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September 22, 2019

The transcriptome of human pluripotent stem cells.

Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify the transcriptome of hESCs. Lately, we developed a new tool (IDP) to integrate the hybrid sequencing data to characterize a more reliable and comprehensive hESC transcriptome with discoveries of many novel transcripts. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform.The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated.We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.

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September 22, 2019

Complete genome sequence of multidrug-resistant Staphylococcus cohnii ssp. urealyticus strain SNUDS-2 isolated from farmed duck, Republic of Korea.

Staphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea.Genomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified.The annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to ß-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain.To the best of our knowledge, this is the first complete genome of S. cohnii, and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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September 22, 2019

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides.

Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness. Copyright © 2015, American Association for the Advancement of Science.

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September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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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-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 evolve and improve, a process that will be more efficient if resources across a community are shared and pooled.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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September 22, 2019

The expressed portion of the barley genome

In this chapter, we refer to the expressed portion of the barley genome as the relatively small fraction of the total cellular DNA that either contains the genes that ultimately produce proteins, or that directly/indirectly controls the level, location and/or timing of when these genes are expressed and proteins are produced. We start by describing the dynamics of tissue and time-dependent gene expression and how common patterns across multiple samples can provide clues about gene networks involved in common biological processes. We then describe some of the complexities of how a single mRNA template can be differentially processed by alternative splicing to generate multiple different proteins or provide a mechanism to regulate the amount of functional gene product in a cell at a given point in time. We extend our analysis, using a number of biological examples, to address how diverse families of small non-coding microRNAs specifically regulate gene expression, and complete our appraisal by looking at the physical/molecular environment around genes that can result in either the promotion or repression of gene expression. We conclude by assessing some of the issues that remain around our ability to fully exploit the depth and power of current approaches for analysing gene expression and propose improvements that could be made using new but available sequencing and bioinformatics technologies.

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September 22, 2019

Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing differential intron retention, and isoform ratio switching.

Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns – a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses.

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September 22, 2019

De novo assembly and characterizing of the culm-derived meta-transcriptome from the polyploid sugarcane genome based on coding transcripts

Sugarcane biomass has been used for sugar, bioenergy and biomaterial production. The majority of the sugarcane biomass comes from the culm, which makes it important to understand the genetic control of biomass production in this part of the plant. A meta-transcriptome of the culm was obtained in an earlier study by using about one billion paired-end (150 bp) reads of deep RNA sequencing of samples from 20 diverse sugarcane genotypes and combining de novo assemblies from different assemblers and different settings. Although many genes could be recovered, this resulted in a large combined assembly which created the need for clustering to reduce transcript redundancy while maintaining gene content. Here, we present a comprehensive analysis of the effect of different assembly settings and clustering methods on de novo assembly, annotation and transcript profiling focusing especially on the coding transcripts from the highly polyploid sugarcane genome. The new coding sequence-based transcript clustering resulted in a better representation of transcripts compared to the earlier approach, having 121,987 contigs, which included 78,052 main and 43,935 alternative transcripts. About 73%, 67%, 61% and 10% of the transcriptome was annotated against the NCBI NR protein database, GO terms, orthologous groups and KEGG orthologies, respectively. Using this set for a differential gene expression analysis between the young and mature sugarcane culm tissues, a total of 822 transcripts were found to be differentially expressed, including key transcripts involved in sugar/fiber accumulation in sugarcane. In the context of the lack of a whole genome sequence for sugarcane, the availability of a well annotated culm-derived meta-transcriptome through deep sequencing provides useful information on coding genes specific to the sugarcane culm and will certainly contribute to understanding the process of carbon partitioning, and biomass accumulation in the sugarcane culm.

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September 22, 2019

Whole genome sequencing of “Faecalibaculum rodentium” ALO17, isolated from C57BL/6J laboratory mouse feces.

Intestinal microorganisms affect host physiology, including ageing. Given the difficulty in controlling for human studies of the gut microbiome, mouse models provide an alternative avenue to study such relationships. In this study, we report on the complete genome of “Faecalibaculum rodentium” ALO17, a bacterium that was isolated from the faeces of a 9-month-old female C57BL/6J mouse. This strain will be utilized in future in vivo studies detailing the relationships between the gut microbiome and ageing.The whole genome sequence of “F. rodentium” ALO17 was obtained using single-molecule, real-time (SMRT) technique on a PacBio instrument. The assembled genome consisted of 2,542,486 base pairs of double-stranded DNA with a GC content of 54.0 % and no plasmids. The genome was predicted to contain 2794 open reading frames, 55 tRNA genes, and 38 rRNA genes. The 16S rRNA gene of ALO17 was 86.9 % similar to that of Allobaculum stercoricanis DSM 13633(T), and the average overall nucleotide identity between strains ALO17 and DSM 13633(T) was 66.8 %. After confirming the phylogenetic relationship between “F. rodentium” ALO17 and A. stercoricanis DSM 13633(T), their whole genome sequences were compared, revealing that “F. rodentium” ALO17 contains more fermentation-related genes than A. stercoricanis DSM 13633(T). Furthermore, “F. rodentium” ALO17 produces higher levels of lactic acid than A. stercoricanis DSM 13633(T) as determined by high-performance liquid chromatography.The availability of the “F. rodentium” ALO17 whole genome sequence will enhance studies concerning the gut microbiota and host physiology, especially when investigating the molecular relationships between gut microbiota and ageing.

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