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Friday, July 19, 2019

The power of Single Molecule Real-Time sequencing technology in the de novo assembly of a eukaryotic genome.

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented.…

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Friday, July 19, 2019

DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals…

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Friday, July 19, 2019

AgIn: Measuring the landscape of CpG methylation of individual repetitive elements.

Determining the methylation state of regions with high copy numbers is challenging for second-generation sequencing, because the read length is insufficient to map reads uniquely, especially when repetitive regions are long and nearly identical to each other. Single-molecule real-time (SMRT) sequencing is a promising method for observing such regions, because it is not vulnerable to GC bias, it produces long read lengths, and its kinetic information is sensitive to DNA modifications.We propose a novel linear-time algorithm that combines the kinetic information for neighboring CpG sites and increases the confidence in identifying the methylation states of those sites. Using a practical…

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Friday, July 19, 2019

Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases.

For the majority of congenital heart diseases (CHDs), the full complexity of the causative molecular network, which is driven by genetic, epigenetic, and environmental factors, is yet to be elucidated. Epigenetic alterations are suggested to play a pivotal role in modulating the phenotypic expression of CHDs and their clinical course during life. Candidate approaches implied that DNA methylation might have a developmental role in CHD and contributes to the long-term progress of non-structural cardiac diseases. The aim of the present study is to define the postnatal epigenome of two common cardiac malformations, representing epigenetic memory, and adaption to hemodynamic alterations,…

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Friday, July 19, 2019

CGG repeat-induced FMR1 silencing depends on the expansion size in human iPSCs and neurons carrying unmethylated full mutations.

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal…

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Friday, July 19, 2019

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast.

BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is…

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Friday, July 19, 2019

Widespread adenine N6-methylation of active genes in fungi.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start…

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Friday, July 19, 2019

Contrasting evolutionary genome dynamics between domesticated and wild yeasts.

Structural rearrangements have long been recognized as an important source of genetic variation, with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation enable precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus shows faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions), whereas S. cerevisiae…

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Friday, July 19, 2019

Centromere evolution and CpG methylation during vertebrate speciation.

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20-345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320-350 million years ago. The centromeres do not all evolve at a similar pace;…

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Friday, July 19, 2019

RNAi is a critical determinant of centromere evolution in closely related fungi.

The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient…

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Friday, July 19, 2019

Identification and analysis of adenine N6-methylation sites in the rice genome.

DNA N6-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes1-8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and…

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Friday, July 19, 2019

Prediction of smoking by multiplex bisulfite PCR with long amplicons considering allele-specific effects on DNA methylation.

Methylation of DNA is associated with a variety of biological processes. With whole-genome studies of DNA methylation, it became possible to determine a set of genomic sites where DNA methylation is associated with a specific phenotype. A method is needed that allows detailed follow-up studies of the sites, including taking into account genetic information. Bisulfite PCR is a natural choice for this kind of task, but multiplexing is one of the most important problems impeding its implementation. To address this task, we took advantage of a recently published method based on Pacbio sequencing of long bisulfite PCR products (single-molecule real-time…

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Sunday, July 7, 2019

It’s more than stamp collecting: how genome sequencing can unify biological research.

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, while the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods…

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Sunday, July 7, 2019

Diversity and evolution of centromere repeats in the maize genome.

Centromere repeats are found in most eukaryotes and play a critical role in kinetochore formation. Though centromere repeats exhibit considerable diversity both within and among species, little is understood about the mechanisms that drive centromere repeat evolution. Here, we use maize as a model to investigate how a complex history involving polyploidy, fractionation, and recent domestication has impacted the diversity of the maize centromeric repeat CentC. We first validate the existence of long tandem arrays of repeats in maize and other taxa in the genus Zea. Although we find considerable sequence diversity among CentC copies genome-wide, genetic similarity among repeats…

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Sunday, July 7, 2019

Tandem repeats in rodents genome and their mapping.

Tandemly-repeated sequences represent a unique class of eukaryotic DNA. Their content in the genome of higher eukaryotes mounts to tens of percents. However, the evolution of this class of sequences is poorly-studied. In our paper, 62 families of Mus musculus tandem repeats are analyzed by bioinformatic methods, and 7 of them are analyzed by fluorescence in situ hybridization. It is shown that the same tandem repeat sets co-occure only in closely related species of mice. But even in such species we observe differences in localization on the chromosomes and the number of individual tandem repeats. With increasing evolutionary distance only…

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