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July 19, 2019

Quality control of the traditional patent medicine Yimu Wan based on SMRT Sequencing and DNA barcoding.

Substandard traditional patent medicines may lead to global safety-related issues. Protecting consumers from the health risks associated with the integrity and authenticity of herbal preparations is of great concern. Of particular concern is quality control for traditional patent medicines. Here, we establish an effective approach for verifying the biological composition of traditional patent medicines based on single-molecule real-time (SMRT) sequencing and DNA barcoding. Yimu Wan (YMW), a classical herbal prescription recorded in the Chinese Pharmacopoeia, was chosen to test the method. Two reference YMW samples were used to establish a standard method for analysis, which was then applied to three different batches of commercial YMW samples. A total of 3703 and 4810 circular-consensus sequencing (CCS) reads from two reference and three commercial YMW samples were mapped to the ITS2 and psbA-trnH regions, respectively. Moreover, comparison of intraspecific genetic distances based on SMRT sequencing data with reference data from Sanger sequencing revealed an ITS2 and psbA-trnH intergenic spacer that exhibited high intraspecific divergence, with the sites of variation showing significant differences within species. Using the CCS strategy for SMRT sequencing analysis was adequate to guarantee the accuracy of identification. This study demonstrates the application of SMRT sequencing to detect the biological ingredients of herbal preparations. SMRT sequencing provides an affordable way to monitor the legality and safety of traditional patent medicines.

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July 19, 2019

Reduction in chromosome mobility accompanies nuclear organization during early embryogenesis in Caenorhabditis elegans.

In differentiated cells, chromosomes are packed inside the cell nucleus in an organised fashion. In contrast, little is known about how chromosomes are packed in undifferentiated cells and how nuclear organization changes during development. To assess changes in nuclear organization during the earliest stages of development, we quantified the mobility of a pair of homologous chromosomal loci in the interphase nuclei of Caenorhabditis elegans embryos. The distribution of distances between homologous loci was consistent with a random distribution up to the 8-cell stage but not at later stages. The mobility of the loci was significantly reduced from the 2-cell to the 48-cell stage. Nuclear foci corresponding to epigenetic marks as well as heterochromatin and the nucleolus also appeared around the 8-cell stage. We propose that the earliest global transformation in nuclear organization occurs at the 8-cell stage during C. elegans embryogenesis.

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July 19, 2019

Detecting AGG interruptions in male and female FMR1 premutation carriers by single-molecule sequencing.

The FMR1 gene contains an unstable CGG repeat in its 5′ untranslated region. Premutation alleles range between 55 and 200 repeat units and confer a risk for developing fragile X-associated tremor/ataxia syndrome or fragile X-associated primary ovarian insufficiency. Furthermore, the premutation allele often expands to a full mutation during female germline transmission giving rise to the fragile X syndrome. The risk for a premutation to expand depends mainly on the number of CGG units and the presence of AGG interruptions in the CGG repeat. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. Here, we demonstrate that single-molecule sequencing enables the determination of not only the repeat size, but also the complete repeat sequence including AGG interruptions in male and female alleles with repeats ranging from 45 to 100 CGG units. We envision this method will facilitate research and diagnostic analysis of the FMR1 repeat expansion. © 2016 WILEY PERIODICALS, INC.

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July 19, 2019

The complete genome sequence of the phytopathogenic fungus Sclerotinia sclerotiorum reveals insights into the genome architecture of broad host range pathogens.

Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a “two-speed” genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit “two-speed” genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale.

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July 19, 2019

Defective HIV-1 proviruses are expressed and can be recognized by cytotoxic T lymphocytes, which shape the proviral landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4(+) T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 19, 2019

Discovery and biosynthesis of gladiolin: A Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

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July 19, 2019

A case study into microbial genome assembly gap sequences and finishing strategies.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

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July 19, 2019

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications.The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

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July 19, 2019

Monitoring microevolution of OXA-48-producing Klebsiella pneumoniae ST147 in a hospital setting by SMRT sequencing.

Carbapenemase-producing Klebsiella pneumoniae pose an increasing risk for healthcare facilities worldwide. A continuous monitoring of ST distribution and its association with resistance and virulence genes is required for early detection of successful K. pneumoniae lineages. In this study, we used WGS to characterize MDR blaOXA-48-positive K. pneumoniae isolated from inpatients at the University Medical Center Göttingen, Germany, between March 2013 and August 2014.Closed genomes for 16 isolates of carbapenemase-producing K. pneumoniae were generated by single molecule real-time technology using the PacBio RSII platform.Eight of the 16 isolates showed identical XbaI macrorestriction patterns and shared the same MLST, ST147. The eight ST147 isolates differed by only 1-25 SNPs of their core genome, indicating a clonal origin. Most of the eight ST147 isolates carried four plasmids with sizes of 246.8, 96.1, 63.6 and 61.0?kb and a novel linear plasmid prophage, named pKO2, of 54.6?kb. The blaOXA-48 gene was located on a 63.6?kb IncL plasmid and is part of composite transposon Tn1999.2. The ST147 isolates expressed the yersinabactin system as a major virulence factor. The comparative whole-genome analysis revealed several rearrangements of mobile genetic elements and losses of chromosomal and plasmidic regions in the ST147 isolates.Single molecule real-time sequencing allowed monitoring of the genetic and epigenetic microevolution of MDR OXA-48-producing K. pneumoniae and revealed in addition to SNPs, complex rearrangements of genetic elements.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 19, 2019

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats. We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntingtontextquoterights disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.

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July 19, 2019

De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii.

Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species.We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm.The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.

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July 19, 2019

The diversity, structure, and function of heritable adaptive immunity sequences in the Aedes aegypti genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 19, 2019

The Aegilops tauschii genome reveals multiple impacts of transposons.

Wheat is an important global crop with an extremely large and complex genome that contains more transposable elements (TEs) than any other known crop species. Here, we generated a chromosome-scale, high-quality reference genome of Aegilops tauschii, the donor of the wheat D genome, in which 92.5% sequences have been anchored to chromosomes. Using this assembly, we accurately characterized genic loci, gene expression, pseudogenes, methylation, recombination ratios, microRNAs and especially TEs on chromosomes. In addition to the discovery of a wave of very recent gene duplications, we detected that TEs occurred in about half of the genes, and found that such genes are expressed at lower levels than those without TEs, presumably because of their elevated methylation levels. We mapped all wheat molecular markers and constructed a high-resolution integrated genetic map corresponding to genome sequences, thereby placing previously detected agronomically important genes/quantitative trait loci (QTLs) on the Ae. tauschii genome for the first time.

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July 19, 2019

Genome and methylome variation in Helicobacter pylori with a cag pathogenicity island during early stages of human infection.

Helicobacter pylori is remarkable for its genetic variation. Yet little isknown about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori strain during experimental infection.We performed a randomized Phase 1 and 2, observer-blind, placebo-controlled, study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n=7) or placebo (n=5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 re-isolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag PAI function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays.The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss; of cag PAI function was observed in 3 re-isolates. In addition, 3 re-isolates from the vaccine; group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of; vacuolization activity from gastric epithelial cells. We observed inter-strain variation in; methylomes due to phase variation in genes encoding methyltransferases.We analyzed adaptation of a fully virulent strain of H pylori to 12 differentvolunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes inthe absence of inter-strain recombination. Our findings indicate that the large amount of; genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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