Menu

Asset Tag: Human Biomedical Research,

July 7, 2019

Methylome diversification through changes in DNA methyltransferase sequence specificity.

Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement–the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

Read more
July 7, 2019

Genome sequences of 228 Shiga toxin-producing Escherichia coli isolates and 12 isolates representing other diarrheagenic E. coli pathotypes.

Shiga toxin-producing Escherichia coli (STEC) are a common cause for food-borne diarrheal illness outbreaks and sporadic cases. Here, we report the availability of the draft genome sequences of 228 STEC strains representing 32 serotypes with known pulsed-field gel electrophoresis (PFGE) types and epidemiological relationships, as well as 12 strains representing other diarrheagenic E. coli pathotypes. Copyright © 2014 Trees et al.

Read more
July 7, 2019

Complete genome sequences of Salmonella enterica serovar Heidelberg strains associated with a multistate food-borne illness investigation.

Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.

Read more
July 7, 2019

Genomic reconnaissance of clinical isolates of emerging human pathogen Mycobacterium abscessus reveals high evolutionary potential.

Mycobacterium abscessus (Ma) is an emerging human pathogen that causes both soft tissue infections and systemic disease. We present the first comparative whole-genome study of Ma strains isolated from patients of wide geographical origin. We found a high proportion of accessory strain-specific genes indicating an open, non-conservative pan-genome structure, and clear evidence of rapid phage-mediated evolution. Although we found fewer virulence factors in Ma compared to M. tuberculosis, our data indicated that Ma evolves rapidly and therefore should be monitored closely for the acquisition of more pathogenic traits. This comparative study provides a better understanding of Ma and forms the basis for future functional work on this important pathogen.

Read more
July 7, 2019

Molecular and biological characterization of a new isolate of guinea pig cytomegalovirus.

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland-adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.

Read more
July 7, 2019

A fault-tolerant method for HLA typing with PacBio data.

Human leukocyte antigen (HLA) genes are critical genes involved in important biomedical aspects, including organ transplantation, autoimmune diseases and infectious diseases. The gene family contains the most polymorphic genes in humans and the difference between two alleles is only a single base pair substitution in many cases. The next generation sequencing (NGS) technologies could be used for high throughput HLA typing but in silico methods are still needed to correctly assign the alleles of a sample. Computer scientists have developed such methods for various NGS platforms, such as Illumina, Roche 454 and Ion Torrent, based on the characteristics of the reads they generate. However, the method for PacBio reads was less addressed, probably owing to its high error rates. The PacBio system has the longest read length among available NGS platforms, and therefore is the only platform capable of having exon 2 and exon 3 of HLA genes on the same read to unequivocally solve the ambiguity problem caused by the “phasing” issue.We proposed a new method BayesTyping1 to assign HLA alleles for PacBio circular consensus sequencing reads using Bayes’ theorem. The method was applied to simulated data of the three loci HLA-A, HLA-B and HLA-DRB1. The experimental results showed its capability to tolerate the disturbance of sequencing errors and external noise reads.The BayesTyping1 method could overcome the problems of HLA typing using PacBio reads, which mostly arise from sequencing errors of PacBio reads and the divergence of HLA genes, to some extent.

Read more
July 7, 2019

Dubowitz syndrome is a complex comprised of multiple, genetically distinct and phenotypically overlapping disorders.

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings’ history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ~ 3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463-65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of = 1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.

Read more
July 7, 2019

Genome sequences of Corynebacterium pseudotuberculosis strains 48252 (human, pneumonia), CS_10 (lab strain), Ft_2193/ 67 (goat, pus), and CCUG 27541.

Here we report the genome sequencess of four Corynebacterium pseudotuberculosis strains. These include a strain isolated from a patient with C. pseudotuberculosis pneumonia (48252), a strain isolated from pus in goat (Ft_2193/67), a laboratory strain originating from strain Ft_2193/67 (CS_10), and the draft genome of an equine reference strain, CCUG 27541. Copyright © 2014 Håvelsrud et al.

Read more
July 7, 2019

First genome sequences of Achromobacter phages reveal new members of the N4 family.

Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far.Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP).In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages.Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.

Read more
July 7, 2019

Characterization of biological pathways associated with a 1.37 Mbp genomic region protective of hypertension in Dahl S rats.

The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood-pressure salt sensitivity in Dahl salt-sensitive (SS) rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high-salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) a linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups and 2) a Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na(+) and K(+) electrochemical gradients and tubular Na(+) transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms. Copyright © 2014 the American Physiological Society.

Read more
July 7, 2019

LUMPY: a probabilistic framework for structural variant discovery.

Comprehensive discovery of structural variation (SV) from whole genome sequencing data requires multiple detection signals including read-pair, split-read, read-depth and prior knowledge. Owing to technical challenges, extant SV discovery algorithms either use one signal in isolation, or at best use two sequentially. We present LUMPY, a novel SV discovery framework that naturally integrates multiple SV signals jointly across multiple samples. We show that LUMPY yields improved sensitivity, especially when SV signal is reduced owing to either low coverage data or low intra-sample variant allele frequency. We also report a set of 4,564 validated breakpoints from the NA12878 human genome. https://github.com/arq5x/lumpy-sv.

Read more
July 7, 2019

FGAP: an automated gap closing tool.

The fast reduction of prices of DNA sequencing allowed rapid accumulation of genome data. However, the process of obtaining complete genome sequences is still very time consuming and labor demanding. In addition, data produced from various sequencing technologies or alternative assemblies remain underexplored to improve assembly of incomplete genome sequences.We have developed FGAP, a tool for closing gaps of draft genome sequences that takes advantage of different datasets. FGAP uses BLAST to align multiple contigs against a draft genome assembly aiming to find sequences that overlap gaps. The algorithm selects the best sequence to fill and eliminate the gap.FGAP reduced the number of gaps by 78% in an E. coli draft genome assembly using two different sequencing technologies, Illumina and 454. Using PacBio long reads, 98% of gaps were solved. In human chromosome 14 assemblies, FGAP reduced the number of gaps by 35%. All the inserted sequences were validated with a reference genome using QUAST. The source code and a web tool are available at http://www.bioinfo.ufpr.br/fgap/.

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.