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Asset Tag: Whole Genome Sequencing,

July 19, 2019

Winding paths to simplicity: genome evolution in facultative insect symbionts.

Symbiosis between organisms is an important driving force in evolution. Among the diverse relationships described, extensive progress has been made in insect-bacteria symbiosis, which improved our understanding of the genome evolution in host-associated bacteria. Particularly, investigations on several obligate mutualists have pushed the limits of what we know about the minimal genomes for sustaining cellular life. To bridge the gap between those obligate symbionts with extremely reduced genomes and their non-host-restricted ancestors, this review focuses on the recent progress in genome characterization of facultative insect symbionts. Notable cases representing various types and stages of host associations, including those from multiple genera in the family Enterobacteriaceae (class Gammaproteobacteria), Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), are discussed. Although several general patterns of genome reduction associated with the adoption of symbiotic relationships could be identified, extensive variation was found among these facultative symbionts. These findings are incorporated into the established conceptual frameworks to develop a more detailed evolutionary model for the discussion of possible trajectories. In summary, transitions from facultative to obligate symbiosis do not appear to be a universal one-way street; switches between hosts and lifestyles (e.g. commensalism, parasitism or mutualism) occur frequently and could be facilitated by horizontal gene transfer. © FEMS 2016.

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July 19, 2019

De novo assembly and phasing of a Korean human genome.

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9?Mb and a scaffold N50 size of 44.8?Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03?Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6?Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

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July 19, 2019

Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.

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July 19, 2019

Multiple origins of the pathogenic yeast Candida orthopsilosis by separate hybridizations between two parental species.

Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.

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July 19, 2019

IncFIIk plasmid harbouring an amplification of 16S rRNA methyltransferase-encoding gene rmtH associated with mobile element ISCR2.

To investigate the resistance mechanisms and genetic support underlying the high resistance level of the Klebsiella pneumoniae strain CMUL78 to aminoglycoside and ß-lactam antibiotics.Antibiotic susceptibility was assessed by the disc diffusion method and MICs were determined by the microdilution method. Antibiotic resistance genes and their genetic environment were characterized by PCR and Sanger sequencing. Plasmid contents were analysed in the clinical strain and transconjugants obtained by mating-out assays. Complete plasmid sequencing was performed with PacBio and Illumina technology.Strain CMUL78 co-produced the 16S rRNA methyltransferase (RMTase) RmtH, carbapenemase OXA-48 and ESBL SHV-12. The rmtH- and blaSHV-12-encoding genes were harboured by a novel ~115 kb IncFIIk plasmid designated pRmtH, and blaOXA-48 by a ~62 kb IncL/M plasmid related to pOXA-48a. pRmtH plasmid possessed seven different stability modules, one of which is a novel hybrid toxin-antitoxin system. Interestingly, pRmtH plasmid harboured a 4-fold amplification of an rmtH-ISCR2 unit arranged in tandem and inserted within a novel IS26-based composite transposon designated Tn6329.This is the first known report of the 16S RMTase-encoding gene rmtH in a plasmid. The rmtH-ISCR2 unit was inserted in a composite transposon as a 4-fold tandem repeat, a scarcely reported organization.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 19, 2019

Ribbon: Visualizing complex genome alignments and structural variation

Visualization has played an extremely important role in the current genomic revolution to inspect and understand variants, expression patterns, evolutionary changes, and a number of other relationships. However, most of the information in read-to-reference or genome-genome alignments is lost for structural variations in the one-dimensional views of most genome browsers showing only reference coordinates. Instead, structural variations captured by long reads or assembled contigs often need more context to understand, including alignments and other genomic information from multiple chromosomes. We have addressed this problem by creating Ribbon (genomeribbon.com) an interactive online visualization tool that displays alignments along both reference and query sequences, along with any associated variant calls in the sample. This way Ribbon shows patterns in alignments of many reads across multiple chromosomes, while allowing detailed inspection of individual reads (Supplementary Note 1). For example, here we show a gene fusion in the SK-BR-3 breast cancer cell line linking the genes CYTH1 and EIF3H. While it has been found in the transcriptome previously, genome sequencing did not identify a direct chromosomal fusion between these two genes. After SMRT sequencing, Ribbon shows that there are indeed long reads that span from one gene to the other, going through not one but two variants, for the first time showing the genomic link between these two genes (Figure 1a). More gene fusions of this cancer cell line are investigated in Supplementary Note 2. Figure 1b shows another complex event in this sample made simple in Ribbon: the translocation of a 4.4 kb sequence deleted from chr19 and inserted into chr16 (Figure 1b). Thus, Ribbon enables understanding of complex variants, and it may also help in the detection of sequencing and sample preparation issues, testing of aligners and variant-callers, and rapid curation of structural variant candidates (Supplementary Note 3). In addition to SAM and BAM files with long, short, or paired-end reads, Ribbon can also load coordinate files from whole genome aligners such as MUMmer. Therefore, Ribbon can be used to test assembly algorithms or inspect the similarity between species. Supplementary Note 4 shows a comparison of gorilla and human genomes using Ribbon, highlighting major structural differences. In conclusion, Ribbon is a powerful interactive web tool for viewing complex genomic alignments.

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July 19, 2019

Rapid functional and sequence differentiation of a tandemly repeated species-specific multigene family in Drosophila.

Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, our understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated, and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, we studied the organization, sequence evolution, and functional attributes of the different Sdic copies. By leveraging long-read sequencing data, we uncovered both copy number and order differences from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, we also detected signatures of two episodes of diversifying selection, which have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. Our results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 19, 2019

The establishment and diversification of epidemic-associated serogroup W meningococcus in the African meningitis belt, 1994 to 2012.

Epidemics of invasive meningococcal disease (IMD) caused by meningococcal serogroup A have been eliminated from the sub-Saharan African so-called “meningitis belt” by the meningococcal A conjugate vaccine (MACV), and yet, other serogroups continue to cause epidemics. Neisseria meningitidis serogroup W remains a major cause of disease in the region, with most isolates belonging to clonal complex 11 (CC11). Here, the genetic variation within and between epidemic-associated strains was assessed by sequencing the genomes of 92 N. meningitidis serogroup W isolates collected between 1994 and 2012 from both sporadic and epidemic IMD cases, 85 being from selected meningitis belt countries. The sequenced isolates belonged to either CC175 (n = 9) or CC11 (n = 83). The CC11 N. meningitidis serogroup W isolates belonged to a single lineage comprising four major phylogenetic subclades. Separate CC11 N. meningitidis serogroup W subclades were associated with the 2002 and 2012 Burkina Faso epidemics. The subclade associated with the 2012 epidemic included isolates found in Burkina Faso and Mali during 2011 and 2012, which descended from a strain very similar to the Hajj (Islamic pilgrimage to Mecca)-related Saudi Arabian outbreak strain from 2000. The phylogeny of isolates from 2012 reflected their geographic origin within Burkina Faso, with isolates from the Malian border region being closely related to the isolates from Mali. Evidence of ongoing evolution, international transmission, and strain replacement stresses the importance of maintaining N. meningitidis surveillance in Africa following the MACV implementation. IMPORTANCE Meningococcal disease (meningitis and bloodstream infections) threatens millions of people across the meningitis belt of sub-Saharan Africa. A vaccine introduced in 2010 protects against Africa’s then-most common cause of meningococcal disease, N. meningitidis serogroup A. However, other serogroups continue to cause epidemics in the region-including serogroup W. The rapid identification of strains that have been associated with prior outbreaks can improve the assessment of outbreak risk and enable timely preparation of public health responses, including vaccination. Phylogenetic analysis of newly sequenced serogroup W strains isolated from 1994 to 2012 identified two groups of strains linked to large epidemics in Burkina Faso, one being descended from a strain that caused an outbreak during the Hajj pilgrimage in 2000. We find that applying whole-genome sequencing to meningococcal disease surveillance collections improves the discrimination among strains, even within a single nation-wide epidemic, which can be used to better understand pathogen spread.

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July 19, 2019

Methylome analysis of two Xanthomonas spp. using Single-Molecule Real-Time Sequencing.

Single-molecule real-time (SMRT) sequencing allows identification of methylated DNA bases and methylation patterns/motifs at the genome level. Using SMRT sequencing, diverse bacterial methylomes including those of Helicobacter pylori, Lactobacillus spp., and Escherichia coli have been determined, and previously unreported DNA methylation motifs have been identified. However, the methylomes of Xanthomonas species, which belong to the most important plant pathogenic bacterial genus, have not been documented. Here, we report the methylomes of Xanthomonas axonopodis pv. glycines (Xag) strain 8ra and X. campestris pv. vesicatoria (Xcv) strain 85-10. We identified N(6)-methyladenine (6mA) and N(4)-methylcytosine (4mC) modification in both genomes. In addition, we assigned putative DNA methylation motifs including previously unreported methylation motifs via REBASE and MotifMaker, and compared methylation patterns in both species. Although Xag and Xcv belong to the same genus, their methylation patterns were dramatically different. The number of 4mC DNA bases in Xag (66,682) was significantly higher (29 fold) than in Xcv (2,321). In contrast, the number of 6mA DNA bases (4,147) in Xag was comparable to the number in Xcv (5,491). Strikingly, there were no common or shared motifs in the 10 most frequently methylated motifs of both strains, indicating they possess unique species- or strain-specific methylation motifs. Among the 20 most frequent motifs from both strains, for 9 motifs at least 1% of the methylated bases were located in putative promoter regions. Methylome analysis by SMRT sequencing technology is the first step toward understanding the biology and functions of DNA methylation in this genus.

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July 19, 2019

Mechanisms of evolution in high-consequence drug resistance plasmids.

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as transposons and integrons have been strongly associated with the rapid spread of genes responsible for antibiotic resistance. Understanding the consequences of their actions allowed us to establish unambiguous evolutionary relationships between plasmids in the analysis set. Copyright © 2016 He et al.

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July 19, 2019

Comprehensive genome analysis of carbapenemase-producing Enterobacter spp.: new insights into phylogeny, population structure and resistance mechanisms.

Knowledge regarding the genomic structure of Enterobacter spp., the second most prevalent carbapenemase-producing Enterobacteriaceae, remains limited. Here we sequenced 97 clinical Enterobacter species isolates that were both carbapenem susceptible and resistant from various geographic regions to decipher the molecular origins of carbapenem resistance and to understand the changing phylogeny of these emerging and drug-resistant pathogens. Of the carbapenem-resistant isolates, 30 possessed blaKPC-2, 40 had blaKPC-3, 2 had blaKPC-4, and 2 had blaNDM-1 Twenty-three isolates were carbapenem susceptible. Six genomes were sequenced to completion, and their sizes ranged from 4.6 to 5.1 Mbp. Phylogenomic analysis placed 96 of these genomes, 351 additional Enterobacter genomes downloaded from NCBI GenBank, and six newly sequenced type strains into 19 phylogenomic groups-18 groups (A to R) in the Enterobacter cloacae complex and Enterobacter aerogenes Diverse mechanisms underlying the molecular evolutionary trajectory of these drug-resistant Enterobacter spp. were revealed, including the acquisition of an antibiotic resistance plasmid, followed by clonal spread, horizontal transfer of blaKPC-harboring plasmids between different phylogenomic groups, and repeated transposition of the blaKPC gene among different plasmid backbones. Group A, which comprises multilocus sequence type 171 (ST171), was the most commonly identified (23% of isolates). Genomic analysis showed that ST171 isolates evolved from a common ancestor and formed two different major clusters; each acquiring unique blaKPC-harboring plasmids, followed by clonal expansion. The data presented here represent the first comprehensive study of phylogenomic interrogation and the relationship between antibiotic resistance and plasmid discrimination among carbapenem-resistant Enterobacter spp., demonstrating the genetic diversity and complexity of the molecular mechanisms driving antibiotic resistance in this genus.Enterobacter spp., especially carbapenemase-producing Enterobacter spp., have emerged as a clinically significant cause of nosocomial infections. However, only limited information is available on the distribution of carbapenem resistance across this genus. Augmenting this problem is an erroneous identification of Enterobacter strains because of ambiguous typing methods and imprecise taxonomy. In this study, we used a whole-genome-based comparative phylogenetic approach to (i) revisit and redefine the genus Enterobacter and (ii) unravel the emergence and evolution of the Klebsiella pneumoniae carbapenemase-harboring Enterobacter spp. Using genomic analysis of 447 sequenced strains, we developed an improved understanding of the species designations within this complex genus and identified the diverse mechanisms driving the molecular evolution of carbapenem resistance. The findings in this study provide a solid genomic framework that will serve as an important resource in the future development of molecular diagnostics and in supporting drug discovery programs. Copyright © 2016 Chavda et al.

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July 19, 2019

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice.

The related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon-like population, referred to as Taxon A, and O. meridionalis-like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short- and long-read next-generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement.© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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July 19, 2019

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

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July 19, 2019

The impact of third generation genomic technologies on plant genome assembly.

Since the introduction of next generation sequencing, plant genome assembly projects do not need to rely on dedicated research facilities or community-wide consortia anymore, even individual research groups can sequence and assemble the genomes they are interested in. However, such assemblies are typically not based on the entire breadth of genomic technologies including genetic and physical maps and their contiguities tend to be low compared to the full-length gold standard reference sequences. Recently emerging third generation genomic technologies like long-read sequencing or optical mapping promise to bridge this quality gap and enable simple and cost-effective solutions for chromosomal-level assemblies.

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July 19, 2019

Chromosomal integration of the Klebsiella pneumoniae carbapenemase gene, blaKPC, in Klebsiella species is elusive but not rare.

Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-?I-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated. Copyright © 2017 Mathers et al.

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