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Auto Tag: RNA-Seq

April 21, 2020  |  

Insights into transcriptional characteristics and homoeolog expression bias of embryo and de-embryonated kernels in developing grain through RNA-Seq and Iso-Seq.

Bread wheat (Triticum aestivum L.) is an allohexaploid, and the transcriptional characteristics of the wheat embryo and endosperm during grain development remain unclear. To analyze the transcriptome, we performed isoform sequencing (Iso-Seq) for wheat grain and RNA sequencing (RNA-Seq) for the embryo and de-embryonated kernels. The differential regulation between the embryo and de-embryonated kernels was found to be greater than the difference between the two time points for each tissue. Exactly 2264 and 4790 tissue-specific genes were found at 14 days post-anthesis (DPA), while 5166 and 3784 genes were found at 25 DPA in the embryo and de-embryonated kernels, respectively. Genes expressed in the embryo were more likely to be related to nucleic acid and enzyme regulation. In de-embryonated kernels, genes were rich in substance metabolism and enzyme activity functions. Moreover, 4351, 4641, 4516, and 4453 genes with the A, B, and D homoeoloci were detected for each of the four tissues. Expression characteristics suggested that the D genome may be the largest contributor to the transcriptome in developing grain. Among these, 48, 66, and 38 silenced genes emerged in the A, B, and D genomes, respectively. Gene ontology analysis showed that silenced genes could be inclined to different functions in different genomes. Our study provided specific gene pools of the embryo and de-embryonated kernels and a homoeolog expression bias model on a large scale. This is helpful for providing new insights into the molecular physiology of wheat.

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April 21, 2020  |  

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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April 21, 2020  |  

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.

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April 21, 2020  |  

Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis, and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing full-length isoforms are essential for functional genomics study. In this study, we performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on single-molecule full-length cDNA sequencing. To phase and analyze full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short read data and identified cases of allele-specific, gene-level, and isoform-level expression. Our results revealed that maize parental and hybrid lines exhibit different splicing activities. After phasing 6,847 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase power and accuracy in studies of allelic expression.

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April 21, 2020  |  

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.

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April 21, 2020  |  

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Long-read RNA sequencing (RNA-seq) is promising to transcriptomics studies, however, the alignment of the reads is still a fundamental but non-trivial task due to the sequencing errors and complicated gene structures. We propose deSALT, a tailored two-pass long RNA-seq read alignment approach, which constructs graph-based alignment skeletons to sensitively infer exons, and use them to generate spliced reference sequence to produce refined alignments. deSALT addresses several difficult issues, such as small exons, serious sequencing errors and consensus spliced alignment. Benchmarks demonstrate that this approach has a better ability to produce high-quality full-length alignments, which has enormous potentials to transcriptomics studies.

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April 21, 2020  |  

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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April 21, 2020  |  

Defining transgene insertion sites and off-target effects of homology-based gene silencing informs the use of functional genomics tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spreads to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for twelve different gene targets indicated that neighbors within 500-nt were often co-silenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, cis-spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not co-silenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.

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April 21, 2020  |  

Complete genome screening of clinical MRSA isolates identifies lineage diversity and provides full resolution of transmission and outbreak events

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3 adults and 18 infants in a neonatal intensive care unit (NICU) lasting 7 months. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its origins and progression, which was characterized by multiple sub-transmissions and likely precipitated by equipment sharing. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance and persistence. This included a DNA-recognition domain recombination in the hsdS gene of a Type-I restriction-modification system that altered DNA methylation. RNA-Seq profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall our findings demonstrate that long-read sequencing substantially improves our ability to characterize accessory genomic elements that impact MRSA virulence and persistence, and provides valuable information for infection control efforts.

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April 21, 2020  |  

Genome rearrangements induce biofilm formation in Escherichia coli C, an old model organism with a new application in biofilm research

Escherichia coli C forms more robust biofilms than the other laboratory strains. Biofilm formation and cell aggregation under a high shear force depends on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for LPS synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the -35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene, which may drive its overexpression in biofilm. And finally, E. coli C encodes an additional sigma70 subunit overexpressed in biofilm and driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions and total expression profiles using RNA-seq analyses comparing planktonic and biofilm envirovars provided insights into understanding this regulatory pathway in E. coli.

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April 21, 2020  |  

A chromosome-level genome of black rockfish, Sebastes schlegelii, provides insights into the evolution of live birth.

Black rockfish (Sebastes schlegelii) is a teleost species where eggs are fertilized internally and retained in the maternal reproductive system, where they undergo development until live birth (termed viviparity). In the present study, we report a chromosome-level black rockfish genome assembly. High-throughput transcriptome analysis (RNA-seq and ATAC-seq), coupled with in situ hybridization (ISH) and immunofluorescence, identify several candidate genes for maternal preparation, sperm storage and release, and hatching. We propose that zona pellucida (ZP) proteins retain sperm at the oocyte envelope, while genes in two distinct astacin metalloproteinase subfamilies serve to release sperm from the ZP and free the embryo from chorion at pre-hatching stage. Finally, we present a model of black rockfish reproduction, and propose that the rockfish ovarian wall has a similar function to the uterus of mammals. Taken together, these genomic data reveal unprecedented insights into the evolution of an unusual teleost life history strategy, and provide a sound foundation for studying viviparity in non-mammalian vertebrates and an invaluable resource for rockfish ecological and evolutionary research. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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April 21, 2020  |  

Lateral transfers of large DNA fragments spread functional genes among grasses.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species. Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020  |  

A comprehensive evaluation of long read error correction methods

Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods, and provide a comprehensive evaluation of the corresponding long read error correction tools. Leveraging recent real sequencing data, we establish benchmark data sets and set up evaluation criteria for a comparative assessment which includes quality of error correction as well as run-time and memory usage. We study how trimming and long read sequencing depth affect error correction in terms of length distribution and genome coverage post-correction, and the impact of error correction performance on an important application of long reads, genome assembly. We provide guidelines for practitioners for choosing among the available error correction tools and identify directions for future research.

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April 21, 2020  |  

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block excedding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

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April 21, 2020  |  

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

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