Menu

Asset Tag: P6-C4 chemistry

April 21, 2020

Complete genome sequence of acetate-producing Klebsiella pneumoniae L5-2 isolated from infant feces.

Acetate is an important metabolite in infants as it can affect metabolism as well as immune and inflammatory responses. However, there have been no studies on acetate production by Klebsiella pneumoniae isolated from infant feces. In this study, we isolated a K. pneumoniae strain, L5-2, from infant feces, and we found it produces acetate. The genome of L5-2 consisted of a 5,237,123-bp single chromosome and a 139,211-bp single plasmid. The G?+?C content was 57.27%. By whole-genome analysis of K. pneumoniae L5-2, we identified seven genes related to acetate production (poxA, pta, eutD, ackA, eutP, eutQ, and adhE). We confirmed acetate production by K. pneumoniae L5-2 by ion chromatography. The aldehyde/alcohol dehydrogenase (adhE) activity of K. pneumoniae L5-2 was significantly higher than that of the K. pneumoniae subsp. ozaenae ATCC 11296. Thus, the acetate-producing ability of K. pneumoniae L5-2 was influenced by the adhE gene. In addition, K. pneumoniae L5-2 had significantly less virulence factor-encoding genes than other K. pneumoniae strains isolated from humans. In conclusion, K. pneumoniae L5-2 isolated from infant feces has less virulence factors and higher adhE activity than other K. pneumoniae strains.

Read more
April 21, 2020

Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal.

Restriction site-associated DNA sequencing (RADseq) has revolutionized the study of wild organisms by allowing cost-effective genotyping of thousands of loci. However, for species lacking reference genomes, it can be challenging to select the restriction enzyme that offers the best balance between the number of obtained RAD loci and depth of coverage, which is crucial for a successful outcome. To address this issue, PredRAD was recently developed, which uses probabilistic models to predict restriction site frequencies from a transcriptome assembly or other sequence resource based on either GC content or mono-, di- or trinucleotide composition. This program generates predictions that are broadly consistent with estimates of the true number of restriction sites obtained through in silico digestion of available reference genome assemblies. However, in practice the actual number of loci obtained could potentially differ as incomplete enzymatic digestion or patchy sequence coverage across the genome might lead to some loci not being represented in a RAD dataset, while erroneous assembly could potentially inflate the number of loci. To investigate this, we used genome and transcriptome assemblies together with RADseq data from the Antarctic fur seal (Arctocephalus gazella) to compare PredRAD predictions with empirical estimates of the number of loci obtained via in silico digestion and from de novo assemblies.PredRAD yielded consistently higher predicted numbers of restriction sites for the transcriptome assembly relative to the genome assembly. The trinucleotide and dinucleotide models also predicted higher frequencies than the mononucleotide or GC content models. Overall, the dinucleotide and trinucleotide models applied to the transcriptome and the genome assemblies respectively generated predictions that were closest to the number of restriction sites estimated by in silico digestion. Furthermore, the number of de novo assembled RAD loci mapping to restriction sites was similar to the expectation based on in silico digestion.Our study reveals generally high concordance between PredRAD predictions and empirical estimates of the number of RAD loci. This further supports the utility of PredRAD, while also suggesting that it may be feasible to sequence and assemble the majority of RAD loci present in an organism’s genome.

Read more
April 21, 2020

The Anaplasma ovis genome reveals a high proportion of pseudogenes.

The genus Anaplasma is made up of organisms characterized by small genomes that are undergoing reductive evolution. Anaplasma ovis, one of the seven recognized species in this genus, is an understudied pathogen of sheep and other ruminants. This tick-borne agent is thought to induce only mild clinical disease; however, small deficits may add to larger economic impacts due to the wide geographic distribution of this pathogen.In this report we present the first complete genome sequence for A. ovis and compare the genome features with other closely related species. The 1,214,674?bp A. ovis genome encodes 933 protein coding sequences, the split operon arrangement for ribosomal RNA genes, and more pseudogenes than previously recognized for other Anaplasma species. The metabolic potential is similar to other Anaplasma species. Anaplasma ovis has a small repertoire of surface proteins and transporters. Several novel genes are identified.Analyses of these important features and significant gene families/genes with potential to be vaccine candidates are presented in a comparative context. The availability of this genome will significantly facilitate research for this pathogen.

Read more
April 21, 2020

Structural variation of centromeric endogenous retroviruses in human populations and their impact on cutaneous T-cell lymphoma, Sézary syndrome, and HIV infection.

Human Endogenous Retroviruses type K HML-2 (HK2) are integrated into 117 or more areas of human chromosomal arms while two newly discovered HK2 proviruses, K111 and K222, spread extensively in pericentromeric regions, are the first retroviruses discovered in these areas of our genome.We use PCR and sequencing analysis to characterize pericentromeric K111 proviruses in DNA from individuals of diverse ethnicities and patients with different diseases.We found that the 5′ LTR-gag region of K111 proviruses is missing in certain individuals, creating pericentromeric instability. K111 deletion (-/- K111) is seen in about 15% of Caucasian, Asian, and Middle Eastern populations; it is missing in 2.36% of African individuals, suggesting that the -/- K111 genotype originated out of Africa. As we identified the -/-K111 genotype in Cutaneous T-cell lymphoma (CTCL) cell lines, we studied whether the -/-K111 genotype is associated with CTCL. We found a significant increase in the frequency of detection of the -/-K111 genotype in Caucasian patients with severe CTCL and/or Sézary syndrome (n?=?35, 37.14%), compared to healthy controls (n?=?160, 15.6%) [p?=?0.011]. The -/-K111 genotype was also found to vary in HIV-1 infection. Although Caucasian healthy individuals have a similar frequency of detection of the -/- K111 genotype, Caucasian HIV Long-Term Non-Progressors (LTNPs) and/or elite controllers, have significantly higher detection of the -/-K111 genotype (30.55%; n?=?36) than patients who rapidly progress to AIDS (8.5%; n?=?47) [p?=?0.0097].Our data indicate that pericentromeric instability is associated with more severe CTCL and/or Sézary syndrome in Caucasians, and appears to allow T-cells to survive lysis by HIV infection. These findings also provide new understanding of human evolution, as the -/-K111 genotype appears to have arisen out of Africa and is distributed unevenly throughout the world, possibly affecting the severity of HIV in different geographic areas.

Read more
April 21, 2020

Sequencing a Juglans regia?×?J. microcarpa hybrid yields high-quality genome assemblies of parental species.

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes, which greatly complicates genome sequence assembly. The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species. We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa?×?J. regia using a novel combination of single-molecule sequencing and optical genome mapping technologies. The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions. Chromosome termini consisted of arrays of telomeric repeats about 8?kb long and heterochromatic subtelomeric regions about 10?kb long. The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes, many of them transcribed. Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes, which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes. Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other. The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes. Given the importance of J. microcarpa?×?J. regia hybrids as potential walnut rootstocks, we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution. We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

Read more
April 21, 2020

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551?bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress.Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

Read more
April 21, 2020

Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing

In recent genome analyses, population-specific reference panels have indicated important. However, reference panels based on short-read sequencing data do not sufficiently cover long insertions. Therefore, the nature of long insertions has not been well documented. Here, we assembled a Japanese genome using single-molecule real-time sequencing data and characterized insertions found in the assembled genome. We identified 3691 insertions ranging from 100?bps to ~10,000?bps in the assembled genome relative to the international reference sequence (GRCh38). To validate and characterize these insertions, we mapped short-reads from 1070 Japanese individuals and 728 individuals from eight other populations to insertions integrated into GRCh38. With this result, we constructed JRGv1 (Japanese Reference Genome version 1) by integrating the 903 verified insertions, totaling 1,086,173 bases, shared by at least two Japanese individuals into GRCh38. We also constructed decoyJRGv1 by concatenating 3559 verified insertions, totaling 2,536,870 bases, shared by at least two Japanese individuals or by six other assemblies. This assembly improved the alignment ratio by 0.4% on average. These results demonstrate the importance of refining the reference assembly and creating a population-specific reference genome. JRGv1 and decoyJRGv1 are available at the JRG website.

Read more
April 21, 2020

A comparative evaluation of hybrid error correction methods for error-prone long reads.

Third-generation sequencing technologies have advanced the progress of the biological research by generating reads that are substantially longer than second-generation sequencing technologies. However, their notorious high error rate impedes straightforward data analysis and limits their application. A handful of error correction methods for these error-prone long reads have been developed to date. The output data quality is very important for downstream analysis, whereas computing resources could limit the utility of some computing-intense tools. There is a lack of standardized assessments for these long-read error-correction methods.Here, we present a comparative performance assessment of ten state-of-the-art error-correction methods for long reads. We established a common set of benchmarks for performance assessment, including sensitivity, accuracy, output rate, alignment rate, output read length, run time, and memory usage, as well as the effects of error correction on two downstream applications of long reads: de novo assembly and resolving haplotype sequences.Taking into account all of these metrics, we provide a suggestive guideline for method choice based on available data size, computing resources, and individual research goals.

Read more
April 21, 2020

Complete Sequences of Multiple-Drug Resistant IncHI2 ST3 Plasmids in Escherichia coli of Porcine Origin in Australia

IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2 and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:- that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26.

Read more
October 23, 2019

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M.?mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-?glf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Read more
October 23, 2019

CRISPR/Cas9-generated p47(phox)-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47(phox)-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (?GT) mutation in p47(phox) encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47(phox)-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47(phox) CGD patients carries ?GT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ?GT cell line reflects the most frequent form of p47(phox)-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Read more
October 23, 2019

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus.This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

Read more
October 23, 2019

CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Read more
October 23, 2019

Development of a Novel Reference Transcriptome for Scleractinian Coral Porites lutea Using Single-Molecule Long-Read Isoform Sequencing (Iso-Seq)

Elevation in seawater temperature associated with global climate change has caused coral bleaching problems and posed a significant threat to coral health and survival worldwide. Several studies have explored the effects of thermal stress on changes in gene expression levels of both coral hosts and their algal endosymbionts and provided evidences suggesting that corals could acclimatize to environmental stressors through differential regulation of their gene expression (Desalvo et al., 2008, 2010; Császár et al., 2009; Rodriguez-Lanetty et al., 2009; Polato et al., 2010; Meyer et al., 2011; Kenkel et al., 2013). Such information is crucial for understanding the adaptive capacity of the coral holobionts (Hughes et al., 2003). The availability of transcriptome data from a number of coral species and their associated Symbiodinium allows us to probe the molecular stress response of the organisms to heat stress (Traylor-Knowles et al., 2011; Moya et al., 2012; Kenkel et al., 2013; Shinzato et al., 2014; Kitchen et al., 2015; Anderson et al., 2016; Davies et al., 2016). Here, we report the first reference transcriptome for a scleractinian coral Porites lutea, one of the dominant reef-builders in the Indo-West Pacific (Yeemin et al., 2009). We applied both short-read Ion S5 RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate transcriptome sequences of P. lutea under normal and heat stress conditions. The key advantage of PacBio’s Iso-seq technology lies within its ability to capture full-length mRNA sequences. These full-length transcripts enable the identification of novel genes/isoforms and the detection of alternative splice variants, which have been shown to be overrepresented in stress responses (Iida et al., 2004; Reddy et al., 2013; Liu and Guo, 2017). We envision that this reference transcriptome will provide a coral research community a valuable resource for investigating changes in gene expression under various biotic/abiotic stress conditions.

Read more
October 23, 2019

Bioengineered viral platform for intramuscular passive vaccine delivery to human skeletal muscle.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.