splicing is frequent. On average, 19 significantly expressed transcript variants are derived from a given aGPCR gene. The domain architecture of the N terminus encoded by transcript variants often differs and N termini without or with an incomplete seven-helix transmembrane anchor as well as separate seven-helix transmembrane domains are frequently derived from aGPCR genes. Experimental analyses of selected aGPCR transcript variants revealed marked functional differences. Our analysis has an impact on a rational design of aGPCR constructs for structural analyses and gene-deficient mouse lines and provides new support for independent functions of both, the large N terminus and the transmembrane domain of aGPCRs.

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April 21, 2020

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

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April 21, 2020

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.

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April 21, 2020

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

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April 21, 2020

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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April 21, 2020

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

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April 21, 2020

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50?bp) and 27,622 SVs (=50?bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

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April 21, 2020

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2?Mb and a contig N50 of 3.3?Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.

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