The draft genome sequence of Malassezia restricta KCTC 27527, a clinical isolate from a patient with dandruff, was previously reported. Using the PacBio Sequel platform, we completed and reannotated the genome of M. restricta KCTC 27527 for a better understanding of the genome of this fungus.Copyright © 2019 Cho et al.
Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.
Development of dental plaque begins with the adhesion of salivary bacteria to the acquired pellicle covering the tooth surface. In this study, we collected in vivo dental plaque formed on hydroxyapatite disks for 6 h from 74 young adults and identified initial colonizing taxa based on full-length 16S rRNA gene sequences. A long-read, single-molecule sequencer, PacBio Sequel, provided 100,109 high-quality full-length 16S rRNA gene sequence reads from the early plaque microbiota, which were assigned to 90 oral bacterial taxa. The microbiota obtained from every individual mostly comprised the 21 predominant taxa with the maximum relative abundance of over 10% (95.8?±?6.2%, mean ± SD), which included Streptococcus species as well as nonstreptococcal species. A hierarchical cluster analysis of their relative abundance distribution suggested three major patterns of microbiota compositions: a Streptococcus mitis/Streptococcus sp. HMT-423-dominant profile, a Neisseria sicca/Neisseria flava/Neisseria mucosa-dominant profile, and a complex profile with high diversity. No notable variations in the community structures were associated with the dental caries status, although the total bacterial amounts were larger in the subjects with a high number of caries-experienced teeth (=8) than in those with no or a low number of caries-experienced teeth. Our results revealed the bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults.IMPORTANCE Selective attachment of salivary bacteria to the tooth surface is an initial and repetitive phase in dental plaque development. We employed full-length 16S rRNA gene sequence analysis with a high taxonomic resolution using a third-generation sequencer, PacBio Sequel, to determine the bacterial composition during early plaque formation in 74 young adults accurately and in detail. The results revealed 21 bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults, which include several streptococcal species as well as nonstreptococcal species, such as Neisseria sicca/Nflava/Nmucosa and Rothia dentocariosa Given that no notable variations in the microbiota composition were associated with the dental caries status, the maturation process, rather than the specific bacterial species that are the initial colonizers, is likely to play an important role in the development of dysbiotic microbiota associated with dental caries. Copyright © 2019 Ihara et al.
Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly.This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.
Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
Cell-free DNA (cfDNA) fragments in maternal plasma contain DNA damage and may negatively impact the sensitivity of noninvasive prenatal testing (NIPT). However, some of these DNA damages are potentially reparable. We aimed to recover these damaged cfDNA molecules using PreCR DNA repair mix.cfDNA was extracted from 20 maternal plasma samples and was repaired and sequenced by the Illumina platform. Size profiles and fetal DNA fraction changes of repaired samples were characterized. Targeted sequencing of chromosome Y sequences was used to enrich fetal cfDNA molecules following repair. Single-molecule real-time (SMRT) sequencing platform was employed to characterize long (>250 bp) cfDNA molecules. NIPT of five trisomy 21 samples was performed.Size profiles of repaired libraries were altered, with significantly increased long (>250 bp) cfDNA molecules. Single nucleotide polymorphism (SNP)-based analyses showed that both fetal- and maternal-derived cfDNA molecules were enriched by the repair. Fetal DNA fractions in maternal plasma showed a small but consistent (4.8%) increase, which were contributed by a higher increment of long fetal cfDNA molecules. z-score values were improved in NIPT of all trisomy 21 samples.Plasma DNA repair recovers and enriches long cfDNA molecules of both fetal and maternal origins in maternal plasma. © 2018 John Wiley & Sons, Ltd.
The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.
Colwellia sp. Arc7-D, a psychrophilic H2O2-resisitant bacterium, was isolated from Arctic Ocean sediment. Here we describe the complete genome of Colwellia sp. Arc7-D. The genome has one circular chromosome of 4,305,442?bp (37.67?mol%?G?+?C content), consisting of 3526 coding genes, 77 tRNA genes, as well as five rRNA operons as 16S–23S-5S rRNA and one rRNA operon as 16S-23S-5S-5S. According to KEGG analysis, strain Arc7-D encodes 23 genes related with antioxidant activity including superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase. However, many additional genes affiliated with anti-oxidative stress were also identified, such as aconitase, thioredoxin and ascorbic acid.
Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity. Copyright © 2019 the Author(s). Published by PNAS.
We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions and three insertions) on the genes tagged with autosomal recessive phenotypes. Among them, a 12.4-kb deletion involving the CLN6 gene was the top candidate because its homozygous abnormalities cause neuronal ceroid lipofuscinosis. This deletion included the initiation codon and was found in a GC-rich region containing multiple repetitive elements. These results indicate the presence of a causal variant in a difficult-to-sequence region and suggest that such variants that remain enigmatic after the application of current whole-exome sequencing technology could be uncovered by unbiased application of long-read whole-genome sequencing.
Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.
The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the Midwestern United States, with both agricultural populations and herbicide resistance only recently emerging in Canada. To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the United States. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically nonagricultural. Moreover, resistance on these local, nonagricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the Midwestern United States show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.Copyright © 2019 the Author(s). Published by PNAS.
Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10?kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV selection of low-coverage WGS data (7×) narrowed down the candidates to only six SVs in a 7.16-Mb region of the BAFME1 locus and correctly determined an approximately 4.6-kb SAMD12 intronic repeat insertion, which is causal of BAFME1. These results indicate that long-read WGS is potentially useful for evaluating all of the known SVs in a genome and identifying new disease-causing SVs in combination with other genetic methods to resolve the genetic causes of currently unexplained diseases.
The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30°?N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.
Streptomyces species are known to produce various bioactive metabolites that can prevent plant diseases. Previously, the Streptomyces strain TFH56 was found to inhibit the gray mold pathogen, Botrytis cinerea, in tomato flower. In this study, the genome sequence of strain TFH56 was acquired using the Pacific Biosciences RS II platform. Three linear sequences (7.67 Mbp in total) were obtained. Based on average nucleotide identity, strain TFH56 was classified as Streptomyces globisporus, which is consistent with the presence of a linear chromosome and linear plasmids. Moreover, as with other examples of S. globisporus, the genome of strain TFH56 included a caryolan-1-ol synthase gene, a conprimycin synthetic gene cluster, and a lidamycin synthetic gene cluster.Copyright © 2019 Cho, Kwak.
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