Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses.

The evolutionary analysis of emerging low frequency HIV-1 CXCR4 using variants through time--an ultra-deep approach.

Large-scale parallel pyrosequencing produces unprecedented quantities of sequence data. However, when generated from viral populations current mapping software is inadequate for dealing with the high levels of variation present, resulting in the potential for biased data loss. In order to apply the 454 Life Sciences' pyrosequencing system to the study of viral populations, we have developed software for the processing of highly variable sequence data.

Analysis of TSC cortical tubers by deep sequencing of TSC1, TSC2 and KRAS demonstrates that small second-hit mutations in these genes are rare events.

Tuberous sclerosis complex (TSC) is an often severe neurocutaneous syndrome. Cortical tubers are the predominant neuropathological finding in TSC, and their number and location has been shown to correlate roughly with the severity of neurologic features in TSC. Past studies have shown that genomic deletion events in TSC1 or TSC2 are very rare in tubers, and suggested the potential involvement of the MAPK pathway in their pathogenesis.

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.

Ultra deep sequencing detects a low rate of mosaic mutations in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by mutations in TSC1 and TSC2. However, 10-15% TSC patients have no mutation identified with conventional molecular diagnostic studies. We used the ultra-deep pyrosequencing technique of 454 Sequencing to search for mosaicism in 38 TSC patients who had no TSC1 or TSC2 mutation identified by conventional methods.

Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing.

Objective: Identification of low-frequency variants is of clinical importance in the identification of preexisting drug resistance. Using 'ultra-deep' sequencing, we address the detection of potential resistance to the chemokine (C-C motif) receptor 5 (CCR5) antagonist, maraviroc, due to the pretreatment presence of low levels of chemokine (CXC motif) receptor 4 (CXCR4)-using virus.

Methods: We present a novel protocol for the phenotyping of HIV based on '454' pyrosequence data and apply this to two large data sets comprised of 104 628 (before treatment, day 1) and 191 637 (after treatment, day 11) reads from the envelope region.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue.

Paired-end mapping reveals extensive structural variation in the human genome.

Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) approximately 3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome.

"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions.

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) alpha-galactosidase activity, and (iii) beta-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis.

The Melanoma Vascular Mimicry Phenotype Defined in Gene Expression and Microsome Sequencing Analysis.

The phenomenon of vasculogenic mimicry in melanoma has been recently described to be an important factor relating to melanoma progression. Large scale gene expression profiling by real-time quantitative RT-QPCR of a panel of 40 normal tissues and 54 cancer cell lines revealed that two genetically related melanoma cell lines, one derived from a primary lesion Hs.688(A) and one derived from a lymph node metastasis Hs.

Simultaneous gene expression analysis of steady-state and actively translated mRNA populations from osteosarcoma MG-63 cells in response to IL-1alpha via an open expression analysis platform.

Pro-inflammatory cytokines play a key role in various forms of metabolic bone diseases, including osteopenia and osteoporosis. Human MG-63 cells treated with IL-1alpha were used as a model system to identify potential marker genes that are differentially expressed. This study is designed to quantitate gene expression of actively translated mRNAs as compared to the steady-state mRNA population.