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July 19, 2019

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

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July 19, 2019

Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli.

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

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July 19, 2019

An incomplete understanding of human genetic variation.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies. Copyright © 2016 by the Genetics Society of America.

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July 19, 2019

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies. Copyright © 2016. Published by Elsevier B.V.

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July 19, 2019

Polymerase specific error rates and profiles identified by single molecule sequencing.

DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 19, 2019

Chromosomal-level assembly of the Asian seabass genome using long sequence reads and multi-layered scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

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July 19, 2019

Nested Russian doll-like genetic mobility drives rapid dissemination of the Carbapenem resistance gene blaKPC

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance. Copyright © 2016 Sheppard et al.

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July 19, 2019

Next generation sequencing of Actinobacteria for the discovery of novel natural products.

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects.

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July 19, 2019

Initial assessment of the molecular epidemiology of blaNDM-1 in Colombia.

We report complete genome sequences of fourblaNDM-1-harboring Gram-negative multidrug resistant (MDR) isolates from Colombia. TheblaNDM-1genes were located 193Kb-Inc FIA, 178Kb-Inc A/C2 and 47Kb (unknown Inc type) plasmids. MLST revealed that isolates belong to ST10 (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumanniiandA. nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid inE. colicontained a novel complex transposon (Tn125and Tn5393with 3 copies ofblaNDM-1) and a recombination “hotspot” for the acquisition of new resistance determinants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Integrating DNA methylation and gene expression data in the development of the soybean-Bradyrhizobium N2-fixing symbiosis.

Very little is known about the role of epigenetics in the differentiation of a bacterium from the free-living to the symbiotic state. Here genome-wide analysis of DNA methylation changes between these states is described using the model of symbiosis between soybean and its root nodule-forming, nitrogen-fixing symbiont, Bradyrhizobium diazoefficiens. PacBio resequencing of the B. diazoefficiens genome from both states revealed 43,061 sites recognized by five motifs with the potential to be methylated genome-wide. Of those sites, 3276 changed methylation states in 2921 genes or 35.5% of all genes in the genome. Over 10% of the methylation changes occurred within the symbiosis island that comprises 7.4% of the genome. The CCTTGAG motif was methylated only during symbiosis with 1361 adenosines methylated among the 1700 possible sites. Another 89 genes within the symbiotic island and 768 genes throughout the genome were found to have methylation and significant expression changes during symbiotic development. Of those, nine known symbiosis genes involved in all phases of symbiotic development including early infection events, nodule development, and nitrogenase production. These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state.

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July 19, 2019

Genome structural diversity among 31 Bordetella pertussis isolates from two recent U.S. whooping cough statewide epidemics

During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B.~pertussis populations.IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B.~pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B.~pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.

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July 19, 2019

Large deletions at the SHOX locus in the pseudoautosomal region are associated with skeletal atavism in Shetland ponies.

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure, impaired movements, and affected foals are usually euthanized. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160-180 kb and 60-80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development. Copyright © 2016 Author et al.

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July 19, 2019

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics.We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data.We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.

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July 19, 2019

Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, we investigated an autosome-Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10–15 Mya. We used single molecule real-time sequencing reads to assemble the D. pseudoobscura dot chromosome, including this Y-to-dot translocation. We find that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ~78 Kb and is not repeat-dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y-to-dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, we do not find a consistent reduction in intron sizes across the Y-to-dot region. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y-to-dot translocation.

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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 read coverage of ~30-fold from an inbred strain medaka (Oryzias latipes), we observed that both the sensitivity and precision of our method on individual CpG sites were ~93.7%. We also observed a high correlation coefficient (R?=?0.884) between our method and bisulfite sequencing, and for 92.0% of CpG sites, methylation levels ranging over [0, 1] were in concordance within an acceptable difference 0.25. Using this method, we characterized the landscape of the methylation status of repetitive elements, such as LINEs, in the human genome, thereby revealing the strong correlation between CpG density and hypomethylation and detecting hypomethylation hot spots of LTRs and LINEs. We uncovered the methylation states for nearly identical active transposons, two novel LINE insertions of identity ~99% and length 6050 base pairs (bp) in the human genome, and 16 Tol2 elements of identity >99.8% and length 4682?bp in the medaka genome.AgIn (Aggregate on Intervals) is available at: https://github.com/hacone/AgIn CONTACT: ysuzuki@cb.k.u-tokyo.ac.jp, moris@cb.k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. © The Author(s) 2016. Published by Oxford University Press.

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