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Asset Tag: P6-C4 chemistry

April 21, 2020

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (~1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. © 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020

Vaccine-induced protection from homologous tier 2 SHIV challenge in nonhuman primates depends on serum-neutralizing antibody titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ~1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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April 21, 2020

Cytotoxic and Antibacterial Cervinomycins B1-4 from a Streptomyces Species.

AntiSMASH analysis of genome DNA of Streptomyces CPCC 204980, a soil isolate with potent antibacterial activity, revealed a gene cluster for polycyclic xanthones. A subsequent chemical study confirmed that the microorganism produced polycyclic xanthone cervinomycin A2 (1) and the new congeners cervinomycins B1-4 (2-5). The structures of 1-5 were determined by comprehensive analyses of MS and NMR data, which indicated that 2-5 featured a common dihydro-D ring in the polycyclic xanthone core moiety of their molecules. 2-5 are toxic to human cancer cells and active against Gram-positive bacteria.

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April 21, 2020

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

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April 21, 2020

Characterization of a novel, type II staphylococcal cassette chromosome mec element from an endemic oxacillin-resistant Staphylococcus lugdunensis clone in a hospital setting.

Staphylococcus lugdunensis is a significant pathogen that causes community-acquired and nosocomial infections. The high prevalence of oxacillin-resistant S. lugdunensis (ORSL) is of major concern. Resistance to ß-lactams is caused by acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. The cassette is highly diverse, both structurally and genetically, among CoNS. Isolates carrying SCCmec II-ST6 are the major persistent clones in hospitals.To investigate the structure and evolutionary origin of a novel type II SCCmec element in an endemic ST6 S. lugdunensis clone.The structure of the SCCmec II element carried by ST6 strain CGMH-SL118 was determined by WGS and compared with those reported previously.A novel 39 kb SCCmec element, SCCmecCGMH-SL118, with a unique mosaic structure comprising 41 ORFs integrated into the 3′ end of the rlmH gene, was observed. Some regions of SCCmecCGMH-SL118 were homologous to SCCmec IIa of the prototype MRSA strain N315. The structure of SCCmecCGMH-SL118 was similar to that of SCCmec IIb of the MRSA strain, JCSC3063, mainly lacking the aminoglycoside resistance determinant pUB110 in the J3 region but containing the insertion sequence IS256 in the J2 region. Notably, SCCmecCGMH-SL118 deletions in the J1 region compared with SCCmec types IIa and IIb, and a high homology to SCCmec elements of Staphylococcus aureus JCSC4610 and Staphylococcus haemolyticus strain 621 were found.The genetic diversity of the type II SCCmec element in ORSL suggests that CoNS is a potential reservoir for interspecies transfer of SCCmec to S. aureus in hospitals. © The Author(s) 2019. 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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April 21, 2020

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. © 2018 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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April 21, 2020

Mate Selection in Self-Compatible Wild Tobacco Results from Coordinated Variation in Homologous Self-Incompatibility Genes.

In flowering plants, intraspecific mate preference is frequently related to mating systems: the rejection of self pollen in self-incompatible (SI) plants that prevents inbreeding is one of the best described examples. However, in other mating systems, more nuanced patterns of pollen rejection occur. In the self-compatible (SC) Nicotiana attenuata, in which SI is not found and all crosses are compatible, certain pollen genotypes are consistently selected in mixed pollinations. However, the molecular mechanisms of this polyandrous mate selection remain unknown. Style-expressed NaS-like-RNases and pollen-expressed NaSLF-like genes, homologous to SI factors in Solanaceae, were identified and examined for a role in N. attenuata’s mate selection. A comparison of two NaS-like-RNases and six NaSLF-like genes among 26 natural accessions revealed specific combinations of co-expression and direct protein-protein interactions. To evaluate their role in mate selection, we silenced the expression of specific NaS-like-RNases and NaSLF-like proteins and conducted diagnostic binary mixed pollinations and mixed pollinations with 14 different non-self pollen donors. Styles expressing particular combinations of NaS-like-RNases selected mates from plants with corresponding NaS-like-RNase expression patterns, while styles lacking NaS-like-RNase expression were non-selective in their fertilizations, which reflected the genotype ratios of pollen mixtures deposited on the stigmas. DNA methylation could account for some of the observed variation in stylar NaS-like-RNase patterns. We conclude that the S-RNase-SLF recognition mechanism plays a central role in polyandrous mate selection in this self-compatible species. These results suggest that after the SI-SC transition, natural variation of SI homologous genes was repurposed to mediate intraspecific mate selection. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Full-Length Transcriptome Analysis of the Genes Involved in Tocopherol Biosynthesis in Torreya grandis.

The seeds of Torreya grandis (Cephalotaxaceae) are rich in tocopherols, which are essential components of the human diet as a result of their function in scavenging reactive oxygen and free radicals. Different T. grandis cultivars (10 cultivars selected in this study were researched, and their information is shown in Table S1 of the Supporting Information) vary enormously in their tocopherol contents (0.28-11.98 mg/100 g). However, little is known about the molecular basis and regulatory mechanisms of tocopherol biosynthesis in T. grandis kernels. Here, we applied single-molecule real-time (SMRT) sequencing to T. grandis (X08 cultivar) for the first time and obtained a total of 97?211 full-length transcripts. We proposed the biosynthetic pathway of tocopherol and identified eight full-length transcripts encoding enzymes potentially involved in tocopherol biosynthesis in T. grandis. The results of the correlation analysis between the tocopherol content and gene expression level in the 10 selected cultivars and different kernel developmental stages of the X08 cultivar suggested that homogentisate phytyltransferase coding gene ( TgVTE2b) and ?-tocopherol methyltransferase coding gene ( TgVTE4) may be key players in tocopherol accumulation in the kernels of T. grandis. Subcellular localization assays showed that both TgVTE2b and TgVTE4 were localized to the chloroplast. We also identified candidate regulatory genes similar to WRI1 and DGAT1 in Arabidopsis that may be involved in the regulation of tocopherol biosynthesis. Our findings provide valuable genetic information for T. grandis using full-length transcriptomic analysis, elucidating the candidate genes and key regulatory genes involved in tocopherol biosynthesis. This information will be critical for further molecular-assisted screening and breeding of T. grandis genotypes with high tocopherol contents.

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April 21, 2020

DNA methylation analysis.

DNA methylation is a process by which methyl groups are added to cytosine or adenine. DNA methylation can change the activity of the DNA molecule without changing the sequence. Methylation of 5-methylcytosine (5mC) is widespread in both eukaryotes and prokaryotes, and it is a very important epigenetic modification event, which can regulate gene activity and influence a number of key processes such as genomic imprinting, cell differentiation, transcriptional regulation, and chromatin remodeling. Profiling DNA methylation across the genome is critical to understanding the influence of methylation in normal biology and diseases including cancer. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC), and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the methylation regulation. Genome-wide analyses such as microarrays and next-generation sequencing technologies have been used to assess large fractions of the methylome. A number of different quantitative approaches have also been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing, pyrosequencing etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this chapter is to provide the methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.

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April 21, 2020

Clonal expansion and spread of the ceftriaxone-resistant Neisseria gonorrhoeae strain FC428, identified in Japan in 2015, and closely related isolates.

Ceftriaxone resistance in Neisseria gonorrhoeae is a major public health concern globally because a high-dose (1?g) injection of ceftriaxone is the only remaining option for empirical monotherapy of gonorrhoea. The ceftriaxone-resistant gonococcal strain FC428, cultured in Osaka in 2015, is suspected to have spread nationally and internationally. We describe the complete finished genomes of FC428 and two closely related isolates from Osaka in 2015, and examine the genomic epidemiology of these isolates plus three ceftriaxone-resistant gonococcal isolates from Osaka and Hyogo in 2016-17 and four ceftriaxone-resistant gonococcal isolates cultured in 2017 in Australia, Canada and Denmark.During 2015-17, we identified six ceftriaxone-resistant gonococcal isolates through our surveillance systems in Kyoto, Osaka and Hyogo. Antimicrobial susceptibility testing (six antimicrobials) was performed using Etest. Complete whole-genome sequences of the first three isolates (FC428, FC460 and FC498) from 2015 were obtained using PacBio RS II and Illumina MiSeq sequencing. The three complete genome sequences and draft genome sequences of the three additional Japanese (sequenced with Illumina MiSeq) and four international ceftriaxone-resistant isolates were compared.Detailed genomic analysis suggested that the Japanese isolates (FC428, FC460, FC498, KU16054, KM383 and KU17039) and the four international MLST ST1903 isolates from Australia, Canada and Denmark formed four linked subclades.Using detailed genomic analysis, we describe the clonal expansion of the ceftriaxone-resistant N. gonorrhoeae strain FC428, initially identified in 2015 in Japan, and closely related isolates. FC428 and its close relatives show some genomic diversity, suggesting multiple genetic subclades are already spreading internationally. © The Author(s) 2019. 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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April 21, 2020

The Genome of C57BL/6J “Eve”, the Mother of the Laboratory Mouse Genome Reference Strain.

Isogenic laboratory mouse strains enhance reproducibility because individual animals are genetically identical. For the most widely used isogenic strain, C57BL/6, there exists a wealth of genetic, phenotypic, and genomic data, including a high-quality reference genome (GRCm38.p6). Now 20 years after the first release of the mouse reference genome, C57BL/6J mice are at least 26 inbreeding generations removed from GRCm38 and the strain is now maintained with periodic reintroduction of cryorecovered mice derived from a single breeder pair, aptly named Adam and Eve. To provide an update to the mouse reference genome that more accurately represents the genome of today’s C57BL/6J mice, we took advantage of long read, short read, and optical mapping technologies to generate a de novo assembly of the C57BL/6J Eve genome (B6Eve). Using these data, we have addressed recurring variants observed in previous mouse genomic studies. We have also identified structural variations, closed gaps in the mouse reference assembly, and revealed previously unannotated coding sequences. This B6Eve assembly explains discrepant observations that have been associated with GRCm38-based analyses, and will inform a reference genome that is more representative of the C57BL/6J mice that are in use today.Copyright © 2019 Sarsani et al.

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April 21, 2020

Microbial community and fermentation characteristic of Italian ryegrass silage prepared with corn stover and lactic acid bacteria.

The bacterial community determined via PacBio single molecule, real-time sequencing technology (SMRT) and the fermentation characteristics of Italian ryegrass (IR, 82% moisture) silage prepared with corn stover (CS) were investigated. A selected strain of Lactobacillus plantarum (L694) and a commercial inoculant stain of Lactobacillus plantarum (LP) were used as additives. Lactic acid bacteria (LAB) effectively improved silage quality. After fermentation, Lactobacillus plantarum was the dominant species in IR?+?LP and IR?+?L694 treatments, which led to higher (P?

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April 21, 2020

Characterization of Mauritian cynomolgus macaque Fc?R alleles using long-read sequencing.

The Fc?Rs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. Fc?Rs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize Fc?R diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM Fc?R genes. We also developed a high-throughput Fc?R genotyping assay, which we used to determine allele frequencies and identify Fc?R haplotypes in more than 500 additional MCMs. We found three alleles for Fc?R1A, seven each for Fc?R2A and Fc?R2B, and four for Fc?R3A; these segregate into eight haplotypes. We also assessed whether different Fc?R alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of Fc?R responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies. Copyright © 2018 by The American Association of Immunologists, Inc.

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