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Auto Tag: Methylation

April 21, 2020

Convergent horizontal gene transfer and cross-talk of mobile nucleic acids in parasitic plants.

Horizontal gene transfer (HGT), the movement and genomic integration of DNA across species boundaries, is commonly associated with bacteria and other microorganisms, but functional HGT (fHGT) is increasingly being recognized in heterotrophic parasitic plants that obtain their nutrients and water from their host plants through direct haustorial feeding. Here, in the holoparasitic stem parasite Cuscuta, we identify 108?transcribed and probably functional HGT events in Cuscuta campestris and related species, plus 42?additional regions with host-derived transposon, pseudogene and non-coding sequences. Surprisingly, 18?Cuscuta fHGTs were acquired from the same gene families by independent HGT events in Orobanchaceae parasites, and the majority are highly expressed in the haustorial feeding structures in both lineages. Convergent retention and expression of HGT sequences suggests an adaptive role for specific additional genes in parasite biology. Between 16 and 20 of the transcribed HGT events are inferred as ancestral in Cuscuta based on transcriptome sequences from species across the phylogenetic range of the genus, implicating fHGT in the successful radiation of Cuscuta parasites. Genome sequencing of C. campestris supports transfer of genomic DNA-rather than retroprocessed RNA-as the mechanism of fHGT. Many of the C. campestris genes horizontally acquired are also frequent sources of 24-nucleotide small RNAs that are typically associated with RNA-directed DNA methylation. One HGT encoding a leucine-rich repeat protein kinase overlaps with a microRNA that has been shown to regulate host gene expression, suggesting that HGT-derived parasite small RNAs may function in the parasite-host interaction. This study enriches our understanding of HGT by describing a parasite-host system with unprecedented gene exchange that points to convergent evolution of HGT events and the functional importance of horizontally transferred coding and non-coding sequences.

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April 21, 2020

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the ~2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

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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

The use of Online Tools for Antimicrobial Resistance Prediction by Whole Genome Sequencing in MRSA and VRE.

The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly-available web-based AMR databases, whole genome sequencing (WGS) offers the capacity for rapid detection of antibiotic resistance genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) clinical isolates using publicly-available tools and databases.Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. Antibiotic resistance gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between WGS-predicted resistance profile and phenotypic susceptibility as well as sensitivity, specificity, positive and negative predictive values (NPV, PPV) were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard.Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3% with a sensitivity, specificity, PPV, NPV of 98.7% (95% CI, 97.2-99.5%), 99.6% (95 % CI, 98.8-99.9%), 99.3% (95% CI, 98.0-99.8%), 99.2% (95% CI, 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4% and the sensitivity to 99.3% (95% CI, 98.2-99.8%) and NPV to 99.4% (95% CI, 98.4-99.8%).WGS can be used as a reliable predicator of phenotypic resistance for both MRSA and VRE using readily-available online tools.Copyright © 2019. Published by Elsevier Ltd.

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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

High satellite repeat turnover in great apes studied with short- and long-read technologies.

Satellite repeats are a structural component of centromeres and telomeres, and in some instances their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50?bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: (1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and (2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males vs. females; using Y chromosome assemblies or FIuorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59?kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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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

Strengths and potential pitfalls of hay-transfer for ecological restoration revealed by RAD-seq analysis in floodplain Arabis species

Achieving high intraspecific genetic diversity is a critical goal in ecological restoration as it increases the adaptive potential and long-term resilience of populations. Thus, we investigated genetic diversity within and between pristine sites in a fossil floodplain and compared it to sites restored by hay-transfer between 1997 and 2014. RAD-seq genotyping revealed that the stenoecious flood-plain species Arabis nemorensis is co-occurring with individuals that, based on ploidy, ITS-sequencing and morphology, probably belong to the close relative Arabis sagittata, which has a documented preference for dry calcareous grasslands but has not been reported in floodplain meadows. We show that hay-transfer maintains genetic diversity for both species. Additionally, in A. sagittata, transfer from multiple genetically isolated pristine sites resulted in restored sites with increased diversity and admixed local genotypes. In A. nemorensis, transfer did not create novel admixture dynamics because genetic diversity between pristine sites was less differentiated. Thus, the effects of hay-transfer on genetic diversity also depend on the genetic makeup of the donor communities of each species, especially when local material is mixed. Our results demonstrate the efficiency of hay-transfer for habitat restoration and emphasize the importance of pre-restoration characterization of micro-geographic patterns of intraspecific diversity of the community to guarantee that restoration practices reach their goal, i.e. maximize the adaptive potential of the entire restored plant community. Overlooking these patterns may alter the balance between species in the community. Additionally, our comparison of summary statistics obtained from de novo and reference-based RAD-seq pipelines shows that the genomic impact of restoration can be reliably monitored in species lacking prior genomic knowledge.

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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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April 21, 2020

Genome Sequences and Methylation Patterns of Natrinema versiforme BOL5-4 and Natrinema pallidum BOL6-1, Two Extremely Halophilic Archaea from a Bolivian Salt Mine.

Two extremely halophilic archaea, namely, Natrinema versiforme BOL5-4 and Natrinema pallidum BOL6-1, were isolated from a Bolivian salt mine and their genomes sequenced using single-molecule real-time sequencing. The GC-rich genomes of BOL5-4 and BOL6-1 were 4.6 and 3.8 Mbp, respectively, with large chromosomes and multiple megaplasmids. Genome annotation was incorporated into HaloWeb and methylation patterns incorporated into REBASE.Copyright © 2019 DasSarma et al.

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