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April 21, 2020

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551?bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress.Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

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April 21, 2020

Differential retention of transposable element-derived sequences in outcrossing Arabidopsis genomes.

Transposable elements (TEs) are genomic parasites with major impacts on host genome architecture and host adaptation. A proper evaluation of their evolutionary significance has been hampered by the paucity of short scale phylogenetic comparisons between closely related species. Here, we characterized the dynamics of TE accumulation at the micro-evolutionary scale by comparing two closely related plant species, Arabidopsis lyrata and A. halleri.Joint genome annotation in these two outcrossing species confirmed that both contain two distinct populations of TEs with either ‘recent’ or ‘old’ insertion histories. Identification of rare segregating insertions suggests that diverse TE families contribute to the ongoing dynamics of TE accumulation in the two species. Orthologous TE fragments (i.e. those that have been maintained in both species), tend to be located closer to genes than those that are retained in one species only. Compared to non-orthologous TE insertions, those that are orthologous tend to produce fewer short interfering RNAs, are less heavily methylated when found within or adjacent to genes and these tend to have lower expression levels. These findings suggest that long-term retention of TE insertions reflects their frequent acquisition of adaptive roles and/or the deleterious effects of removing nearly neutral TE insertions when they are close to genes.Our results indicate a rapid evolutionary dynamics of the TE landscape in these two outcrossing species, with an important input of a diverse set of new insertions with variable propensity to resist deletion.

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April 21, 2020

Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing

In recent genome analyses, population-specific reference panels have indicated important. However, reference panels based on short-read sequencing data do not sufficiently cover long insertions. Therefore, the nature of long insertions has not been well documented. Here, we assembled a Japanese genome using single-molecule real-time sequencing data and characterized insertions found in the assembled genome. We identified 3691 insertions ranging from 100?bps to ~10,000?bps in the assembled genome relative to the international reference sequence (GRCh38). To validate and characterize these insertions, we mapped short-reads from 1070 Japanese individuals and 728 individuals from eight other populations to insertions integrated into GRCh38. With this result, we constructed JRGv1 (Japanese Reference Genome version 1) by integrating the 903 verified insertions, totaling 1,086,173 bases, shared by at least two Japanese individuals into GRCh38. We also constructed decoyJRGv1 by concatenating 3559 verified insertions, totaling 2,536,870 bases, shared by at least two Japanese individuals or by six other assemblies. This assembly improved the alignment ratio by 0.4% on average. These results demonstrate the importance of refining the reference assembly and creating a population-specific reference genome. JRGv1 and decoyJRGv1 are available at the JRG website.

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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

Retrotranspositional landscape of Asian rice revealed by 3000 genomes.

The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.

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April 21, 2020

CAMISIM: simulating metagenomes and microbial communities.

Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required.We describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series, and differential abundance studies, includes real and simulated strain-level diversity, and generates second- and third-generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes, we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT, and metaSPAdes, on several thousand small data sets generated with CAMISIM.CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with standards of truth for method evaluation. All data sets and the software are freely available at https://github.com/CAMI-challenge/CAMISIM.

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April 21, 2020

Whole-genome sequencing of Klebsiella pneumoniae isolates to track strain progression in a single patient with recurrent urinary tract infection.

Klebsiella pneumoniae is an important uropathogen that increasingly harbors broad-spectrum antibiotic resistance determinants. Evidence suggests that some same-strain recurrences in women with frequent urinary tract infections (UTIs) may emanate from a persistent intravesicular reservoir. Our objective was to analyze K. pneumoniae isolates collected over weeks from multiple body sites of a single patient with recurrent UTI in order to track ordered strain progression across body sites, as has been employed across patients in outbreak settings. Whole-genome sequencing of 26 K. pneumoniae isolates was performed utilizing the Illumina platform. PacBio sequencing was used to create a refined reference genome of the original urinary isolate (TOP52). Sequence variation was evaluated by comparing the 26 isolate sequences to the reference genome sequence. Whole-genome sequencing of the K. pneumoniae isolates from six different body sites of this patient with recurrent UTI demonstrated 100% chromosomal sequence identity of the isolates, with only a small P2 plasmid deletion in a minority of isolates. No single nucleotide variants were detected. The complete absence of single-nucleotide variants from 26 K. pneumoniae isolates from multiple body sites collected over weeks from a patient with recurrent UTI suggests that, unlike in an outbreak situation with strains collected from numerous patients, other methods are necessary to discern strain progression within a single host over a relatively short time frame.

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April 21, 2020

Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids.

Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids.We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58 insertions that were absent from the reference assembly. Insertions were found throughout the dog genome including within and near gene models. By comparison of orthologous occupied sites, we characterized element prevalence across 332 genomes including all nine extant canid species, revealing evolutionary patterns of ERV-Fc segregation among species as well as subpopulations.Sequence analysis revealed common disruptive mutations, suggesting a predominant form of ERV-Fc spread by trans complementation of defective proviruses. ERV-Fc activity included multiple circulating variants that infected canid ancestors from the last 20 million to within 1.6 million years, with recent bursts of germline invasion in the sublineage leading to wolves and dogs.

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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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October 23, 2019

ParLECH: Parallel Long-Read Error Correction with Hadoop

Long-read sequencing is emerging as a promising sequencing technology because it can tackle the short length limitation of second-generation sequencing, which has dominated the sequencing market in past years. However, it has substantially higher error rates compared to short-read sequencing (e.g., 13% vs. 0.1%), and its sequencing cost per base is typically more expensive than that of short-read sequencing. To address these limitations, we present a distributed hybrid error correction framework, called ParLECH, that is scalable and cost-efficient for PacBio long reads. For correcting the errors in the long reads, ParLECH utilizes the Illumina short reads that have the low error rate with high coverage at low cost. To efficiently analyze the high-throughput Illumina short reads, ParLECH is equipped with Hadoop and a distributed NoSQL system. To further improve the accuracy, ParLECH utilizes the k-mer coverage information of the Illumina short reads. Specifically, we develop a distributed version of the widest path algorithm, which maximizes the minimum k-mer coverage in a path of the de Bruijn graph constructed from the Illumina short reads. We replace an error region in a long read with its corresponding widest path. Our experimental results show that ParLECH can handle large-scale real-world datasets in a scalable and accurate manner. Using ParLECH, we can process a 312 GB human genome PacBio dataset, with a 452 GB Illumina dataset, on 128 nodes in less than 29 hours.

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October 23, 2019

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.

Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers.We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells.We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs.These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications.

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October 23, 2019

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M.?mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-?glf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

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October 23, 2019

CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation. Copyright © 2017 American Society of Human Genetics. All rights reserved.

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October 23, 2019

Transmission, evolution, and endogenization: Lessons learned from recent retroviral invasions.

Viruses of the subfamily Orthoretrovirinaeare defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and “fossil” endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions. Copyright © 2017 American Society for Microbiology.

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October 23, 2019

Development of a Novel Reference Transcriptome for Scleractinian Coral Porites lutea Using Single-Molecule Long-Read Isoform Sequencing (Iso-Seq)

Elevation in seawater temperature associated with global climate change has caused coral bleaching problems and posed a significant threat to coral health and survival worldwide. Several studies have explored the effects of thermal stress on changes in gene expression levels of both coral hosts and their algal endosymbionts and provided evidences suggesting that corals could acclimatize to environmental stressors through differential regulation of their gene expression (Desalvo et al., 2008, 2010; Császár et al., 2009; Rodriguez-Lanetty et al., 2009; Polato et al., 2010; Meyer et al., 2011; Kenkel et al., 2013). Such information is crucial for understanding the adaptive capacity of the coral holobionts (Hughes et al., 2003). The availability of transcriptome data from a number of coral species and their associated Symbiodinium allows us to probe the molecular stress response of the organisms to heat stress (Traylor-Knowles et al., 2011; Moya et al., 2012; Kenkel et al., 2013; Shinzato et al., 2014; Kitchen et al., 2015; Anderson et al., 2016; Davies et al., 2016). Here, we report the first reference transcriptome for a scleractinian coral Porites lutea, one of the dominant reef-builders in the Indo-West Pacific (Yeemin et al., 2009). We applied both short-read Ion S5 RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate transcriptome sequences of P. lutea under normal and heat stress conditions. The key advantage of PacBio’s Iso-seq technology lies within its ability to capture full-length mRNA sequences. These full-length transcripts enable the identification of novel genes/isoforms and the detection of alternative splice variants, which have been shown to be overrepresented in stress responses (Iida et al., 2004; Reddy et al., 2013; Liu and Guo, 2017). We envision that this reference transcriptome will provide a coral research community a valuable resource for investigating changes in gene expression under various biotic/abiotic stress conditions.

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