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Asset Tag: RNA-seq

September 22, 2019  |  

Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing.

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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September 22, 2019  |  

Long reads: their purpose and place.

In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

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September 22, 2019  |  

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass.

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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September 22, 2019  |  

Divergent brain gene expression profiles between alternative behavioural helper types in a cooperative breeder.

Juveniles of the cooperatively breeding cichlid fish Neolamprologus pulcher either consistently provide help in form of alloparental egg care (“cleaners”) or consistently abstain from helping (“noncleaners”). These phenotypes are not based on heritable genetic differences. Instead, they arise during ontogeny, which should lead to differences in brain structure or physiology, a currently untested prediction. We compared brain gene expression profiles of cleaners and noncleaners in two experimental conditions, a helping opportunity and a control condition. We aimed to identify (a) expression differences between cleaners and noncleaners in the control, (b) changes in gene expression induced by the opportunity and (c) differences in plasticity of gene expression between cleaners and noncleaners. Control cleaners and noncleaners differed in the expression of a single gene, irx2, which regulates neural differentiation. During the opportunity, cleaners and noncleaners had three upregulated genes in common, which were implicated in neuroplasticity, hormonal signalling and cell proliferation. Thus, the stimulus in the opportunity was sufficiently salient. Cleaners also showed higher expression of seven additional genes that were unique to the opportunity. One of these cleaner-specific genes is implicated in neuropeptide metabolism, indicating that this process is associated with cleaning performance. This suggests that the two types employed different pathways to integrate social information, preparing them for accelerated reaction to future opportunities. Interestingly, three developmental genes were downregulated between the control and the opportunity in cleaners only. Our results indicate that the two behavioural types responded differently to the helping opportunity and that only cleaners responded by downregulating developmental genes.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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September 22, 2019  |  

Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing.

Zea mays is an important genetic model for elucidating transcriptional networks. Uncertainties about the complete structure of mRNA transcripts limit the progress of research in this system. Here, using single-molecule sequencing technology, we produce 111,151 transcripts from 6 tissues capturing ~70% of the genes annotated in maize RefGen_v3 genome. A large proportion of transcripts (57%) represent novel, sometimes tissue-specific, isoforms of known genes and 3% correspond to novel gene loci. In other cases, the identified transcripts have improved existing gene models. Averaging across all six tissues, 90% of the splice junctions are supported by short reads from matched tissues. In addition, we identified a large number of novel long non-coding RNAs and fusion transcripts and found that DNA methylation plays an important role in generating various isoforms. Our results show that characterization of the maize B73 transcriptome is far from complete, and that maize gene expression is more complex than previously thought.

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September 22, 2019  |  

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.

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September 22, 2019  |  

ISOdb: A comprehensive database of full-length isoforms generated by Iso-Seq.

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

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September 22, 2019  |  

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

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September 22, 2019  |  

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

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September 22, 2019  |  

Event analysis: Using transcript events to improve estimates of abundance in RNA-seq data.

Alternative splicing leverages genomic content by allowing the synthesis of multiple transcripts and, by implication, protein isoforms, from a single gene. However, estimating the abundance of transcripts produced in a given tissue from short sequencing reads is difficult and can result in both the construction of transcripts that do not exist, and the failure to identify true transcripts. An alternative approach is to catalog the events that make up isoforms (splice junctions and exons). We present here the Event Analysis (EA) approach, where we project transcripts onto the genome and identify overlapping/unique regions and junctions. In addition, all possible logical junctions are assembled into a catalog. Transcripts are filtered before quantitation based on simple measures: the proportion of the events detected, and the coverage. We find that mapping to a junction catalog is more efficient at detecting novel junctions than mapping in a splice aware manner. We identify 99.8% of true transcripts while iReckon identifies 82% of the true transcripts and creates more transcripts not included in the simulation than were initially used in the simulation. Using PacBio Iso-seq data from a mouse neural progenitor cell model, EA detects 60% of the novel junctions that are combinations of existing exons while only 43% are detected by STAR. EA further detects ~5,000 annotated junctions missed by STAR. Filtering transcripts based on the proportion of the transcript detected and the number of reads on average supporting that transcript captures 95% of the PacBio transcriptome. Filtering the reference transcriptome before quantitation, results in is a more stable estimate of isoform abundance, with improved correlation between replicates. This was particularly evident when EA is applied to an RNA-seq study of type 1 diabetes (T1D), where the coefficient of variation among subjects (n = 81) in the transcript abundance estimates was substantially reduced compared to the estimation using the full reference. EA focuses on individual transcriptional events. These events can be quantitate and analyzed directly or used to identify the probable set of expressed transcripts. Simple rules based on detected events and coverage used in filtering result in a dramatic improvement in isoform estimation without the use of ancillary data (e.g., ChIP, long reads) that may not be available for many studies. Copyright © 2018 Newman et al.

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September 22, 2019  |  

The genomic and functional landscapes of developmental plasticity in the American cockroach.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its adaptation to urban environments and developmental plasticity. In comparison with other insects, expansions of gene families in P. americana exist for most core gene families likely associated with environmental adaptation, such as chemoreception and detoxification. Multiple pathways regulating metamorphic development are well conserved, and RNAi experiments inform on key roles of 20-hydroxyecdysone, juvenile hormone, insulin, and decapentaplegic signals in regulating plasticity. Our analyses reveal a high level of sequence identity in genes between the American cockroach and two termite species, advancing it as a valuable model to study the evolutionary relationships between cockroaches and termites.

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September 22, 2019  |  

SMRT sequencing of full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt).

This study was aimed at generating the full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt) using single-molecule real-time (SMRT) sequencing. Four developmental stages of A. hygrophila, including eggs, larvae, pupae, and adults were harvested for isolating total RNA. The mixed samples were used for SMRT sequencing to generate the full-length transcriptome. Based on the obtained transcriptome data, alternative splicing event, simple sequence repeat (SSR) analysis, coding sequence prediction, transcript functional annotation, and lncRNA prediction were performed. Total 9.45?Gb of clean reads were generated, including 335,045 reads of insert (ROI) and 158,085 full-length non-chimeric (FLNC) reads. Transcript clustering analysis of FLNC reads identified 40,004 consensus isoforms, including 31,015 high-quality ones. After removing redundant reads, 28,982 transcripts were obtained. Total 145 alternative splicing events were predicted. Additionally, 12,753 SSRs and 16,205 coding sequences were identified based on SSR analysis. Furthermore, 24,031 transcripts were annotated in eight functional databases, and 4,198 lncRNAs were predicted. This is the first study to perform SMRT sequencing of the full-length transcriptome of A. hygrophila. The obtained transcriptome may facilitate further exploration of the genetic data of A. hygrophila and uncover the interactions between this insect and the ecosystem.

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September 22, 2019  |  

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.

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September 22, 2019  |  

Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids, derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding a total of 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

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