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.


September 22, 2019  |  

Alternative TSSs are co-regulated in single cells in the mouse brain.

Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS usage is a regulated process, and the correlation between two TSSs expressed in single cells of the same cell type is surprisingly high. Our findings indicate that TSSs generally are regulated by common factors rather than being independently regulated or stochastically expressed.© 2017 The Authors. Published under the terms of the CC BY 4.0 license.


September 22, 2019  |  

Defining a personal, allele-specific, and single-molecule long-read transcriptome.

Personal transcriptomes in which all of an individual’s genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes =3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV–in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome.


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.


September 22, 2019  |  

Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts.

Polyploidization contributes to the complexity of gene expression, resulting in numerous related but different transcripts. This study explored the transcriptome diversity and complexity of the tetraploid Arabica coffee (Coffea arabica) bean. Long-read sequencing (LRS) by Pacbio Isoform sequencing (Iso-seq) was used to obtain full-length transcripts without the difficulty and uncertainty of assembly required for reads from short-read technologies. The tetraploid transcriptome was annotated and compared with data from the sub-genome progenitors. Caffeine and sucrose genes were targeted for case analysis. An isoform-level tetraploid coffee bean reference transcriptome with 95 995 distinct transcripts (average 3236 bp) was obtained. A total of 88 715 sequences (92.42%) were annotated with BLASTx against NCBI non-redundant plant proteins, including 34 719 high-quality annotations. Further BLASTn analysis against NCBI non-redundant nucleotide sequences, Coffea canephora coding sequences with UTR, C. arabica ESTs, and Rfam resulted in 1213 sequences without hits, were potential novel genes in coffee. Longer UTRs were captured, especially in the 5?UTRs, facilitating the identification of upstream open reading frames. The LRS also revealed more and longer transcript variants in key caffeine and sucrose metabolism genes from this polyploid genome. Long sequences (>10 kilo base) were poorly annotated. LRS technology shows the limitation of previous studies. It provides an important tool to produce a reference transcriptome including more of the diversity of full-length transcripts to help understand the biology and support the genetic improvement of polyploid species such as coffee.© The Authors 2017. Published by Oxford University Press.


September 22, 2019  |  

Bayesian nonparametric discovery of isoforms and individual specific quantification.

Most human protein-coding genes can be transcribed into multiple distinct mRNA isoforms. These alternative splicing patterns encourage molecular diversity, and dysregulation of isoform expression plays an important role in disease etiology. However, isoforms are difficult to characterize from short-read RNA-seq data because they share identical subsequences and occur in different frequencies across tissues and samples. Here, we develop BIISQ, a Bayesian nonparametric model for isoform discovery and individual specific quantification from short-read RNA-seq data. BIISQ does not require isoform reference sequences but instead estimates an isoform catalog shared across samples. We use stochastic variational inference for efficient posterior estimates and demonstrate superior precision and recall for simulations compared to state-of-the-art isoform reconstruction methods. BIISQ shows the most gains for low abundance isoforms, with 36% more isoforms correctly inferred at low coverage versus a multi-sample method and 170% more versus single-sample methods. We estimate isoforms in the GEUVADIS RNA-seq data and validate inferred isoforms by associating genetic variants with isoform ratios.


September 22, 2019  |  

PacBio full-length transcriptome profiling of insect mitochondrial gene expression.

In this study, we sequenced the first full-length insect transcriptome using the Erthesina fullo Thunberg based on the PacBio platform. We constructed the first quantitative transcription map of animal mitochondrial genomes and built a straightforward and concise methodology to investigate mitochondrial gene transcription, RNA processing, mRNA maturation and several other related topics. Most of the results were consistent with the previous studies, while to the best of our knowledge some findings were reported for the first time in this study. The new findings included the high levels of mitochondrial gene expression, the 3′ polyadenylation and possible 5′ m(7)G caps of rRNAs, the isoform diversity of 12S rRNA, the polycistronic transcripts and natural antisense transcripts of mitochondrial genes et al. These findings could challenge and enrich fundamental concepts of mitochondrial gene transcription and RNA processing, particularly of the rRNA primary (sequence) structure. The methodology constructed in this study can also be used to study gene expression or RNA processing of nuclear genomes.


September 22, 2019  |  

A comprehensive analysis of alternative splicing in paleopolyploid maize.

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis-sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans-sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.


September 22, 2019  |  

Extensive alternative splicing of KIR transcripts.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.


September 22, 2019  |  

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.


September 22, 2019  |  

Widespread polycistronic transcripts in fungi revealed by single-molecule mRNA sequencing.

Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.


September 22, 2019  |  

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification.

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.© 2018 Tardaguila et al.; Published by Cold Spring Harbor Laboratory Press.


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.


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


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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