Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.     

Environmental and occupational biomonitoring using the Comet assay

Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a “single cell gel” (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.     

MetaGeniE: Characterizing Human Clinical Samples Using Deep Metagenomic Sequencing

With the decreasing cost of next-generation sequencing, deep sequencing of clinical samples provides unique opportunities to understand host-associated microbial communities. Among the primary challenges of clinical metagenomic sequencing is the rapid filtering of human reads to survey for pathogens with high specificity and sensitivity. Metagenomes are inherently variable due to different microbes in the samples and their relative abundance, the size and architecture of genomes, and factors such as target DNA amounts in tissue samples (i.e. human DNA versus pathogen DNA concentration). This variation in metagenomes typically manifests in sequencing datasets as low pathogen abundance, a high number of host reads, and the presence of close relatives and complex microbial communities. In addition to these challenges posed by the composition of metagenomes, high numbers of reads generated from high-throughput deep sequencing pose immense computational challenges. Accurate identification of pathogens is confounded by individual reads mapping to multiple different reference genomes due to gene similarity in different taxa present in the community or close relatives in the reference database. Available global and local sequence aligners also vary in sensitivity, specificity, and speed of detection. The efficiency of detection of pathogens in clinical samples is largely dependent on the desired taxonomic resolution of the organisms. We have developed an efficient strategy that identifies “all against all” relationships between sequencing reads and reference genomes. Our approach allows for scaling to large reference databases and then genome reconstruction by aggregating global and local alignments, thus allowing genetic characterization of pathogens at higher taxonomic resolution. These results were consistent with strain level SNP genotyping and bacterial identification from laboratory culture.     

Identification and resolution of artifacts in bisulfite sequencing

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Clinical aspects for the use of DNA image cytometry in detection of bladder cancer: a valuable tool?

The employment of DNA flow or image cytometry for oncological diagnostic procedures is favored because of its high correlation to tumor biological behavior. Prognostic statements and therapeutic strategies therefore are based on the high validity of DNA cytometric measurements. Using 151 bladder washings from patients suspected of bladder cancer for this study, we examined the clinical value of various common methods of DNA single cell (SCI) and stemline interpretations (SLI). Comparing the specificity and sensitivity of DNA image cytometry in detection of bladder tumors, we found 81 and 52%, respectively, for SCI of Boecking, 84 and 45% for SLI of Boecking, 61 and 58% for SLI of Fu, and 82 and 40% for conventional stemline interpretation. To improve diagnostic and prognostic validity of DNA image cytometry, we designed our own method of interpretation. In consequence, we identified six single DNA parameters out of all recorded measurements that correlated most to histopathological grading (G1-G3). Creating reference values at random and rating by points, we used a cytometric grading system for ranking. In detection of bladder cancer specificity and sensitivity ultimately arrived at almost 70% in application of our method. Thus, by this study, we were able to show that sensitivity of DNA examination can be increased by combining various DNA parameters. Apart from our own scheme, the discrepancy in interpretation of DNA image cytometry does not allow us to recommend this procedure as the only diagnostic in detection of bladder cancer. However, in regard to prognostic statements, particularly tumor biological behavior, DNA image cytometry appears to be useful.     

Array-based comparative genomic hybridization and copy number variation in cancer research

Array-based comparative genomic hybridization (aCGH) is a molecular cytogenetic technique used in detecting and mapping DNA copy number alterations. aCGH is able to interrogate the entire genome at a previously unattainable, high resolution and has directly led to the recent appreciation of a novel class of genomic variation: copy number variation (CNV) in mammalian genomes. All forms of DNA variation/polymorphism are important for studying the basis of phenotypic diversity among individuals. CNV research is still at its infancy, requiring careful collation and annotation of accumulating CNV data that will undoubtedly be useful for accurate interpretation of genomic imbalances identified during cancer research.     

Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.     

Evidence That Inconsistent Gene Prediction Can Mislead Analysis of Dinoflagellate Genomes

Comparative algal genomics often relies on predicted genes from de novo assembled genomes. However, the artifacts introduced by different gene-prediction approaches, and their impact on comparative genomic analysis remain poorly understood. Here, using available genome data from six dinoflagellate species in the Symbiodiniaceae, we identified methodological biases in the published genes that were predicted using different approaches and putative contaminant sequences in the published genome assemblies. We developed and applied a comprehensive customized workflow to predict genes from these genomes. The observed variation among predicted genes resulting from our workflow agreed with current understanding of phylogenetic relationships among these taxa, whereas the variation among the previously published genes was largely biased by the distinct approaches used in each instance. Importantly, these biases affect the inference of homologous gene families and synteny among genomes, thus impacting biological interpretation of these data. Our results demonstrate that a consistent gene-prediction approach is critical for comparative analysis of dinoflagellate genomes.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

Applications of mass spectrometry for quantitation of DNA adducts

DNA adducts are formed when electrophilic molecules or free radicals attack DNA. 32P-postlabeling has been the most commonly used assay for quantitation of DNA adducts due mainly to its excellent sensitivity that allows quantitation at concentrations as low as approximately 1 adduct per 10(9) normal bases. Such methods, however, do not have the specificity desired for accurate and reliable quantitation, and are prone to produce false positives and artifacts. In the last decade, mass spectrometry in combination with liquid and gas chromatography has presented itself as a good alternative to these techniques since it can satisfy the need for specificity and reliability through the use of stable isotope-labeled internal standards and highly specific detection modes such as selected reaction monitoring and high-resolution mass spectrometry. In this article, the contribution of mass spectrometry to the quantitation of DNA adducts is reviewed with special emphasis on unique applications of mass spectrometry in the area of DNA adduct quantitation and recent applications with improvements in sensitivity.     

Straight Walk: A modified method of ligation-mediated genome walking for plant species with large genomes

Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to clone unknown genomic regions flanking known sequences. However, these methods are typically problematic when applied to highly complex DNA templates isolated from plants with large genomes. Here we describe a reliable and efficient genome walking method that is particularly effective for plants with large genomes. Our ligation-mediated PCR method, Straight Walk, has improved sensitivity and specificity due to optimization of sequences of adaptors and adaptor primers. Successful genome walking in lily, which has one of the largest genomes in plants, indicates that Straight Walk is applicable for most plant species.     

Étude de l’impact d’une correction d’atténuation par tomodensitométrie en scintigraphie myocardique

Myocardial perfusion SPECT is a routine for the assessment of patients with coronary artery disease (CAD). However, attenuation artifacts may decrease the specificity of the test. These artifacts can be corrected with an attenuation correction. We prospectively included 70 patients who underwent myocardial perfusion SPECT with (IRAC) and without (IRNC) attenuation correction using transmission CT imaging integrated in the acquisition system in patients with low prevalence of CAD. Automatic quantitative analysis with summed stress score (SSS) and rest (SRS) and summed difference score (SDS) was used as interpretation criteria. The results showed a specificity of 80% for IRAC and 56% for IRNC, a positive predictive value of 40% for IRAC and 23% for IRNC, without any significant change in sensitivity. An unpaired t-test showed no significant difference between the overall population and one where an artifact was corrected for heart rate and breath rate during the acquisition effort, the body mass index, chest and abdomen circumferences, and the ratio of these two parameters. Attenuation correction significantly improves the specificity of myocardial scintigraphy with no significant difference in sensitivity. The majority of corrections were for artifacts from the inferior wall in men. There is no correlation between the anthropomorphic and physiological parameters and the occurrence of an artifact of attenuation corrected with CT data.     

Measurement of locus copy number by hybridisation with amplifiable probes

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.     

Modified moving average analysis of T-wave alternans to predict ventricular fibrillation with high accuracy.

T-wave alternans is a marker of cardiac electrical instability with the potential for arrhythmia risk stratification. The modified moving average method was developed to measure alternans in settings with artifacts, noise, and nonstationary data. Algorithms were developed and performance characteristics were validated with simulated electrocardiograms (ECGs). Experimental laboratory ECGs with dynamically changing alternans values were analyzed. Alternans values estimated by modified moving average analysis correlated strongly with input alternans values (r(2) = 0.9999). Rapidly changing alternans levels and phase reversals did not perturb the measurement. When heart rate was increased from 60 to 180 beats/min, with T-wave alternans apex moving from 237 to 103 ms after the R wave, the measured alternans peak varied <5% from input value. Simulated 50- to 1,000-microV motion artifact spikes typical of treadmill ECGs produced inaccuracies <2%. Alternans values in experimental laboratory study using standard electrodes tracked vulnerability to myocardial ischemia-induced ventricular fibrillation with 100% sensitivity and specificity at a cut point of 0.75 mV. Modified moving average analysis is a robust method that precisely measures T-wave alternans in settings with artifacts, noise, and nonstationary data typical of clinical ECGs and yields an accurate estimate of risk for ventricular fibrillation.     

芯片技术与肿瘤中DNA甲基化研究

DNA甲基化是表观遗传学的一个重要部分。它参与基因转录调控, X染色体失活, 发育调控及细胞分化的过程。异常的DNA甲基化与癌症的发生密切相关。芯片技术的发展为高通量研究DNA甲基化提供了新的方法。各种芯片技术以不同的DNA预处理方法为基础, 包括免疫沉淀和限制性内切酶等。免疫沉淀方法特异性高, 而限制性酶的方法具有较高的灵敏度。虽然每种方法都有一定的局限性, 但是它们为在基因组范围研究癌症的甲基化谱提供了更多的选择。