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July 19, 2019

Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae.

Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics.

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July 19, 2019

Variable genetic architectures produce virtually identical molecules in bacterial symbionts of fungus-growing ants.

Small molecules produced by Actinobacteria have played a prominent role in both drug discovery and organic chemistry. As part of a larger study of the actinobacterial symbionts of fungus-growing ants, we discovered a small family of three previously unreported piperazic acid-containing cyclic depsipeptides, gerumycins A-C. The gerumycins are slightly smaller versions of dentigerumycin, a cyclic depsipeptide that selectively inhibits a common fungal pathogen, Escovopsis. We had previously identified this molecule from a Pseudonocardia associated with Apterostigma dentigerum, and now we report the molecule from an associate of the more highly derived ant Trachymyrmex cornetzi. The three previously unidentified compounds, gerumycins A-C, have essentially identical structures and were produced by two different symbiotic Pseudonocardia spp. from ants in the genus Apterostigma found in both Panama and Costa Rica. To understand the similarities and differences in the biosynthetic pathways that produced these closely related molecules, the genomes of the three producing Pseudonocardia were sequenced and the biosynthetic gene clusters identified. This analysis revealed that dramatically different biosynthetic architectures, including genomic islands, a plasmid, and the use of spatially separated genetic loci, can lead to molecules with virtually identical core structures. A plausible evolutionary model that unifies these disparate architectures is presented.

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July 19, 2019

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS. We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

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July 19, 2019

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.

Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits.PacBio SMRT sequencing was used to determine the whole genome seqeunces of two B. longum subsp. longum strains. The B. longum pan-genome was computed using PGAP v1.2 and the core B. longum phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0. M.blmNCII was cloned in E. coli and an internal fragment if arfBarfB was cloned into pORI19 for insertion mutagenesis.In this study we present the complete genome sequences of two Bifidobacterium longum subsp. longum strains. Comparative analysis with thirty one publicly available B. longum genomes allowed the definition of the B. longum core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the B. longum subspecies infantis, longum and suis. Furthermore, phylogenetic analysis of the B. longum core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of B. longum subsp. longum NCIMB 8809 to a level that was shown to permit site-directed mutagenesis.Comparative genomic analysis of thirty three B. longum representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the B. longum core genome also provides evidence for a novel fifth B. longum subspecies. Finally, we improved genetic accessibility for the strain B. longum subsp. longum NCIMB 8809, which allowed the generation of a mutant of this strain.

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July 19, 2019

SMRT Sequencing for parallel analysis of multiple targets and accurate SNP phasing.

Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses. Copyright © 2015 Guo et al.

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July 19, 2019

Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships.

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33-35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.

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July 19, 2019

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16?kilobases) reads with random errors, we assembled 99% (244?megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4?megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

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July 19, 2019

Fc? receptors: genetic variation, function, and disease.

Fc? receptors (Fc?Rs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal Fc?R engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the Fc?R gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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July 19, 2019

A supergene determines highly divergent male reproductive morphs in the ruff.

Three strikingly different alternative male mating morphs (aggressive ‘independents’, semicooperative ‘satellites’ and female-mimic ‘faeders’) coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

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July 19, 2019

Lineage-specific methyltransferases define the methylome of the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone. Copyright © 2015 Forde et al.

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July 19, 2019

Long-read Single-Molecule Real-Time (SMRT) full gene sequencing of cytochrome P450-2D6 (CYP2D6).

The CYP2D6 enzyme metabolizes ~25% of common medications, yet homologous pseudogenes and copy-number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single-molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run non-reference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement (CYP2D6*36+*41). Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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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. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

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July 19, 2019

DNA methylation assessed by SMRT Sequencing is linked to mutations in Neisseria meningitidis isolates.

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.

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July 19, 2019

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC’s genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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