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July 19, 2019  |  

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

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July 19, 2019  |  

Detecting DNA modifications from SMRT sequencing data by modeling sequence context dependence of polymerase kinetic.

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.

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July 19, 2019  |  

The epigenomic landscape of prokaryotes.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

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July 19, 2019  |  

Separate F-type plasmids have shaped the evolution of the H30 subclone of Escherichia coli sequence type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

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July 19, 2019  |  

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

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July 19, 2019  |  

Revealing complete complex KIR haplotypes phased by long-read sequencing technology

The killer cell immunoglobulin-like receptor (KIR) region of human chromosome 19 contains up to 16 genes for natural killer (NK) cell receptors that recognize human leukocyte antigen (HLA)/peptide complexes and other ligands. The KIR proteins fulfill functional roles in infections, pregnancy, autoimmune diseases and transplantation. However, their characterization remains a constant challenge. Not only are the genes highly homologous due to their recent evolution by tandem duplications, but the region is structurally dynamic due to frequent transposon-mediated recombination. A sequencing approach that precisely captures the complexity of KIR haplotypes for functional annotation is desirable. We present a unique approach to haplotype the KIR loci using single-molecule, real-time (SMRT) sequencing. Using this method, we have—for the first time—comprehensively sequenced and phased sixteen KIR haplotypes from eight individuals without imputation. The information revealed four novel haplotype structures, a novel gene-fusion allele, novel and confirmed insertion/deletion events, a homozygous individual, and overall diversity for the structural haplotypes and their alleles. These KIR haplotypes augment our existing knowledge by providing high-quality references, evolutionary informers, and source material for imputation. The haplotype sequences and gene annotations provide alternative loci for the KIR region in the human genome reference GrCh38.p8.

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July 19, 2019  |  

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

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July 19, 2019  |  

Comparative genomic analyses of Clavibacter michiganensis subsp. insidiosus and pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,930 genes, with 401 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

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July 19, 2019  |  

A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium.

Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance ofMycobacterium aviumsubsp.avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, includingMycobacterium abscessusandMycobacterium chelonaeOverall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.IMPORTANCEAn extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTMrpoBgenes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance ofMycobacterium aviumsubsp.aviumincreased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species. Copyright © 2018 Haig et al.

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July 19, 2019  |  

A near-complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high interhaplotype diversity.

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis. Copyright © 2018 Schwessinger et al.

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July 19, 2019  |  

Single copy transgene integration in a transcriptionally active site for recombinant protein synthesis.

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019  |  

A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop.

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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July 7, 2019  |  

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii.

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Complete genome of Geobacter pickeringii G13T, a metal-reducing isolate from sedimentary kaolin deposits.

We used PacBio sequencing to assemble the genome of the pristine freshwater isolate Geobacter pickeringii G13(T) into a single 3,618,700-bp circular chromosome polished to 99.999% accuracy (quality value [QV], 50). This isolate shares several features with other Geobacter spp., including genes for degradation of aromatics and an abundance of multiheme c-type cytochromes. Copyright © 2015 Badalamenti and Bond.

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