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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  |  

Transcriptome profiling in the spathe of Anthurium andraeanum ‘Albama’ and its anthocyanin-loss mutant ‘Xueyu’.

Anthurium andraeanum is a popular tropical ornamental plant. Its spathes are brilliantly coloured due to variable anthocyanin contents. To examine the mechanisms that control anthocyanin biosynthesis, we sequenced the spathe transcriptomes of ‘Albama’, a red-spathed cultivar of A. andraeanum, and ‘Xueyu’, its anthocyanin-loss mutant. Both long reads and short reads were sequenced. Long read sequencing produced 805,869 raw reads, resulting in 83,073 high-quality transcripts. Short read sequencing produced 347.79?M reads, and the subsequent assembly resulted in 111,674 unigenes. High-quality transcripts and unigenes were quantified using the short reads, and differential expression analysis was performed between ‘Albama’ and ‘Xueyu’. Obtaining high-quality, full-length transcripts enabled the detection of long transcript structures and transcript variants. These data provide a foundation to elucidate the mechanisms regulating the biosynthesis of anthocyanin in A. andraeanum.

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September 22, 2019  |  

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives.

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

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September 22, 2019  |  

Evaluating approaches to find exon chains based on long reads.

Transcript prediction can be modeled as a graph problem where exons are modeled as nodes and reads spanning two or more exons are modeled as exon chains. Pacific Biosciences third-generation sequencing technology produces significantly longer reads than earlier second-generation sequencing technologies, which gives valuable information about longer exon chains in a graph. However, with the high error rates of third-generation sequencing, aligning long reads correctly around the splice sites is a challenging task. Incorrect alignments lead to spurious nodes and arcs in the graph, which in turn lead to incorrect transcript predictions. We survey several approaches to find the exon chains corresponding to long reads in a splicing graph, and experimentally study the performance of these methods using simulated data to allow for sensitivity/precision analysis. Our experiments show that short reads from second-generation sequencing can be used to significantly improve exon chain correctness either by error-correcting the long reads before splicing graph creation, or by using them to create a splicing graph on which the long-read alignments are then projected. We also study the memory and time consumption of various modules, and show that accurate exon chains lead to significantly increased transcript prediction accuracy.The simulated data and in-house scripts used for this article are available at http://www.cs.helsinki.fi/group/gsa/exon-chains/exon-chains-bib.tar.bz2.

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September 22, 2019  |  

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

neoantigenR: An annotation based pipeline for tumor neoantigen identification from sequencing data

Studies indicate that more than 90% of human genes are alternatively spliced, suggesting the complexity of the transcriptome assembly and analysis. The splicing process is often disrupted, resulting in both functional and non-functional end-products (Sveen et al. 2016) in many cancers. Harnessing the immune system to fight against malignant cancers carrying aberrantly mutated or spliced products is becoming a promising approach to cancer therapy. Advances in immune checkpoint blockade have elicited adaptive immune responses with promising clinical responses to treatments against human malignancies (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Emerging data suggest that recognition of patient-specific mutation-associated cancer antigens (i.e. from alternative splicing isoforms) may allow scientists to dissect the immune response in the activity of clinical immunotherapies (Schumacher and Schreiber 2015). The advent of high-throughput sequencing technology has provided a comprehensive view of both splicing aberrations and somatic mutations across a range of human malignancies, allowing for a deeper understanding of the interplay of various disease mechanisms. Meanwhile, studies show that the number of transcript isoforms reported to date may be limited by the short-read sequencing due to the inherit limitation of transcriptome reconstruction algorithms, whereas long-read sequencing is able to significantly improve the detection of alternative splicing variants since there is no need to assemble full-length transcripts from short reads. The analysis of these high-throughput long-read sequencing data may permit a systematic view of tumor specific peptide epitopes (also known as neoantigens) that could serve as targets for immunotherapy (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Currently, there is no software pipeline available that can efficiently produce mutation-associated cancer antigens from raw high-throughput sequencing data on patient tumor DNA (The Problem with Neoantigen Prediction 2017). In addressing this issue, we introduce a R package that allows the discoveries of peptide epitope candidates, which are the tumor-specific peptide fragments containing potential functional neoantigens. These peptide epitopes consist of structure variants including insertion, deletions, alternative sequences, and peptides from nonsynonymous mutations. Analysis of these precursor candidates with widely used tools such as netMHC allows for the accurate in-silico prediction of neoantigens. The pipeline named neoantigeR is currently hosted in https://github.com/ICBI/neoantigeR.

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September 22, 2019  |  

Improving PacBio long read accuracy by short read alignment.

The recent development of third generation sequencing (TGS) generates much longer reads than second generation sequencing (SGS) and thus provides a chance to solve problems that are difficult to study through SGS alone. However, higher raw read error rates are an intrinsic drawback in most TGS technologies. Here we present a computational method, LSC, to perform error correction of TGS long reads (LR) by SGS short reads (SR). Aiming to reduce the error rate in homopolymer runs in the main TGS platform, the PacBio® RS, LSC applies a homopolymer compression (HC) transformation strategy to increase the sensitivity of SR-LR alignment without scarifying alignment accuracy. We applied LSC to 100,000 PacBio long reads from human brain cerebellum RNA-seq data and 64 million single-end 75 bp reads from human brain RNA-seq data. The results show LSC can correct PacBio long reads to reduce the error rate by more than 3 folds. The improved accuracy greatly benefits many downstream analyses, such as directional gene isoform detection in RNA-seq study. Compared with another hybrid correction tool, LSC can achieve over double the sensitivity and similar specificity.

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September 22, 2019  |  

The state of long non-coding RNA biology.

Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as organizational and guide molecules: most lncRNAs are nuclear-localized and chromatin-associated, with some involved in the formation of specialized subcellular domains. I suggest that a reassessment of the conceptual framework of genetic information and gene expression in the 4-dimensional ontogeny of spatially organized multicellular organisms is required. Together with this and further studies on their biology, the key challenges now are to determine the structure?function relationships of lncRNAs, which may be aided by emerging evidence of their modular structure, the role of RNA editing and modification in enabling epigenetic plasticity, and the role of RNA signaling in transgenerational inheritance of experience.

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September 22, 2019  |  

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

A near complete snapshot of the Zea mays seedling transcriptome revealed from ultra-deep sequencing.

RNA-sequencing (RNA-seq) enables in-depth exploration of transcriptomes, but typical sequencing depth often limits its comprehensiveness. In this study, we generated nearly 3 billion RNA-Seq reads, totaling 341 Gb of sequence, from a Zea mays seedling sample. At this depth, a near complete snapshot of the transcriptome was observed consisting of over 90% of the annotated transcripts, including lowly expressed transcription factors. A novel hybrid strategy combining de novo and reference-based assemblies yielded a transcriptome consisting of 126,708 transcripts with 88% of expressed known genes assembled to full-length. We improved current annotations by adding 4,842 previously unannotated transcript variants and many new features, including 212 maize transcripts, 201 genes, 10 genes with undocumented potential roles in seedlings as well as maize lineage specific gene fusion events. We demonstrated the power of deep sequencing for large transcriptome studies by generating a high quality transcriptome, which provides a rich resource for the research community.

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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  |  

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

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September 22, 2019  |  

Carbohydrate staple food modulates gut microbiota of Mongolians in China.

Gut microbiota is a determining factor in human physiological functions and health. It is commonly accepted that diet has a major influence on the gut microbial community, however, the effects of diet is not fully understood. The typical Mongolian diet is characterized by high and frequent consumption of fermented dairy products and red meat, and low level of carbohydrates. In this study, the gut microbiota profile of 26 Mongolians whom consumed wheat, rice and oat as the sole carbohydrate staple food for a week each consecutively was determined. It was observed that changes in staple carbohydrate rapidly (within a week) altered gut microbial community structure and metabolic pathway of the subjects. Wheat and oat favored bifidobacteria (Bifidobacterium catenulatum, Bifodobacteriumbifidum, Bifidobacterium adolescentis); whereas rice suppressed bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) and wheat suppresses Lactobaciilus, Ruminococcus and Bacteroides. The study exhibited two gut microbial clustering patterns with the preference of fucosyllactose utilization linking to fucosidase genes (glycoside hydrolase family classifications: GH95 and GH29) encoded by Bifidobacterium, and xylan and arabinoxylan utilization linking to xylanase and arabinoxylanase genes encoded by Bacteroides. There was also a correlation between Lactobacillus ruminis and sialidase, as well as Butyrivibrio crossotus and xylanase/xylosidase. Meanwhile, a strong concordance was found between the gastrointestinal bacterial microbiome and the intestinal virome. Present research will contribute to understanding the impacts of the dietary carbohydrate on human gut microbiome, which will ultimately help understand relationships between dietary factor, microbial populations, and the health of global humans.

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