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Background Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. Results We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. Conclusion The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.

The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed "fragment recruitment," addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed "extreme assembly," made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2-0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730 xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

It is now clear that tyrosine kinases represent attractive targets for therapeutic intervention in cancer. Recent advances in DNA sequencing technology now provide the opportunity to survey mutational changes in cancer in a high-throughput and comprehensive manner. Here we report on the sequence analysis of members of the receptor tyrosine kinase (RTK) gene family in the genomes of glioblastoma brain tumors. Previous studies have identified a number of molecular alterations in glioblastoma, including amplification of the RTK epidermal growth factor receptor. We have identified mutations in two other RTKs: ( i ) fibroblast growth receptor 1, including the first mutations in the kinase domain in this gene observed in any cancer, and ( ii ) a frameshift mutation in the platelet-derived growth factor receptor-α gene. Fibroblast growth receptor 1, platelet-derived growth factor receptor-α, and epidermal growth factor receptor are all potential entry points to the phosphatidylinositol 3-kinase and mitogen-activated protein kinase intracellular signaling pathways already known to be important for neoplasia. Our results demonstrate the utility of applying DNA sequencing technology to systematically assess the coding sequence of genes within cancer genomes.

Current HTTP servers process requests using a first-come first-serve queuing policy. What this implies is that the WWW server must process each request as it arrives. The result is that the more requests a client makes, the more replies the server will generate in response. Unfortunately, the bandwidth of the network and the processing capabilities of the server are often limited resulting in an aggressive client, or sets of clients, consuming the majority of the server's resources, limiting other clients' ability to use their fair allocation. While the traditional behavior of a Web server works efficiently for a Web site that is non-discriminating towards all clients, guaranteeing service for preferred clients from the server itself is not yet possible. This paper describes the algorithm we have designed and implemented on the Apache HTTP server, which has been shown to be effective in allocating configurable fixed percentages of bandwidth across numerous simultaneous clients, independent of the aggressiveness of the clients' requests.

The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

To the editor: In the July 29, 2010, issue of Blood , Pillay et al presented a novel study on the kinetics of neutrophils in humans.[1][1] Their study used deuterium in heavy water to label proliferating cells in the marrow. Using a first-order model, the authors found that the neutrophil

BACKGROUND: In patients with chronic lymphocytic leukemia (CLL), the Bruton tyrosine kinase(BTK) inhibitor ibrutinib induces a rapid reduction in lymphadenopathy, accompanied by a transient lymphocytosis. Continuation of therapy leads to a reduction in peripheral blood disease burden with achievement of objective responses in a majority of patients over time. Inhibition of B cell receptor (BCR) and chemokine receptor signaling are considered the mechanisms for redistribution lymphocytosis and CLL cell death. However, these mechanisms of action have not been formally documented directly in patients. To address this, before initiating therapy with ibrutinib, we labeled the DNA of proliferating CLL cells with deuterium (2H) by asking patients to drink deuterated water (2H2O) and then determined the effects of ibrutinib on leukemia cell kinetics (proliferation and death rates) and mobilization of cells from lymphoid tissues (trafficking) after ibrutinib administration. METHODS: 30 previously untreated CLL/SLL patients were enrolled between December 2012 and June 2013 at MD Anderson Cancer Center. Patients required treatment per iwCLL guidelines, had adequate hematopoietic (platelets > 50,000/μL, ANC > 750/μL) and organ functions. Patients drank 50 mL of 70% 2H2O 3 times a day for 5 days, followed by 60 mL daily for a total of 4 weeks (“labeling phase”). After a 6-12 week “washout phase”, patients started once-daily ibrutinib 420 mg continuously on 28 day cycles. Study objectives were to determine the impact on CLL cell proliferation and death rates, before and after ibrutinib, and on CLL cell trafficking after ibrutinib. PATIENT CHARACTERISTICS: Median age of the 30 patients was 64 years (range 48–78) with 19 males. 16 patients exhibited early stage (Rai stage 0-2), and 14 advanced stage (Rai stage 3-4) disease. 17 patients expressed unmutated IGHVs, 11 patients had mutated IGHVs, and 2 had inconclusive IGHV results. 8 were CD38+ and 15 ZAP-70 + by immunohistochemistry. FISH cytogenetics revealed 11 patients with del13q, 6 with trisomy 12, 3 with del17p or TP53 mutation, 4 with del11q, and 6 without abnormalities. Median β2 microglobulin level was 2.8 mg/L (1.6 - 7.4). RESPONSE TO THERAPY: At a median follow up of 13 months, 28 of 30 patients continued on therapy without disease progression. All patients were evaluable for response assessment per 2008 IWCLL guidelines: 28 (93%) achieved partial remission, 1 (3%) complete remission, and 1 (3%) had stable disease, yielding an ORR of 97%. Two patients came off study at 184 and 480 days, respectively. CLL CELL KINETICS AND TRAFFICKING: The average CLL cell birth rate determined before ibrutinib therapy was 0.42% per day (range 0.32 – 1.42%). After initiating ibrutinib, there was a rapid increase in absolute lymphocyte count in the blood in 26 of 30 patients; this contained previously labeled CLL cells rather than newly-divided unlabeled cells. Over time, the proportion of circulating CLL cells that were labeled did not decrease appreciably despite a concomitant fall in circulating lymphocytes, indicating that newly divided cells were not entering the circulation and strongly suggesting that ibrutinib had a major inhibitory effect on leukemia cell proliferation. In 3 of the 4 patients in whom there was a rapid influx of unlabeled cells with the onset of ibrutinib treatment, maintenance of circulating labeled cells concomitant with a significant fall in circulating lymphocytes suggested proliferation of CLL cells was inhibited with extended treatment. Prior to ibrutinib therapy, the measured proliferation rate of CLL cells was 0.42 % per day. Because after ibrutinib therapy both cell proliferation and exiting from the blood were markedly reduced, the elimination rate should equal the death rate. Notably, with treatment the elimination rate of CLL cells was much faster than before ibrutinib, revealing a “true” death rate of 1.48 % of the clone per day. CONCLUSIONS: Using 2H-labelling of dividing leukemic cells, we have shown for the first time directly in CLL patients that ibrutinib significantly inhibits leukemia cell proliferation, leads to an efflux of CLL cells from tissues into the blood, and promotes a remarkably high death rate of CLL cells in an apparently indirect manner, likely due to the interruption of survival signals from the BCR and other receptors that are engaged in lymphoid tissues. Disclosures Burger: Pharmacyclics, Inc.: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Li:KineMed, Inc.: Employment, Equity Ownership. O'Brien:Pharmacyclics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Turner:KineMed, Inc.: Employment, Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc.: Research Funding; KineMed: Equity Ownership.

Summary: JCVI Metagenomics Reports (METAREP) is a Web 2.0 application designed to help scientists analyze and compare annotated metagenomics datasets. It utilizes Solr/Lucene, a high-performance scalable search engine, to quickly query large data collections. Furthermore, users can use its SQL-like query syntax to filter and refine datasets. METAREP provides graphical summaries for top taxonomic and functional classifications as well as a GO, NCBI Taxonomy and KEGG Pathway Browser. Users can compare absolute and relative counts of multiple datasets at various functional and taxonomic levels. Advanced comparative features comprise statistical tests as well as multidimensional scaling, heatmap and hierarchical clustering plots. Summaries can be exported as tab-delimited files, publication quality plots in PDF format. A data management layer allows collaborative data analysis and result sharing. Availability: Web site http://www.jcvi.org/metarep; source code http://github.com/jcvi/METAREP Contact: syooseph@jcvi.org Supplementary information: Supplementary data are available at Bioinformatics online.

In this presentation we describe the methodology and clinical implementation of a mutual information-based automated image registration system for conformal radiotherapy treatment planning. This system estimates the transformation that relates the coordinate systems of diagnostic MR or PET/SPECT image datasets with the treatment planning x-ray CT dataset. This transformation can then be applied to map volumes of interest, such as tumor volumes, defined on one dataset to the reference. Unlike many other studied registration systems, this system is not limited to affine transformations [1]. Instead, the option of a thin-plate spline warping transformation can also be applied to model possible distortions as a result of tissue deformations, as well as those that are imaging device related. This feature allows a wider range of imaging studies to be incorporated into the treatment planning process. Since the system uses the native image data rather than extracted surfaces or ridges, very little user interaction is required to perform the registrations.

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S217 containing 50 % shells and 50% seeds (by weight) and the other version containing 70% of shells and 30% of seeds (by weight). All animals 16 dogs and 16 horses were treated for a time period of three month, the dose was 0.3 g pr kg daily, and data was lumped together. The human studies also tested two different versions of the same shell and seed containing Rose-hip remedy: The daily dose of one version was 1.2 g seeds and a similar amount of shells (A version) and the daily dose of the version (B) was 0.2 g seeds combined with 1.8 g shells. The human study was performed in two groups of middle aged patients with modest osteoarthritis. A seasonal variation is reported for CRP (higher during winter), and both groups started treatment late autumn. A number of 43 patients were given high dose seed version and 40 other patients were given corresponding placebo. Another group of 32 patients (B group) were given the low seed version and corresponded to 30 patients on placebo. Data given are mean +/SD. Wilcoxon test for matched pairs was used within groups and Mann-Whitney when comparing placebo and active treatment. A p level less than 0.050 was regarded as statistically significant. Results: Treatment with the high seed level resulted in a statistically significant reduction in chemotaxis from 28.6 +/13.5 to 5.6 +/3.4 in a group of eight animals (p 50mm. 40 patients completed the study, 21 had been randomly selected to receive 80 mg daily of Oralvisc , and the remaining 19 had identical appearing placebos. Each month they were evaluated for VAS and WOMAC pain and joint function. A subset of 10 subjects per treatment group began a 2-day long oral administration of heavy water (35 mL H2O TID for 2 days) at the conclusion of the treatment phase (Week 12), followed by a synovial fluid aspiration in the affected knee. Synovial fluid lavage samples were analyzed for 2H-labeling of component GAGs by gas chromatography/ mass spectrometry (GC/MS) to obtain fractional synthesis rates of HA and CS in SF. Results: The age, sex, race, BMI, KL scores, as well as VAS pain and WOMAC function were balanced between groups at the beginning of the trial. Treatment with oral HA during 3 months resulted in a significant improvement in VAS pain (p1⁄40.0035), WOMAC pain (p1⁄40.0259) and WOMAC function (p1⁄40.0132) compared to placebo. The stableisotope-mass spectrometry method was successfully implemented for the clinical study of SF GAG kinetics. The rate of HA turnover was 0.78 0.42 / day (i.e., 78% per day) in placebo-treated OA patients (n1⁄410; Fig. 1). With 12 weeks of oral HA treatment, the mean rate of HA turnover declined by 45% to 0.42 0.24 / day (p1⁄4 0.046). While we have not directly compared the rate of HA turnover in normal vs. osteoarthritic knees, comparison of the current results with data from a previous study in ACL patients suggests that these osteoarthritis patients (0.78 / d) have elevated HA turnover relative to “normal” subjects (0.25 / d), and that oral HA partially normalized the HA turnover rate. CS molecules in the SF were also highly enriched, with the majority of the patients recording the maximum measureable turnover rate for this assay (>140% / day). Thus, virtually all of the SF-derived CS was recently synthesized, suggesting a defect in the retention of potential repair molecules in osteoarthritic cartilage. Conclusions: This is the first study of its kind to show that the use of an oral HA (Oralvisc ) agent in knee OA patients can lead to a significant impact on HA turnover in synovial fluid. The normalization in HA turnover was paralleled by an overall decrease in pain scores and improvement in joint function. Further studies are needed to investigate its mechanism but this novel form of oral HA may provide a safe alternative treatment to correct the homeostatic imbalances in the progression of OA. Figure 1. Turnover rate of HA in the knee synovial fluid of osteoarthritic patients. Bar shows the group mean (ANOVA p 1⁄4 0.046). 410 SULFORAPHANE REPRESSES MATRIX-DEGRADING PROTEASES AND PROTECTS CARTILAGE FROM DESTRUCTION IN VITRO AND IN VIVO R.K. Davidson y, O. Jupp y, R. de Ferrars y, C.D. Kay y, K.L. Culley y, R. Norton y, C. Driscoll z, T.L. Vincent z, S.T. Donell y, Y. Bao y, I.M. Clark y. yUniv. of East Anglia, Norwich, United Kingdom; zKennedy Inst. for Rheumatology, Oxford, United Kingdom Purpose: Broccoli is rich in glucoraphanin (a glucosinolate). When broccoli is cut or chewed, glucoraphanin is converted by an enzyme, myrosinase, to sulforaphane (an isothiocyanate). Similar compounds are derived from other cruciferous vegetables and these compounds are therefore accessible via the diet. Sulforaphane (SFN) has been reported to regulate signalling pathways relevant to chronic diseases. Our study investigated whether sulforaphane can abrogate cartilage destruction in osteoarthritis and examined mechanism of action in chondrocytes. Methods: The bovine nasal cartilage explant model (BNC) and destabilisation of medial meniscus (DMM) murine model of osteoarthritis were used to study chondroprotection by SFN. Histone acetylation, gene expression, transcription factors nuclear factor (erythroid-derived 2)like 2 (Nrf2) and nuclear factor kappaB (NF-kB) signalling were

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

HPC systems have shifted to burst buffer storage and high radix interconnect topologies in order to meet the challenges of large-scale, data-intensive scientific computing. Both of these technologies have been studied in detail independently, but the interaction between them is not well understood. I/O traffic and communication traffic from concurrently scheduled applications may interfere with each other in unexpected ways, and this behavior may vary considerably depending on resource allocation, scheduling, and routing policies.In this work, we analyze I/O and network traffic interference on burst-buffer-equipped dragonfly-based systems using the high-resolution packet-level simulations provided by the CODES storage and interconnect simulation framework. The analysis is performed using realistic I/O workload sizes, a variety of resource allocation and network routing strategies employed in production environments, and a dragonfly network configuration modeled after current vendor options. We analyze the impact of interference on both I/O and communication traffic.We observe that although average network packet latency is stable across a wide variety of configurations, the maximum network packet latency in the presence of concurrent I/O traffic is highly sensitive to subtle policy changes. Our simulations reveal a worst-case single packet latency of 4,700 times the average latency for sub-optimal configurations. While a topology-aware mapping of compute nodes to burst buffer storage nodes can minimize the variation in maximum packet latency, it can slow down the I/O traffic by creating contention on the burst buffer nodes. Overall, balancing I/O and network performance requires careful selection of routing, data placement, and job placement policies.