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April 21, 2020  |  

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, with a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed and many new generally shorter transcripts were detected by normalization. For the same input cDNA and the same data yield, the normalized library recovered more total transcript isoforms, number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ~1.25 kb, more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ~52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ~80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

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April 21, 2020  |  

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.

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April 21, 2020  |  

Genome of lethal Lepiota venenata and insights into the evolution of toxin-biosynthetic genes.

Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway.The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes a-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the house-keeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome.To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.

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April 21, 2020  |  

Comparative Phylogenomics, a Stepping Stone for Bird Biodiversity Studies

Birds are a group with immense availability of genomic resources, and hundreds of forthcoming genomes at the doorstep. We review recent developments in whole genome sequencing, phylogenomics, and comparative genomics of birds. Short read based genome assemblies are common, largely due to efforts of the Bird 10K genome project (B10K). Chromosome-level assemblies are expected to increase due to improved long-read sequencing. The available genomic data has enabled the reconstruction of the bird tree of life with increasing confidence and resolution, but challenges remain in the early splits of Neoaves due to their explosive diversification after the Cretaceous-Paleogene (K-Pg) event. Continued genomic sampling of the bird tree of life will not just better reflect their evolutionary history but also shine new light onto the organization of phylogenetic signal and conflict across the genome. The comparatively simple architecture of avian genomes makes them a powerful system to study the molecular foundation of bird specific traits. Birds are on the verge of becoming an extremely resourceful system to study biodiversity from the nucleotide up.

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April 21, 2020  |  

Systematic analysis of dark and camouflaged genes reveals disease-relevant genes hiding in plain sight.

The human genome contains “dark” gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions with few mappable reads that we call dark by depth, and others that have ambiguous alignment, called camouflaged. We assess how well long-read or linked-read technologies resolve these regions.Based on standard whole-genome Illumina sequencing data, we identify 36,794 dark regions in 6054 gene bodies from pathways important to human health, development, and reproduction. Of these gene bodies, 8.7% are completely dark and 35.2% are =?5% dark. We identify dark regions that are present in protein-coding exons across 748 genes. Linked-read or long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduce dark protein-coding regions to approximately 50.5%, 35.6%, and 9.6%, respectively. We present an algorithm to resolve most camouflaged regions and apply it to the Alzheimer’s Disease Sequencing Project. We rescue a rare ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in disease cases but not in controls.While we could not formally assess the association of the CR1 frameshift mutation with Alzheimer’s disease due to insufficient sample-size, we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

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April 21, 2020  |  

Whole genomes and transcriptomes reveal adaptation and domestication of pistachio.

Pistachio (Pistacia vera), one of the most important commercial nut crops worldwide, is highly adaptable to abiotic stresses and is tolerant to drought and salt stresses.Here, we provide a draft de novo genome of pistachio as well as large-scale genome resequencing. Comparative genomic analyses reveal stress adaptation of pistachio is likely attributable to the expanded cytochrome P450 and chitinase gene families. Particularly, a comparative transcriptomic analysis shows that the jasmonic acid (JA) biosynthetic pathway plays an important role in salt tolerance in pistachio. Moreover, we resequence 93 cultivars and 14 wild P. vera genomes and 35 closely related wild Pistacia genomes, to provide insights into population structure, genetic diversity, and domestication. We find that frequent genetic admixture occurred among the different wild Pistacia species. Comparative population genomic analyses reveal that pistachio was domesticated about 8000?years ago and suggest that key genes for domestication related to tree and seed size experienced artificial selection.Our study provides insight into genetic underpinning of local adaptation and domestication of pistachio. The Pistacia genome sequences should facilitate future studies to understand the genetic basis of agronomically and environmentally related traits of desert crops.

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April 21, 2020  |  

The Anaplasma ovis genome reveals a high proportion of pseudogenes.

The genus Anaplasma is made up of organisms characterized by small genomes that are undergoing reductive evolution. Anaplasma ovis, one of the seven recognized species in this genus, is an understudied pathogen of sheep and other ruminants. This tick-borne agent is thought to induce only mild clinical disease; however, small deficits may add to larger economic impacts due to the wide geographic distribution of this pathogen.In this report we present the first complete genome sequence for A. ovis and compare the genome features with other closely related species. The 1,214,674?bp A. ovis genome encodes 933 protein coding sequences, the split operon arrangement for ribosomal RNA genes, and more pseudogenes than previously recognized for other Anaplasma species. The metabolic potential is similar to other Anaplasma species. Anaplasma ovis has a small repertoire of surface proteins and transporters. Several novel genes are identified.Analyses of these important features and significant gene families/genes with potential to be vaccine candidates are presented in a comparative context. The availability of this genome will significantly facilitate research for this pathogen.

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April 21, 2020  |  

Progression of the canonical reference malaria parasite genome from 2002-2019.

Here we describe the ways in which the sequence and annotation of the Plasmodium falciparum reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the P. falciparum research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpretating intra-species sequence diversity. We therefore describe in this publication a population reference genome of P. falciparum, called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. P. falciparum 3D7 will be continued to be maintained with ongoing curation ensuring continual improvements in annotation quality.

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April 21, 2020  |  

The complexity of the Fragaria x ananassa (octoploid) transcriptome by single-molecule long-read sequencing.

Strawberry (Fragaria x ananassa) is an allopolyploid species with diverse and complex transcripts. The regulatory mechanisms of fruit development and maturation have been extensively studied; however, little is known about the signaling mechanisms that direct this process in octoploid strawberry (Fragaria x ananassa). Here, we used long-read sequencing (LRS) technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry. We obtained a reference transcriptome with 119,897 unique full-length isoforms, including 2017 new isoforms and 2510 long noncoding RNAs. Based on the genome of the plausible progenitor (Fragaria vesca), 20,229 alternative splicing (AS) events were identified. Using this transcriptome, we found 17,485 differentially expressed transcripts during strawberry fruit development, including 527 transcription factors (TFs) belonging to 41 families. The expression profiles of all members of the auxin, ABA pathway, and anthocyanin biosynthesis gene families were also examined, and many of them were highly expressed at the ripe fruit stage, strongly indicating that the role of those genes is in the regulation of fruit ripening. We produce a high-quality reference transcriptome for octoploid strawberry, including much of the full-length transcript diversity, to help understand the regulatory mechanisms of fruit development and maturation of polyploid species, particularly via elucidation of the biochemical pathways involved in auxin, ABA, and anthocyanin biosynthesis.

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April 21, 2020  |  

Horizontal transfer of a retrotransposon between parasitic nematodes and the common shrew.

As the genomes of more metazoan species are sequenced, reports of horizontal transposon transfers (HTT) have increased. Our understanding of the mechanisms of such events is at an early stage. The close physical relationship between a parasite and its host could facilitate horizontal transfer. To date, two studies have identified horizontal transfer of RTEs, a class of retrotransposable elements, involving parasites: ticks might act as vector for BovB between ruminants and squamates, and AviRTE was transferred between birds and parasitic nematodes.We searched for RTEs shared between nematode and mammalian genomes. Given their physical proximity, it was necessary to detect and remove sequence contamination from the genome datasets, which would otherwise distort the signal of horizontal transfer. We developed an approach that is based on reads instead of genomic sequences to reliably detect contamination. From comparison of 43 RTEs across 197 genomes, we identified a single putative case of horizontal transfer: we detected RTE1_Sar from Sorex araneus, the common shrew, in parasitic nematodes. From the taxonomic distribution and evolutionary analysis, we show that RTE1_Sar was horizontally transferred.We identified a new horizontal RTE transfer in host-parasite interactions, which suggests that it is not uncommon. Further, we present and provide the workflow a read-based method to distinguish between contamination and horizontal transfer.

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April 21, 2020  |  

Transcriptome, proteome and draft genome of Euglena gracilis.

Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts.We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500?Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids.Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the ‘shopping bag’ hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

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April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.

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