text:
Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events.
We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem.
The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package.
Malaria; Sequencing; Genome; Polymorphism; VariantBackground
The detection of single nucleotide and copy number variants (SNVs and CNVs) conferring resistance to drug and vaccine candidates provides researchers with a powerful tool to choose the best combination of agents to treat infectious diseases such as malaria in specific regions, to study pathogen population dynamics and transmission, as well as to engineer new treatments that cannot be easily evaded. In addition, in organisms in which genetic complementation or backcrosses may be difficult or time consuming, whole genome sequencing (WGS) offers an opportunity to determine if second-site mutations may have been inadvertently introduced after transfection or transformation, and contribute to an observed phenotype.
With the reduction in price and increased power of current short-read high-throughput WGS methods and the wide dispersal of a variety of sequencing platforms and accompanying support, full genome sequence data is now relatively easy to generate. Recent advances in the algorithmic and programmatic analysis of WGS data have led to a number of standards, especially the use of the Genome Analysis Toolkit (GATK) [1], being used in the analyses of human genomic data to detect SNVs and CNVs. However, there are opportunities for more comprehensive analyses of the genomes of simpler eukaryotes such as the ~23.5 Mb genome of Plasmodium falciparum, the apicomplexan parasite and etiological agent of human malaria, which has also served as a model for eukaryotic pathogen genomics since the completion and full assembly of its genome sequence in 2002 [2]. Full genome sequencing at 30-40X coverage is now readily achieved [3-6]. Such coverage allows for the identification of recombination events, the description of SNVs in sequences other than in the exomes, and the detection of small structural variants, including short-length insertion or deletion events. P. falciparum is responsible for up to a million deaths annually [7], and although its haploid genome is worthy of investigation for this reason alone, it also serves as an ideal test system because heterozygous calls generally do not need to be considered in sequence analysis validation (although mixed infections are a real concern) and a fully assembled reference genome is available [2]. Furthermore, the parasite can be sub-cloned and readily cultured in vitro within white-cell depleted, anucleated human erythrocytes [8], mitigating host DNA contamination.
In this manuscript, we introduce a validated pipeline for the comprehensive analysis of short-read WGS data in Plasmodium spp.. The pipeline, which can be readily adapted to other small eukaryotes, integrates well-known alignment tools and custom filtration options so that SNV or structural variant data can be easily generated and understood. We believe that the pipeline will work well, once adapted, with species of any ploidy (indeed, it has been used already in Arabidopsis analysis) and genomes of size up to 75 Mbp have been tested. As well, we introduce improved algorithms for utilizing depth of coverage to call CNVs, improving on current GC bias normalization methods [9]. This pipeline is implemented in a stand-alone program called "Platypus", for open distribution and collaboration among research groups. We validate the pipeline using data from 26 P. falciparum samples with known SNVs and CNVs (Table 1), demonstrating both its accuracy and precision. This pipeline should allow those generating WGS data to not only find all SNVs and structural variants detected by other methods (as well as novel ones) but to eliminate all or almost all false positives, reducing ambiguity and potentially allowing WGS to substitute for complementation, Southern blotting, or other genetic methods designed to link phenotype to genotype.
Table 1. Whole-genome sequencing statistics
Current genotyping programs are generally designed to be conservative and as a consequence, return a large number of false positive variant calls. These programs, including GATK [1] and the sequence/alignment map toolbox (SAMTools) [13], typically allow the user to set a number of stringency filters such as the quality of the read alignment or bias towards a specific strand, that can theoretically be used to separate false from true positives. However, the actual threshold values for each filter are not pre-determined, and as such, it is left to the researcher to decide how to best utilize each metric, creating barriers for the novice user. Thus, we set out to create a set of empirically-derived filters for Plasmodium WGS data that could be used as a reference point for future SNV analyses.
To identify a robust set of filtering parameters we began with a list of 15,145 known SNVs identified using traditional Sanger resequencing of Dd2 to 7X coverage [14] and deposited in PlasmoDB (http://plasmodb.org) webcite[15]. These distinguish the multidrug-resistant P. falciparum laboratory Indochina strain, Dd2, from the African drug-sensitive reference strain, 3D7. We then compared a P. falciparum Dd2 strain WGS short-read sequence obtained in our lab to the P. falciparum reference (3D7 strain) sequence. Our Dd2 sequence was generated with 70 bp paired-end reads on an Illumina Genome Analyzer II to a mean of 31X coverage with 96.4% of bases being covered by 5 reads or more. We considered the 15,145 curated SNVs to be true positives. All other SNVs detected were considered false positives, although it is likely that some of the novel SNVs are indeed true genetic differences (genetic diversity, especially in the subtelomeric regions, is extremely high approaching 90% diversity in at least one base position between field samples) [16]. We then worked to identify a set of filtering parameters, which would have the sensitivity to detect at least 90% of the known SNVs, while eliminating as many 'novel' SNVs as possible.
Because the entire mathematical domain of all commonly used filtering parameters (17 characteristics of SNVs and their combinations, see Table 2) is too large to search exhaustively in efficient computational time, we developed a genetic searching optimization algorithm that searched over the entire domain of the 17 filtering parameters that characterize WGS data. This 'genetic' class of algorithm implements an objective function ('fitness') that is defined by several filters, and tries to minimize the value of the objective function over the entire domain (the lowest and highest possible values) of filters (every possible combination of filter choices) [17]. In our case we forced the sensitivity at various levels (0-100% in increments of 0.5%) and searched for the maximum specificity at each of those levels. Our objective function was a simple linear combination of all possible quality metrics for SNV data generated by GATK [1] and Picard (http://picard.sourceforge.net webcite), with a varying polynomial coefficient matrix.
Table 2. Optimized filtering parameters applied by Platypus
We chose a population of 10,000 parameter combinations to run through 100 evolutionary iterations. The algorithm we implemented included a low crossover rate (0.5) and high mutation rate (0.1) as well as a tournament pattern parental determination strategy with a tournament size of 100, and with a guaranteed 10 elite children using the MatLab Global Optimization toolbox. These settings were dynamically determined to give consistency and robustness across a variety of sensitivity ranges. Iterating through a forced sensitivity level in 1% increments yielded a smooth progression along a similar combination of filtering parameters.
The list of 10,000 randomly chosen parameter combinations was assessed for both sensitivity and specificity. Each set of filtering parameters sorted the true positives into two categories ("called" or "not called") and similarly sorted the false positives; these calls were then evaluated for accuracy. Filtering sets that provided high specificity for a given level of sensitivity were carried over to the next round. The filtering parameters were then varied slightly within all successful sets, and individual parameters swapped between sets. After 100 iterative cycles, the most successful sets of filters converged on a single result – a theoretical optimal filtering set. We then added a further set of criteria based on the quality of the sequencing reads. The final optimized set excluded all SNV calls that met any of the following criteria listed in Table 2.
Using the optimal filtering set we detected 95.0% of the known SNVs with a specificity of 75.6% (8,315 total novel calls), and by lowering the total quality threshold we obtained a sensitivity of 90% of known SNVs, with a specificity of 85.1% (5,077 total novel calls). We generated a receiver operating characteristic curve (Figure 1) using optimal parameter sets at each sensitivity level, labeling three sensitivity threshold points of interest. Principal component analysis did not yield any statistically significant common genomic features (e.g. position on chromosome, position within gene, base pair transition) of the false positives detected, but 55% (4,598 calls) were found in the 12% of the genome we define as sub-telomeric (within 100,000 bp of the chromosomal end) and which likely lie in regions that were not adequately covered by the 7X Dd2 Sanger sequencing [14]. We also searched over a space of multi-dimensional filters (those depending on more than one quality metric in a nonlinear relationship). This included all multiplicative combinations of two and three parameters, as well as exponential, power, logarithmic, and quadratic functions of single parameters, but these filters were unable to find results that were as good as a combined one-dimensional approach (that is, better than the optimal specificity and any sensitivity level), possibly due to the computational complexity of searching over multi-dimensional filters. This set of filtering parameters (Table 2) is thus implemented in our pipeline and was used for all subsequent analyses.
Figure 1. Receiver operating characteristic for SNV optimization. An ROC for the filtering parameters used in SNV detection optimization. Red points indicate the calculated specificity for certain fixed sensitivity levels. The blue line indicates the calculated specificity and sensitivity in 0.5% increments along the entire range of parameters. The optimal set of parameters for the GATK is given in Table 2. SNVs were then divided into either a 'called or 'not called' category based on these metrics and the current set of filters being used. The following metrics were obtained from the Unified Genotyper: read depth, strand bias, haplotype score, homopolymer run length, total positional mapping quality, number of null mapping qualities, total positional quality, total quality by depth, and genotype quality (DP, FS, HaplotypeScore, HRun, MQ, MQ0, QUAL, QD, GQ). These SNVs were then hard filtered using the Variant Filtration Walker according to the given set of parameters, as in Table 2.
CNVs contribute substantially to drug resistance in Plasmodium and other eukaryotic pathogens [18-21]. The current methods for calling CNVs in Plasmodium spp. WGS data, like most pathogenic eukaryotes, rely on smoothing the depth of coverage data (e.g. number of reads aligned to the reference) [22-24]. Smoothing is needed because sequencing depends on multiple stochastic processes and there can be great variability in the actual coverage over a given stretch of genomic DNA. Users are thus required to guess the appropriate smoothing parameters such as the number of base pairs to be averaged, meaning that the user already needs to know the approximate size of the CNV. Furthermore, it is known that there is also a non-stochastic bias in the depth of coverage due to the tendency of areas of high and low GC content to be sequenced less efficiently and this must also be accounted for, especially as P. falciparum is extremely AT-rich (81%). Because we found that the current algorithms produced a large number of false calls when applied to our WGS data, we sought to address this problem by developing our own CNV calling algorithm.
The first improvement we made to the standard method was to improve GC bias correction. GC-content bias describes the dependence between fragment count (read coverage) and GC content found in high-throughput sequencing assays, particularly the Illumina Genome Analyzer technology. This bias can dominate the signal of interest for analyses that focus on measuring fragment abundance within a genome, such as copy number estimation and is not consistent between samples. We therefore analyzed regularities in the GC-bias patterns, and generated a closed-form compact description for this curve family. It is the GC content of the full DNA fragment (generally 100-200 bp), not only the sequenced read (the 50-100 bp sequenced on each end), that most influences this bias [9]. This GC bias distribution is unimodal: both GC rich fragments and AT rich fragments are under-represented in the sequencing results. Based on these findings, we devised a new method to calculate predicted coverage and correct for the bias.In order to implement the correction we first calculate the GC content of the sequenced fragment (for paired end reads, both reads plus the insert) after aligning the reads to the reference genome (PlasmoDB 7.1 for imidazolopiperazines, 10.1 for the remainder), assuming that the insert matches the reference genome between the reads exactly. While this assumption does not account for potential point mutations, we have tested the effect of this on the bias normalization algorithm, and it is imperceptible, because point mutations occur so infrequently in a long insert size that the total GC content is not affected. After GC normalization CNVs become much more apparent than they were in the raw data (compare Figure 2A and B).
Figure 2. CNV detection in a cladosporin resistant Dd2 line. A. Raw depth of coverage data for chromosome 13 from the CladoR_clone3, which contains a microarray-verified amplification conferring resistance to cladosporin [10]. B. Depth of coverage normalized after GC bias correction. C. Normalized depth of coverage after the Platypus smoothing and edge-calling algorithm, with the actual copy number prediction overlaid. D. Theoretical minimum detectable variant size for a range of total genome coverage. Deletions are easier to detect and can be identified with less total coverage and in smaller sizes. Similarly, 3x amplifications are easier to detect than 2x.
Secondly, after GC content normalization, the depth of coverage must be 'smoothed' so that true CNVs can be detected and random fluctuations in the data can be identified and discarded. However, smoothing algorithms tend to blur the divisions between otherwise sharp edges. Thus we also developed a simple iterative process of smoothing and edge detection that would identify these boundaries. This step integrates an iterative Weierstrass transform followed by edge detection through convolution with a first-derivative Gaussian kernel [25]. These can be described as follows: Let Dn be the smoothed depth of coverage of a chromosome of length l normalized after n iterations. Then, with a Gaussian kernel G with arbitrary coefficient k, we have, for all positions j on the chromosome, χj:
Which we can expand to:
We can detect the edges of this data by finding all solutions:
Which is expanded to:
An example of the output of this algorithm is demonstrated in Figure 2C.
To save computational time, we applied the convolution theorem to take these operators in the Fourier space and as such, reduce all operations to point-wise multiplication. After each Weierstrass transform, edges are detected by the above formula. The total number of convolution iterations was set to be variable in the first in silico tests, ending only when no new edges had appeared in the last 10 iterations, but was eventually held constant at 5 because in practice no new edges appeared after the 2nd or 3rd iterations of the algorithm. We must treat the mitochondrial and apicoplast genome separately, as the depth of coverage of these is usually very different than the other Plasmodium chromosomes, even by an order of magnitude. The depth of coverage in each region (i.e. between each edge) is then compared to the sample mean, and those that are statistically higher or lower are assigned an amplification number based on their increase (or decrease) relative to the mean.
Recombination contributes substantially to the virulence of many eukaryotic pathogens such as P. falciparum and T. bruceii where genome encoded virulence factors are located in hyper-recombinogenic sections of the genome. In addition, such rearrangements could contribute to a phenotype if no causative SNV or clear dosage effect in a likely target is found. We thus sought to implement a program to find these recombination events.
Our strategy was to identify fragments with mated pairs that had abnormal insert sizes when they were aligned to a reference genome, especially ones with mated pairs that aligned to two different chromosomes or to vastly distant parts of the same chromosome. To find the initial events, all reads which had a mates aligning farther than 10 kb away from each other or on another chromosome were extracted and each region with a group of 10 or more overlapping reads with this property was de novo assembled using PRICE [26] with 20 cycles and otherwise standard settings (-icf $x 1 1 5, -nc 20, -dbmax 100, -maxHp 7, -lenf 2500 20). These contigs were then aligned against the reference genome using ClustalW to discover the origin of each part of the contig. Figure 3A, step IVc outlines the recombination detection process.
Figure 3. Schematic of the Platypus data processing pipeline. Diagram of all the external and internally developed programs involved in processing Plasmodium samples as implemented by the Platypus pipeline. The SNV analysis is coded as a sequence of C programs, combining programs written by a variety of other research groups in Java and C. These programs used are the Burrows-Wheeler Aligner [27], Sequence Alignment Map Tools [13], the Genome Analysis Toolkit [1], and the Picard tool set. Each script encodes a single step of the data cleaning and analysis pipeline, including error checking and customization of the program. The GC bias normalization algorithms and the CNV detection algorithms were written in MATLAB (The Mathworks) and coded in C after export, and are integrated into the pipeline as shell scripts as well. Reads are aligned to the P. falciparum 3D7 reference genome version using the Burrow-Wheelers Aligner. Alignment files are then converted to a binary map and sorted and indexed using SAMTools. (steps I-II, A) Sequencing run statistics for each sample, including GC bias metrics and quality score distribution statistics, are then collected using a number of Picard programs. Read group identifiers are then added, and unmapped reads are removed from the alignment using SAMTools. Next optical and PCR duplicates are removed using Picard, and the entire alignment is realigned around possible insertion-deletion events using GATK. Base quality scores are recalibrated using GATK, and the depth of coverage at every base pair position is calculated. (step III, A) The alignment is then indexed and is ready for normalization and analysis. A. Schematic of entire workflow, with color-coding corresponding to the steps in B. Diagram of every program and action used in the Platypus pipeline, with file types traced passing between programs written in between. We request that all users of the Platypus acknowledge both this manuscript and the referenced manuscripts for the other programs included in the manual. Please see the instruction manual, available on the website, for more information.
Results and discussion
To further test the validity of our optimized SNV filtration, and to test the accuracy of the predicted sensitivity (90%) and specificity (85%), we next evaluated the algorithm's ability to find rare mutations in isogenic lines created in our laboratory. We gathered WGS data from five separate chemical resistance experiments involving known and experimental antimalarial compounds (Table 1). In these experiments a clonal 3D7 or Dd2 line was exposed to sub-lethal concentrations of a small molecule inhibitor for 2–4 months. Parasites were then sub-cloned by limiting dilution and genomic DNA (gDNA) was sequenced, along with the gDNA of their isogenic parental clone. All strain genotype data were compared to the parent so that only genomic changes arising after chemical selection pressure would be identified. These strains have demonstrated resistances to a variety of small molecule inhibitors (atovaquone [6], cladosporin [10], spiroindolones [11] and imidazolopiperazines [12]). Most of these strains were initially characterized using a custom Affymetrix P. falciparum tiling microarray [28] and confirmed using Sanger sequencing or qPCR but some had been whole-genome sequenced previously, and some were sequenced specifically for this study (cladosporin resistant line). Altogether we analyzed data from 26 clones with their respective 2 parents. All were sequenced to 25-83X coverage using paired-end reads with ~150 bp fragment size on the Illumina Genome Analyzer II and Illumina HiSeq platforms (Tables 1 and 3).
Table 3. Total number of SNVs detected using Platypus compared to simple filtering
For each of the 26 whole genome sequencing sample datasets, the Platypus pipeline was able to analyze and filter the sequencing data from tens of thousands of SNVs (examples of which are shown in Figure 4A) to a median of 6 final SNV calls (Figure 4B,C). The total number of raw SNVs, the number after filtering, and the number of these that were nonsynonymous for each of the experiments, are given in Table 3. These results included 100% sensitivity concordance with the respective microarray and sequencing results, including all of the 20 unique SNVs that had been previously implicated in drug resistance (Table 3) as well as 127 additional mutations. More importantly, Platypus detected all SNVs that conferred the respective drug resistance phenotype and highlighted those which would cause a nonsynonymous change in an amino acid and alter protein function. Altogether, 63 SNVs in the atovaquone, spiroindolone, and cladosporin resistant samples that were output by the Platypus program were confirmed as true hits by Sanger sequencing. In addition, 52 SNVs in the atovaquone resistant samples that were closest to the cutoff line, but still excluded, were chosen as validation targets of a true negative result. These regions were sequenced and found to be insertion/deletion events, which our program is not currently designed to detect. These results confirm that the program is robust and does not misinterpret sequencing data for Plasmodium spp.. In addition, the power of the program is clear – in each sample the Platypus filtered out everything (to the best of our knowledge) except that which was most relevant to the experiment. The time and effort saved by removing the long process of manual curation will have a major impact on the analysis of WGS data for Plasmodium and eukaryotic pathogen genomes in general. These methods can be directly applied to a variety of other pathogens as well, and we hope to see collaborative efforts to expand the pipeline to other organisms.
Figure 4. SNV filtering diagram for the Evo Samples. A. Read pileup of a region from EvoR1a2 including miscalled nucleotides within a read (1), misaligned reads (2 and 3), and false SNV calls due to a homopolymer run followed by a repetitive region. This region is an example of the false positive that the Platypus seeks to eliminate. B. Read pileup of a region from EvoR1a2 including a true SNV (4) but still including a number of misaligned reads (5). The Platypus seeks to extract these true positive SNVs from their surroundings. C. Shows the number of raw SNV calls (blue) and the total number after filtration (red) and the subset of nonsynonymous SNVs (green) that may cause a change in the drug efficacy profile. Scale is logarithmic – the Platypus produces a 103-104x reduction in SNV calls.
We note that the Platypus reduces the total number of SNVs from raw data by a factor of approximately 103-104 (Table 3). While we cannot comprehensively genotype the tens of thousands of SNVs called initially by GATK or SAMTools, we have verified in atovaquone, spiroindolone, and cladosporin resistant lines that 63 of the SNVs called by Platypus are true genetic variants, and none of the 52 sites from the atovaquone resistant samples were excluded erroneously.
We also see (Table 3) that a comparison to the Q30 metric [29] identifies the Platypus as having significant gains over this simpler metric, reducing the number of SNVs called by a factor of approximately 1.6x. The sites called by the Q30 metric and excluded by the Platypus constituted 48 of the 52 sites that were Sanger sequenced and subsequently discovered to be not true, validating their exclusion by the Platypus.
There is no standard set of filtering parameters to use with GATK, but we can compare to a set of published filter values for a comparable project [30]. Using Bright et al. as a comparison point, we can adapt their filters into our current pipeline. Doing so yields a 91% sensitivity level with a specificity of 45%. We can see that these heuristically chosen values have a reasonable sensitivity threshold but do not hold up to empirically designed filters in terms of specificity.
The assessment of a false positive and false negative rate can of course never be perfected, but in all cases we have detected plausible drug resistance genes in all cases. Comparison with known values and with extensive Sanger sequencing data confirms our calls, and even indicates that these sets of filters may be too lenient – that we may be detecting nonexistent SNVs rather than missing true ones.
To determine the sensitivity and specificity of the CNV detection algorithm, we sought to detect eight known amplifications from WGS data of the 26 strains with known structural variants, all of which had been discovered using our custom tiling microarray (Table 4). Figure 2A shows the raw depth of coverage data for a known amplification conferring cladosporin resistance on chromosome 13, and also indicates both the depth of coverage data after applying the detection algorithm (Figure 2B) and shows the copy number variant called from the data (Figure 2C).
Table 4. CNVs detected using WGS and genomic microarrays
Altogether Platypus identified all 8 unique CNVs that were known to exist in our strains. Our algorithm identified the large ~100 kb CNV surrounding the P. falciparum multidrug resistance protein-1 gene (pfmdr1, PF3D7_0523000) in the 13 Dd2 derived strains [20,24] and the 5 kb GTP cyclohydrolase amplifications in 13 Dd2 (pfgch, PF3D7_1224000) derived strains as well as the smaller 1.6 kb amplification GTP cyclohydrolase in 13 3D7 derived strains [19]. We were also able to identify several independent larger amplifications that included lysyl tRNA synthetase (pfkrs1, PF3D7_1350100) in 3 strains that are resistant to cladosporin (a drug which targets lysyl-tRNA synthetase) [10], and an amplification on chromosome 1 in the EvoR5 strain that was grown in the presence of atovaquone, both confirmed by microarray as well [6]. We were also able to detect an amplification on chromosome 12 (containing pfatp4) in 3 of the spiroindolone resistant samples [6,11]. Although there was some ambiguity as to the number of copies (i.e. duplication or triplication), the Platypus also reported a SNV in one copy of pfatp4 but not in the other copies of the gene. Furthermore, we discovered no spurious or novel amplification or deletion events, i.e. CNVs that were not detected by tiling microarray.
In addition, the boundaries for the respective CNV called by the algorithm for each sample correspond closely to the boundaries detected by microarray – the edges of the CNV algorithm lie, almost exclusively, within 100 bp of the probes near the amplification boundaries. To further validate these data we also examined the read pileup near the predicted boundaries (Figure 5) for some cases (CladoR_clone1 shown). The read pileup allowed us to investigate the orientation of the amplification event. In the Dd2 lines mentioned above (those with detected amplifications surrounding pfmdr1, pfgch, and pfkrs1, pfatp4), 46-58% of the paired-end reads at the beginning of each amplification mated to the end of that amplification with an abnormally large insert size and in the reverse read orientation (Figure 5, inset). This indicates that the amplification event is not only on the same chromosome but also in tandem – that is, the amplified genomic region is adjacent to the originating sequence (Table 4). Interestingly, we found all amplifications that we examined were in a tandem orientation.To test the theoretical limits of the CNV detection algorithm we also generated simulated amplifications and deletions by producing a depth of coverage from a random distribution that had a mean twice the sample mean (amplifications), or by completely eliminating certain reads (deletions). Deletions could be detected with low coverage and in very small sizes, with a theoretical limit around 3x coverage and 500 bp (with 100 bp reads). Amplifications were harder to detect, but the limit is approximately 1000 bp at 20x total genomic coverage. This limit is accurate for all deletions and for amplifications up to four times, after which point the algorithm can only detect the presence of a variant and estimate its copy number (an estimate of 6x indicates a 5x-7x CNV). Figure 2D demonstrates the theoretical capabilities of the algorithm in detecting both deletion and amplification events for various total genome coverage levels.
Figure 5. Detecting genome amplification events. Depth of coverage data for all 14 chromosomes of a Dd2 clonal line with evolved resistance to cladosporin (CladoR_clone3) [10]. The curve represents the output of the CNV detection algorithm, while the black line is the actual copy number call by the program. The two visible amplifications detected are the one encompassing the pfmdr1 locus on chromosome 5 and the amplification associated with cladosporin-resistance on chromosome 13 [10]. A third amplification on chromosome 12 containing pfgch is visible at higher magnification. The inset shows a read pileup for the region containing an amplification on chromosome 13. The green colored reads indicate those with an abnormal insert size with mated reads mapping back-to-back rather than front-to-front. This is evidence that the amplification is in tandem with the original copy on the chromosome.
We compared our CNV calling algorithm to BreakDancer, a similar program used to detect both copy number and recombination events using the default [31]. Using a set of parameters equivalent to those published in Chen et al., (4 standard deviation threshold, Q>39, MQ>35) we see that BreakDancer is fully able to detect all CNVs present in our samples (those detected by microarray and/or whole genome sequencing), but it also identifies 73 other CNVs ranging from 434 bp to 11639 bp that we do not detect by any method. Indeed, qPCR amplification of these speculated regions indicates no change in copy number in any of these regions not detected by other high throughout methods.
Our algorithm identified 15 of these potential interchromosomal events in 13 3D7 Evo strains that resulted in partial gene deletion/duplication paired events, which also registered as deletion/amplification events in the CNV algorithm. In order to validate that these were indeed recombination events, the PRICE assembler was seeded with a single read from the suspected recombination region, and then de novo assembly was carried out using all reads that surrounded the alignment of the seeded read and its mate pair, as well as their respective mates. Altogether we were able to validate all 15 of these likely recombination events despite the hypervariability and high AT content (as high as 95% in these regions) that makes the specificity and uniqueness of these sequences very low. Figure 6 shows the sequence of one of the predicted regions as well as the matching sequence of the PCR product. To seek further evidence of genomic rearrangement, the 15 reassembled recombination events were validated by Sanger sequencing using primers that spanned the region. We found that the sequence of the de novo assembled fragment matched that of the Sanger sequencing product to within a one base pair difference in all 15 events. These assemblies spanned chromosomal sub-telomeric regions, and spliced together related genes. The PCR sequences of these assemblies and their clustal alignments are included in the supplemental information, and the mapping of each recombination event to each isolate is included in the supplement (see Additional file 1: Table S1). BLAST/CLUSTALOmega analysis verifies that these regions map to two separate chromosomes as expected.
Figure 6. Recombination events detected in long term culture samples. A. Reassembled sequences from recombination events indicate a splicing of 56,000 base pairs from the left sub-telomeric region of chromosome 1 on to the right sub-telomeric region of chromosome 10. The center sequence is the assembled recombination from the sequencing data, while the top and bottom are the respective reference sequences for each of the indicated chromosomal regions. Note the clear transition between alignments. B. Read pileup for the two regions containing the predicted recombination. Note the deletion (region 1) on chromosome 1 next to the breakpoint (region 2) and subsequent normal coverage (region 3). Similarly, chromosome 10 shows an amplification event (region 4) next to the breakpoint (region 5) and then subsequent normal alignment (region 6).
Additional file 1: Table S1. Presence (1) or absence (0) of potential recombination events in various isolates from the evolution experiments (Bopp et al, 2013) [6].
Format: PDF Size: 88KB Download file
This file can be viewed with: Adobe Acrobat Reader
A problem with using WGS is that it may be inaccessible to laboratories that are not strong in bioinformatics. To address this issue we integrated these modules into a program that we call Platypus (Figure 3). The pipeline integrates a number of other software programs, and these are referenced in full in this manuscript and in the software documentation. Platypus takes as input either unaligned FastA/FastQ sequencing data, or aligned data in the BAM format. SNVs, CNVs, and potential recombination events are output as annotated text files which can be cross-referenced with PlasmoDB or similar databases.
The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We have also tested it against both computational samples and actual data with known CNVs (both deletions and amplifications as verified by microarray) and it can detect the size and boundaries of these CNVs with a high degree of accuracy. The success of the Platypus software in both detecting real genetic variants and avoiding the reporting of false positives over a number of parasite lines can be attributed to its basis on first principles. The SNV detection was specifically designed only to use filters that accurately segregated true and false positives, and the robustness of this approach is evident, as there is a completely smooth transition between sensitivity/specificity levels when varying over the ideal filter set. The CNV detection was based on the fundamental theorem of digital signal processing, and indeed the assumptions of this field applies directly to the signals coming off a next-generation whole genome sequencer, complete with random and systematic biases. This streamlined package offers an initial starting point for the field to analyze and report these data in a consistent manner.
Availability and requirements
The program is platform independent and can be run on ordinary desktop computers: In our case all analysis and computer programming was done using Mac OSX 10.7.3 on a Mac Pro with 12 multi-threaded processors on 2 cores and 32Gb of 1066 MHz DDR2 RAM. Altogether 24Gb of RAM was made available to Java while the Platypus was running. We have made Platypus freely available as an open-source package at <http://sourceforge.net/projects/platypusmga/ webcite>.
Bp: Base pair; CNV: Copy number variant; DNA: Deoxyribonucleic acid; FastA: Fast (all) format; FastQ: Fast (all) quality format; GATK: Genome analysis toolkit; gDNA: Genomic DNA; Mb: Mega- base pair; PlasmoDB: Plasmodium database; Platypus: Pathogen lovers automated type uncovering software; SAM: Sequence alignment map; SNV: Single nucleotide variant; spp: Species pluralis; WGS: Whole genome sequencing.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MJM designed the program, aligned the sequences, wrote the computer algorithms, ran the validations, performed the human analysis, and wrote the initial drafts. SSS performed human analysis and ran validation experiments. ELF provided extensive manuscript preparation and program testing. SERB cultured parasites and analyzed the data for the evolution experiment. VCC assisted in program validations and aligning sequences and assisted in manuscript preparation. ATB assisted in the program development and validation. CWM cultured parasites and assisted in the analysis of the cladosporin experiment. JRW sequenced all the samples and assisted in the initial analysis. EAW helped design the program, assisted in validation strategies, and assisted with writing the manuscript. All authors read, reviewed, critiqued, and approved the full manuscript.
The authors would like to acknowledge Stephan Meister and Shinji Okitsu for advice on the manuscript. This work was supported by 7 R01 AI090141-02 and by the Bill and Melinda Gates Foundation (OPP1054480) and MJM was supported by funding from the University of California San Diego Medical Scientist Training Program, the University of California San Diego Genetics Training Grant, and fellowship from the Hertz Foundation.
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.
Genome Res 2010, 20:1297-1303. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, et al.: Genome sequence of the human malaria parasite Plasmodium falciparum.
Nature 2002, 419:498-511. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A, Doumbo O, Zongo I: Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.
Nature 2012, 487(7407):375-379. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA, Amaratunga C, Lim P, Suon S, Sreng S, et al.: Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.
Nat Genet 2013, 45(6):648-655. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Samarakoon U, Regier A, Tan A, Desany BA, Collins B, Tan JC, Emrich SJ, Ferdig MT: High-throughput 454 resequencing for allele discovery and recombination mapping in Plasmodium falciparum.
BMC Genomics 2011, 12:116. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Bopp SER, Manary MJ, Bright AT, Johnston GL, Dharia NV, Luna FL, McCormack S, Plouffe D, McNamara CW, Walker JR, et al.: Mitotic evolution of Plasmodium falciparum shows a stable core Genome but recombination in antigen families.
PLoS Genet 2013, 9:e1003293. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, Fullman N, Naghavi M, Lozano R, Lopez AD: Global malaria mortality between 1980 and 2010: a systematic analysis.
Lancet 2012, 379:413-431. PubMed Abstract | Publisher Full Text
Trager W, Jensen JB: Human malaria parasites in continuous culture.
Science 1976, 193:673-675. PubMed Abstract | Publisher Full Text
Benjamini Y, Speed T: Estimation and correction for GC-content bias in high throughput sequencing. Berkeley, CA USA: Tech Rep; 2011.
Hoepfner D, McNamara CW, Lim CS, Studer C, Riedl R, Aust T, McCormack SL, Plouffe DM, Meister S, Schuierer S, et al.: Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.
Cell Host Microbe 2012, 11:654-663. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rottmann M, McNamara C, Yeung BK, Lee MC, Zou B, Russell B, Seitz P, Plouffe DM, Dharia NV, Tan J, et al.: Spiroindolones, a potent compound class for the treatment of malaria.
Science 2010, 329:1175-1180. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Meister S, Plouffe DM, Kuhen KL, Bonamy GM, Wu T, Barnes SW, Bopp SE, Borboa R, Bright AT, Che J, et al.: Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.
Science 2011, 334:1372-1377. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Subgroup GPDP: The sequence alignment/map format and SAMtools.
Bioinformatics 2009, 25:2078-2079. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Volkman SK, Sabeti PC, DeCaprio D, Neafsey DE, Schaffner SF, Milner DA Jr, Daily JP, Sarr O, Ndiaye D, Ndir O, et al.: A genome-wide map of diversity in Plasmodium falciparum.
Nat Genet 2007, 39:113-119. PubMed Abstract | Publisher Full Text
Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS: PlasmoDB: a functional genomic database for malaria parasites.
Nucleic Acids Res 2009, 37:D539-D543. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Barry AE, Leliwa-Sytek A, Tavul L, Imrie H, Migot-Nabias F, Brown SM, McVean GA, Day KP: Population genomics of the immune evasion (var) genes of Plasmodium falciparum.
PLoS Pathog 2007, 3:e34. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Weise T: Global optimization algorithms–theory and application. La Jolla, CA USA: Self-Published; 2009.
Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ: Adaptive copy number evolution in malaria parasites.
PLoS Genet 2008, 4:e1000243. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kidgell C, Volkman SK, Daily J, Borevitz JO, Plouffe D, Zhou Y, Johnson JR, Le Roch K, Sarr O, Ndir O, et al.: A systematic map of genetic variation in Plasmodium falciparum.
PLoS Pathog 2006, 2:e57. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wilson CM, Serrano AE, Wasley A, Bogenschutz MP, Shankar AH, Wirth DF: Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum.
Science 1989, 244:1184-1186. PubMed Abstract | Publisher Full Text
Singh A, Rosenthal PJ: Selection of cysteine protease inhibitor-resistant malaria parasites is accompanied by amplification of falcipain genes and alteration in inhibitor transport.
J Biol Chem 2004, 279:35236-35241. PubMed Abstract | Publisher Full Text
Medvedev P, Fiume M, Dzamba M, Smith T, Brudno M: Detecting copy number variation with mated short reads.
Genome Res 2010, 20:1613-1622. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Yoon S, Xuan Z, Makarov V, Ye K, Sebat J: Sensitive and accurate detection of copy number variants using read depth of coverage.
Genome Res 2009, 19:1586-1592. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Robinson T, Campino SG, Auburn S, Assefa SA, Polley SD, Manske M, MacInnis B, Rockett KA, Maslen GL, Sanders M: Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.
PLoS One 2011, 6:e23204. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Smith SW: The Scientist and Engineer's Guide to Digital Signal Processing. In 1999. PO Box: California Technical Publishing; 2009.
Ruby JG, Bellare P, DeRisi JL: PRICE: Software for the targeted assembly of components of (Meta) Genomic sequence data.
G3 (Bethesda) 2013, 3:865-880. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Li H, Ruan J, Durbin R: Mapping short DNA sequencing reads and calling variants using mapping quality scores.
Genome Res 2008, 18:1851-1858. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dharia NV, Sidhu AB, Cassera MB, Westenberger SJ, Bopp SE, Eastman RT, Plouffe D, Batalov S, Park DJ, Volkman SK, et al.: Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.
Genome Biol 2009, 10:R21. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Neafsey DE, Galinsky K, Jiang RH, Young L, Sykes SM, Saif S, Gujja S, Goldberg JM, Young S, Zeng Q, Chapman SB, Dash AP, Anvikar AR, Sutton PL, Birren BW, Escalante AA, Barnwell JW, Carlton JM: The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum.
Nat Genet 2012, 44(9):1046-50. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dharia NV, Bright AT, Westenberger SJ, Barnes SW, Batalov S, Kuhen K, Borboa R, Federe GC, McClean CM, Vinetz JM, Neyra V, Llanos-Cuentas A, Barnwell JW, Walker JR, Winzeler EA: Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes.
Proc Natl Acad Sci U S A 2010, 107(46):20045-50. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS, McGrath SD, Wendl MC, Zhang Q, Locke DP: BreakDancer: an algorithm for high-resolution mapping of genomic structural variation.
Nat Methods 2009, 6:677-681. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


text:
This security rail is a useful addition to the Perch Bunk Bed. The rail provides protection from night-time falls, which allows a a younger child to skip the toddler bed and move straight from a crib to the lower bunk of the Perch.
Sustainably produced in Europe Made of Baltic birch plywood Finishes are non toxic and free of VOC health hazards Made under European environmental standards Packaging made of recycled cardboard Responsible recycling through licensed collecting facilities Other info: The mattress for the lower bunk must be between 5-8"d when using the guard rail Please note that this security rail is not for use with the Sparrow Twin Bed

subset: commoncrawl


text:
Hey hey, purchased the truck I've wanted for a while, well, one of them. 1986 Chevy C10 Silverado V8 5.0 305 auto and loaded. I'm attempting to work on it myself, will see how it goes. My Dad was always under hoods. Brings back memories! I'm glad I paid attention a little. I'll be asking a ton of questions since he isn't there to answer mine anymore. Thanks!
Welcome to the club!!! Any questions just post in the appropriate forum and usually someone will have an answer.
We are the Borg. Lift your vehicle and surrender your keys. We will add your biological and technological distinctiveness to our own. Resistance is futile. Erm, I mean ... welcome to GM Truck Club! :great:


text:
News and Views From Around the World | About Us Africa Americas Asia-Pacific Europe Middle East Front Page |
World Newspapers and MagazinesOne of the web's largest and most comprehensive directories of World Newspapers and Magazines, sorted by Country, region, and political affiliation. DenmarkNewspapers and Magazines OnlineÅrhus Stiftstidende Berlingske Tidende Bornholms Tidende Børsen Copenhagen Post, The Ekstra Bladet Information Politiken Weekendavisen Is this information incomplete or inaccurate? |
Copyright © 1997-2015 Worldpress.org. All Rights Reserved. Privacy Notice Terms & Conditions Front Page |


text:
Tea heals the soul.�
A gift to make tea-lovers rejoice! This gift offers a wide variety of teatime edibles, and an assortment of fragrant teas for brewing. Items are packaged in a lovely handled fabric basket. Includes complimentary scissors for easy opening and arrives decorated with a satin ribbon.
This gift includes the following: Matching ceramic teapot & cup, organic teas in several enticing flavors such as white tea, green tea, black tea, jasmine, and peppermint, assorted gourmet cookie tea accompaniments, green tea caramels. Measures 12" x 9" x 8".
Tea Time Basket has been purchased most frequently by our customers for the following occasions - Get Well, and Thinking of You.
If any substitutions are necessary, they will be made with items of equal or greater value.
With over ten years in the online gourmet food and gift basket industry, Gourmet Grocery Online has created a solid name of higher quality gourmet food gift than you will find anywhere online. We want to offer you the very best gourmet gift and basket that are sure to make an impact on your recipient. Nationwide delivery to all 50 states. Shop our gourmet gifts! Send gourmet food, gift baskets, fruit baskets, wine and gourmet food gifts. The best online source for mail order gourmet specialty dinners, food gifts & flowers. We will gladly send your mail order gift purchase to any US city. We offer free shipping and same day delivery on select items. Delivery available in all US states.
© 2004 -2015 Gourmet Grocery Online All Rights Reserved


text:
Welcome, Sign In
Pope Francis Calls for 'Peace and Concord' in Middle East
Description:At St. Peters in the Vatican, an outdoor mass to mark the end of what the church is calling it's 'Year of Faith.' Church leaders from throughout the Middle East gathered to pray for peace in their region. (1:03)
Source: Reuters
Tags: middle east violence , Pope Francis , reuters , reuters news


text:
I have to do a demonstrative speech for public speaking on June 16th. I want to do it on something photography. Any suggestions?
Some various ideas I have so far is stuff like:
-How to do a color splash picture
-How to set up a camera for a session
(2nd is lame, I know this)
Tags: demonstrative, photography, public, speaking, speech
Depends on what equipment you have available, how large the group is, and if they care about photography or not. Unless of course your job it to bore them.
If you have a projector attached to a computer (or a small enough group they can just huddle around a monitor) you could do all sorts of things.
Depth of field,
Painting with light,
Flash vs. no flash portraits, or fill flash, or reflectors,
Long vs. short lenses.
How to do macro photography,
There are lots of possible topics.
@CameraClicker- not really interested in photography. but it has to be a 15 minute speech (YIKES!) thanks for the ideas so far
@Christopher good idea but not sure if I can make that into a 15 minute discussion
Hey Jessica,
Most everyone has a digital camera and they go to weddings or take pictures of their friends, so maybe just a simple introductory, not too technical speech about composition? Or another way to say it is INTENT. A picture should tell a story.
I think that one of the things that is so interesting to me is what makes an interesting picture, and there are a few things that you can teach about that can possibly revolutionize even one person's creativity or spark an interest in photography. I remember one of the most powerful illustrations was in a basic class I went to where they showed a picture of a capitol building with a girl on the steps. It didn't really tell a story and you couldn't really tell what it was about but then when you zoomed in and saw that the girl was looking at the person taking the picture, it became an interesting picture. That's something that could be shown on the projector or power point, too. Just an idea ....
I hope you find just the right topic that you are passionate about and that is easily grasped by your audience. If you're passionate, chances are they will be very interested, right? Good luck!
We offer free daily photography tips for all experience levels. Click Here to Subscribe Now!.
© 2015 Created by PictureSocial.com.


text:
ServicesWe've been providing our members with a full range of financial services since 1956!
We're a non-profit cooperative financial institution completely controlled and owned by the members who save, borrow and conduct other financial business here.
Because we're non-profit, you share in the earnings of the credit union through higher dividends on your savings and reduced rates on your loans. Members pool their savings - and from these savings, make loans to each other with the help of the credit union staff.
Extended Forecast for Narrows, VA US
Celco Federal Credit Union PO BOX 361 Narrows, Virginia 24124 (540)921-2700


text:
Isaac Bennett ran for a career-high 240 yards and three touchdowns as Pittsburgh held off Old Dominion 35-24 on Saturday night.
The Panthers (4-2) overcame an early 10-point deficit behind Bennett. Making his first start of the season, Bennett scored three times in a 12-minute span in the second quarter. Quarterback Tom Savage passed for 104 yards and a touchdown and also ran for a score as Pitt finally shook the Monarchs (4-3) late.
ODU quarterback Taylor Heinicke threw for 312 yards and two touchdowns but couldn't lead the Monarchs to their first win over a Bowl Championship Series opponent. ODU cut an 18-point deficit to four late in the third quarter, but Savage's 5-yard touchdown run gave Pitt enough cushion to survive.
It was hardly pretty on a night as the teams combined for 24 penalties and 221 penalty yards.
The Monarchs are in the midst of transitioning from the Football Championship Subdivision to the Football Bowl Subdivision. They'll be in Conference USA next fall, a leap the school insists it's ready to make even though the program didn't even exist five years ago.
For long stretches on Saturday, it certainly looked like it.
Pitt appeared to be firmly in control when Savage hit Manasseh Garner for a 21-yard touchdown that gave the Panthers a 28-10 lead. The Monarchs rallied behind Heinicke. The reigning Walter Payton Award winner - given annually to the best player in the FCS - found Blair Roberts for a 47-yard score to make it 28-17.
ODU executed a perfect onside kick, with kicker Jarod Brown falling on the ball after it rolled the required 10 yards. Five plays later Antonio Vaughn sprinted into the end zone to pull the Monarchs within 28-24 with 4:35 left in the third quarter.
The Panthers, however, steadied themselves behind Savage. The senior endured eight sacks in a loss to Virginia Tech last week and while he was hardly crisp - completing 11 of 18 passes - he did enough to lead Pitt on a five-play, 47-yard touchdown drive. He covered the final 5 yards himself, spinning between two defenders for his second rushing touchdown in as many weeks.
It was a score the Panthers needed.
ODU raced to a quick 10-0 lead behind Heinicke, who hit Marquel Thomas with a 15-yard touchdown pass to put the Monarchs in front. Brown then drilled a 54-yard field goal through the notoriously tricky winds at Heinz Field, the longest kick in the stadium's 12-year history.
The beginning was eerily similar to Pitt's humbling 31-17 loss to FCS-member Youngstown State in Paul Chryst's head coaching debut last year. The ending, however, was different.
Rather than panic or press, the Panthers turned to Bennett. The junior appeared to have lost any shot at grabbing the starting spot earlier in the season after James Conner's hot start. Conner, however, struggled in recent weeks and was limited in practice all week due to a shoulder injury sustained in last week's loss to Virginia Tech. Conner dressed but spent the entire night on the sideline watching Bennett go to work.
Bennett bolted up the middle for a 25-yard touchdown to get Pitt on the board early in the second quarter. A 56-yard punt return by Kevin Weatherspoon set up a 3-yard burst to give the Panthers the lead with 7:40 remaining in the half. Working behind a line that made sure he was rarely touched until he was into the secondary, Bennett put Pitt firmly in control late in the half, capping a 63-yard drive by darting up the middle for a 16-yard touchdown to make it 21-10 at the break.
Follow Will Graves at www.twitter.com/WillGravesAP
Related Stories
Member Comments
Please note by clicking on "Post comment" you acknowledge that you have read the Terms of Use and the comment you are posting is in compliance with such terms. Be Polite. Inappropriate posts may be removed by the moderator.
NCAA FB Headlines
Fox Sports Store
itemName | itemURL | imageSrc | price | itemDescription |
Nike Florida Gators Gridiron 2012 Team Issue T-Shirt - Royal Blue | http://shop.foxsports.com/FOX_Florida_Gators/browse/featuredproduct/759777/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_759000/ff_759777_xl.jpg&w=100 | 24.95 | Nike Florida Gators Gridiron 2012 Team Issue T-Shirt - Royal Blue |
Nike Michigan State Spartans Football 2012 Team Issue T-Shirt - White | http://shop.foxsports.com/FOX_Michigan_State_Spartans/browse/featuredproduct/760171/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_760000/ff_760171_xl.jpg&w=100 | 24.95 | Nike Michigan State Spartans Football 2012 Team Issue T-Shirt - White |
Nike USC Trojans Fight On My School T-Shirt - Cardinal | http://shop.foxsports.com/FOX_USC_Trojans/browse/featuredproduct/761373/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_761000/ff_761373_xl.jpg&w=100 | 24.95 | Nike USC Trojans Fight On My School T-Shirt - Cardinal |
Nike Alabama Crimson Tide Elite Force Coaches Sideline Performance Polo - Crimson | http://shop.foxsports.com/FOX_Alabama_Crimson_Tide/browse/featuredproduct/829709/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_829000/ff_829709_xl.jpg&w=100 | 64.95 | Nike Alabama Crimson Tide Elite Force Coaches Sideline Performance Polo - Crimso.. |
Nike LSU Tigers Elite Force Coaches Sideline Performance Polo - Purple | http://shop.foxsports.com/FOX_LSU_Tigers/browse/featuredproduct/829740/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_829000/ff_829740_xl.jpg&w=100 | 49.99 | Nike LSU Tigers Elite Force Coaches Sideline Performance Polo - Purple |
Nike Oklahoma Sooners Legacy 91 Dri-FIT Adjustable Coaches Cap - Crimson | http://shop.foxsports.com/FOX_Oklahoma_Sooners/browse/featuredproduct/830935/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_830000/ff_830935_xl.jpg&w=100 | 21.95 | Nike Oklahoma Sooners Legacy 91 Dri-FIT Adjustable Coaches Cap - Crimson |
adidas Notre Dame Fighting Irish Hot Sideline Jacket - Navy Blue | http://shop.foxsports.com/FOX_Notre_Dame_Fighting_Irish/browse/featuredproduct/861243/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_861000/ff_861243_xl.jpg&w=100 | 69.95 | adidas Notre Dame Fighting Irish Hot Sideline Jacket - Navy Blue |
adidas Nebraska Cornhuskers Sideline Hot Quarter Zip Jacket - Scarlet | http://shop.foxsports.com/FOX_Nebraska_Cornhuskers/browse/featuredproduct/861247/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_861000/ff_861247_xl.jpg&w=100 | 69.95 | adidas Nebraska Cornhuskers Sideline Hot Quarter Zip Jacket - Scarlet |
Nike Alabama Crimson Tide 2012 BCS National Champions Players Locker Room Snapback Hat - Charcoal/Black | http://shop.foxsports.com/FOX_Alabama_Crimson_Tide/browse/featuredproduct/1087345/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_1087000/ff_1087345_xl.jpg&w=100 | 29.95 | Nike Alabama Crimson Tide 2012 BCS National Champions Players Locker Room Snapba.. |
Nike Alabama Crimson Tide 2012 BCS National Champions Locker Room T-Shirt - Ash | http://shop.foxsports.com/FOX_Alabama_Crimson_Tide/browse/featuredproduct/1093431/source/foxsports_prsp_ca | http://images.footballfanatics.com/FFImage/thumb.aspx?i=/productImages/_1093000/ff_1093431_xl.jpg&w=100 | 24.95 | Nike Alabama Crimson Tide 2012 BCS National Champions Locker Room T-Shirt - Ash |


text:
Insects Get a Tasty Makeover February 29, 2008 Finding a bug in your meal is usually not a pleasant experience. In a popular restaurant in L.A., however, creepy crawlers are becoming haute cuisine. Listen Loading… Download Playlist
Business Starbucks Baristas Relearn Art of Coffee-Making February 27, 2008 Starbucks closed all its U.S. stores for three and a half hours Tuesday to give employees a refresher course in the art of making coffee. Listen Loading… Download Playlist
Kitchen Window The Shad Are Running February 27, 2008 The forsythia is blooming, and that, fishermen say, means just one thing: The shad are in the river — and at the market. Bonny Wolf loves the fish, but she's just wild about the roe. She shares her favorite ways to prepare the seasonal delicacy.
Dinner Decodes the Mystery of Taste February 26, 2008 Scientists Stuart Firestein and Terry Acree host "How and Why Things Taste the Way They Do" — meals where diners learn the latest discoveries in olfactory science as they eat. Listen Loading… Download Playlist
The Amazing Power of the Beet February 26, 2008 One of the most e-mailed stories at NPR.org is about beets. The root vegetables aren't just for salad anymore — they are also being used for melting snow and ice. Reporter Amanda Rabinowitz at WKSU in Kent, Ohio, explains. Listen Loading… Download Playlist
Law NYC Eateries Fight Law Requiring Calories Be Posted February 26, 2008 Restaurants in New York City are challenging a law that goes into effect next month that requires them to post calories on their menu boards. The restaurants say the law would violate the First Amendment by forcing them to put what amounts to a message on their menu boards. Listen Loading… Download Playlist
Taking the Pain Out of Cooking with Onions February 25, 2008 Cooks who love onions but hate what the vegetables can do to their eyes may soon find relief, thanks to a group of scientists in New Zealand. The team says it has genetically altered an onion so it's missing an enzyme that's essential for an onion's pungent punch. Listen Loading… Download Playlist
The Family Dinner Deconstructed February 25, 2008 NPR reporter Alix Spiegel explores the ritual of the family meal. Listen Loading… Download Playlist
The 80th Annual Academy Awards And the Winner for Best Hors d'oeuvre Is ... February 24, 2008 At Sunday's Oscars, actors, directors, and production teams will be awarded for their hard work and talent. But Weekend Edition Sunday's food commentator Bonny Wolf thinks food should be remembered when praising supporting roles. Listen Loading… Download Playlist
Environment Haagen-Dazs Gives $250K for Bee Research February 21, 2008 Haagen-Dazs is donating a quarter of a million dollars to the University of California and Penn State to study the disappearance of honeybees. Forty percent of the ice cream company's flavors require the work of bees. Listen Loading… Download Playlist
Brits Asked to Ease Migrant Rules to Save Curry February 21, 2008 The favorite takeout dish in Britain is chicken tikka masala. But a new law restricting migrant workers from South Asia is putting Indian restaurants out of business. Authorities are being urged to ease the restrictions to avert a crisis in the restaurant industry. Listen Loading… Download Playlist
Books 'Fortune Cookie' Offers New Taste of America February 20, 2008 Growing up, Chinese-American writer Jennifer 8. Lee noticed the food at Chinese restaurants differed greatly from what her mother served at home, and an obsession was born. The result is a book called The Fortune Cookie Chronicles. Listen Loading… Download Playlist
The Science of Beating Eggs February 20, 2008 One of the most e-mailed stories at NPR.org is this look at the the kitchen chemistry of souffles. Listen Loading… Download Playlist
Kitchen Window When Pizza Gets All Dressed Up February 20, 2008 Why mess with the simplicity and inexpensiveness of Domino's? Because getting creative with unconventional ingredients means infinite ways to enjoy one of the world's most appealing foods. After all, it's just pizza. It's easy as pie.
Recipes from the President's Kitchen February 19, 2008 Hercules, a slave of George Washington, and James Hemings, owned by Thomas Jefferson, began a long connection of presidents and their African-American cooks. Read recipes (and a story) from some of those cooks.


text:
108 comments found
Great work!
I noticed that the portfolio and gallery seem to behave a bit odd IMO .
When either page first shows up, you see all the images. Then about 1 second later, the ajax spinner shows up on all the images and reloads even though they were already visibly loaded. This behavior is easily reproducible by navigating between the portfolio / gallery pages.
I then realized that it seems like all the images in the site that are being lazily loaded are exhibiting this inefficient, annoying behavior. However if it's intended to work this way, please disregard.
Hi there,
The first time when you visit a page it works like it's intended but if you visited the pages before the images are not preloaded anymore because they're already downloaded in the cache and they display right away. That's why you see this.
Of course. However, my point was that if they're already loaded / visible to the visitor, they shouldn't need to be loaded again. If they do for whatever reason, the images should at least be hidden from view. After that, the ajax animation can be shown until the images appear. Otherwise, this will likely be confusing to clients / visitors. Regardless, it seems that you're aware of this behavior and that it's intended to work this way so I'll trouble you no more.
Good luck with sales!
No trouble at all. I'm always keen to listen. This way I can find bugs and fix my themes . Anyway, I'll def. fix this in the next update.
nice work…
Thank you.
Fantastic theme – and fantastic support! Designer has been incredibly helpful in sorting out technical issues (of my own doing – not the theme!). Thank you so much for everything.
Sweet stuff. Keep rollin them out.
Thank you.
Nice work!
Care to share the access to docs?
Oops Does it come a site builder? OR I am asking too much?
Hi there,
The theme documentation can be found in the "help" folder.
Could you please explain a little bit more what do you mean by "site builder"?
I don't have a 'help' folder because I am yet to buy the theme.
The site builder is basically part of the backend where most of the site could be built by simply drag and drop and no shortcodes used. If I can see the help doc I might be able to see how easy it is to work with the theme.
Also: 1.when I view it on my smartphone (Windows), it does not respond.
2. It crashed on Chrome and reloads. Works fine with Firefox but a lot of the content including the slider is missing in IE! Any clue?
Hi there,
I understand now, but no, the theme does not have a drag & drop page builder.
1. Unfortunately I have no Win. mobile environment to check right now, but I'lm looking forward to find a solution.
2. This is weird. I've double checked on most browsers including in IE down to IE7 and it works fine for me on my PC, laptop, iphone and ipad.
Beautiful theme… Does it come with an XML file for import?
Hi there and thank you for your interest.
Yes, it comes with the demo page .xml dump file.
I couldn't wait, so I bought it last night I'll be using it shortly!
Have a quick question before I purchase. On the portfolio page I noticed you have to roll over an image which brings up a little white bar in the bottom portion. Then you have to click on the little icons to get to the page or posting for it. The bar can stay or whatever but my question is can I just make the whole image clickable for ease of use to my visitors?
Reason being is I would want to use the portfolio section for a product page where you just click and image to get to the next page to download a spec sheet and get more info on it.
Also, to add I love how the portfolio section is sortable. Would not want to lose that functionality. Do you have a support forums for your theme?
Hi there and thank you for your interest.
Yes, it's possible to add a link to the whole image. It requires some minor changes in the code, but I can do it for you.
I offer support through this comments section and often through email.
Great so if I purchase this would I just email you about making that change? Thanks for you help!
Yup, just send me a message through the contact form on my profile page and I'll reply you on your email.
Where do i find help/documentation.
> How to do Homepage customization? > I want to make Blog Posts wider
Hi there,
1. You can find the docs. in the archive you've downloaded > help > index
2. Do you want to customize the homepage or the blog page?
Hello Ginaalessia,
Can you send me email too as voxana ask
I have additional questions :
1. When i install disqus plug-in -> its working but comment value its not working in blogpage
2. Can you guide me to Clone "Lastest Work Section" block – i want add another one like "our work, our client and etc."
Thank you verymuch
Hi there,
Go to the theme's folder and edit the file named "three-columns-portfolio_page.php".
On line 73 you'll find this code:
<img src="<?php echo $image_url; ?>" alt="" title="" width="250" height="145" />
Replace it with this:
<a href="<?php the_permalink(); ?>"><img src="<?php echo $image_url; ?>" alt="" title="" width="250" height="145" />
1. WordPress is pulling the comments count from it's commenting system so if you're using a comment plugin it won't show the comments count.
2. If you want to add additional sections like that you need to have so php/html/css skills. The theme only supports one portfolio section so you must make another one (keep just the styling of the existing one) and set the "other" sections to pull the items from the portfolio newly created.
This theme both looks very nice and appears to have a lot of flexibility. Question – is there a capability to do any sort of mega dropdowns with this theme.
Hi there and thank you for your interest.
The theme does not support mega dropdowns by default but it could work with a plugin. Though, I'm not 100% sure and I do not guarantee it because I've never tested it.
I am wondering why are the slider images distorted when you re-size the page? Is there any way to fix this?
Hi there,
What do you mean by distorted? Could you send me a screenshot?
The URL of the actual installation is http://dmrgraphics.net/tarryhere. Here is a screen shot of what I am talking about.
http://dmrgraphics.net/tarryhere/screenshot.jpgIf you compare the slider images, the re-sized page is out of proportion.
This is another question relating to this, is there a way when the page is re-sized for the slider image to be aligned to the left instead of centered?
Thanks so much.
Oh, I understand. This is a feature of the theme. It is called "responsive design" – it adapts the content on different screen sizes (especially for mobile devices like phones and tablets).
If you don't want this feature let me know and I'll tell you how to remove it.
ps. you did a nice job customizing the theme.
Thanks for the quick response. I'm not sure your following me. I want the responsive design, the images in the slider are distorted (stretched, see screen shot http://dmrgraphics.net/tarryhere/screenshot.jpg), this is also the case in the demo of the theme.
I think it might be either a problem with the theme or something I did. Do you know what I am talking about when I say the images in the slider are stretched?
Thanks so much.
Sorry when I pasted the screenshot URL in the last post it was wrong… http://dmrgraphics.net/tarryhere/screenshot.jpg
I ended up using a responsive slider plugin that will work much better for what I need the slider to do (http://devpress.com/plugins/responsive-slider/). I'm not able to use video but this will work for me right now.
Hi, can I have paging on my gallery page? Could You please explain how do I use portfolio? I can't get i work:-(
Hi there,
1. WP currently doesn't support gallery pagination. Though, you can use a plugin to make it work. Here you can find more details:
http://wp.tutsplus.com/articles/tips-articles/quick-tip-paginate-your-wordpress-gallery/2. Step 1
Create a new page (eg. Portfolio) and on the right of the page you'll see a box called "Page Attributes". From the option "Templates" select "3 Columns Portfolio". Save and publish the page blank.
Step 2
Add items to the portfolio. Go to your WP dashboard > Portfolio > Add New. Insert your content and select the type of the item (image, audio or video). Set up a featured image on the right of the page. Publish. Done.
Please check your email
I have regarding question about 2 unique small slide box in homepage.
Cheers too
Hi Gina! I wanted to see if you could tell me how to increase the space for a logo. I am using a logo that is larger then your demo, and when uploading, the logo now sits over top of the navigation bar. If you can let me know where I can increase the space so the logo fits properly I would greatly apprecaite it! Also.. is there a way I can add my own custom backgrounds instead of the ones that are included?
Thanks so much for your help!
Hi there,
1. To change the height of the header go to the theme options page > Styling Options and paste this in the "Custom CSS " box:
#header {
height: 140px;
140px is the default height. Adjust it to fit your needs.
2. For the logo margins paste this in the "Custom CSS " box:
#site_title {
top: 35px;
left: 40px;
These are the default values. Adjust it to your needs.
3. To change the bg. you need to edit the images responsible for it. In the theme's folder go to "images" folder and edit the files named: "body_bg.jpg" (for header and footer) and "html_bg.jpg" for the content bg. You can also find the css code responsible for the bg in the "styles" folder and editing the style-sheet you're using (eg. blue.css) just at the beginning of the file.
Perfect! Thank you so much!!!
looking to add a title and divider line (just like the regular page title and divider line) on a page about half way down to separate the services. Is this possible? was looking for a divider line shortcode but the two divider shortcodes both seem to just be empty spaces. Please let me know. Thanks for your help!
Go to the theme's folder > styles and edit the style-sheet you're using (eg. blue.css) Look for this code on line 2035:
background: url(../images/image_divider.png) repeat-x;
Replace it with this:
border-bottom: 1px solid #E8E8E8;
Save. Done.
Now, simply use the [divider type="image"] shortcode.
Sorry to keep bothering you, but one last thing I am coming across. My home page is not showing. I've added the text from the file in the help folder, I've changes the template to homepage, and I've changed my settings> reading to show a static frontpage as home, but nothing is showing still. any thoughts on what I am doing wrong? thank you so much again for your help!!!
Make sure that on the theme options page > Homepage Options you use the "Homepage Layout Manager" to enable/sort the content of the page.
sorry i missed that! That did it!
I added in my own icons for the home page intro area, but they are showing up as ""/"/"/" instead of the icon. when I add copy and paste the shortcode to a page, it showing up no problem. Any idea why it is showing as /"/" on the home page instead of the icon/picture? PM me if you need urls
That's happening because your php config. has the "Magic Quotes" enabled.
Just replied to your email with more detailed instructions & info.
You are wonderful!!! Thank you SO MUCH !!!!
I wasn't sure how to disable the magic quotes, but the line of code you sent me to replace did the trick!! Thank you so much again! Awesome theme! I'll be getting your Gotham theme for my next project


text:
Sponsored by the U.S. Department of Energy Human Genome Program
Genome News Archive Edition |
Available in PDF In this issue... DOE '99 Oakland Highlights
Genome Project In the News
Microbial Genomics
Ethical, Legal, and Social Issues Informatics Web, Other Resources, Publications
Funding Meeting Calendars & Acronyms
Microbial GenomicsThermotoga Sequence PresentedEarly Analysis Suggests Gene Exchange Between Kingdoms In May, researchers led by Karen Nelson (The Institute for Genomic Research) reported obtaining the complete 1.8-Mb genomic sequence of the heat-loving bacterium Thermotoga maritima, first isolated from geothermally heated marine sediment in Vulcano, Italy. Early analysis reveals some unusual features that could affect our understanding of how earth's simplest life forms evolved. The three major life groups or kingdoms are eubacteria and archaea, which include the simplest life forms lacking a central nucleus; and the more complex eucaryota, which include animals and plants. T. maritima has been considered one of the deepest and most slowly evolving lineages in the eubacteria kingdom. Authors of the May 27 article in Nature (399, 323-29) reported that almost a quarter of T. maritima's genes are similar to those found in archaea, with 81 archaeal-style genes clustered in 15 genomic regions. These results pose new questions about defining organisms that have mosaic-like genomes, with features shared across two domains. The authors note that these findings do not necessarily reflect a closely shared common ancestor but could point instead to lateral gene transfers. Because of growing evidence for a high frequency of gene transfers and a lack of agreement in different phylogenetic (evolutionary) analyses on individual genes, the authors suggest that sequence comparisons of individual genes may be inaccurate indicators of organismal evolution. Relationships among the eubacteria and archaea willbe understood better as other microbial genomes are fully sequenced and analyzed. The electronic form of the newsletter may be cited in the following
style: |
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.


text:
Binary package "xkbsel" in ubuntu jaunty
Tool for defining, selecting, and indicating XKB keyboards.
xkbsel is a framework for defining, selecting, and indicating XKB keyboard
mappings, without the need to modify X11 distribution in any way.
This package contains the xkbsel basic files and binaries, including the
Athena keyboard switching applet.
Homepage: http://


text:
Expect to have a nervous brickdown while playing this Brick Break esque game! This game is filled with 135 levels and 10 completely different game modes, and all of them test your smarts and speed! This is a great and addicting pick up and play game for everyone!Official URL
Official Site- Genre: Puzzle Games
- Developer: Arkedo Studio
- Publisher: Eidos
Released North America Jun 26, 2007 Europe September 2007


text:
Voted Pittsburgh's #1 Exotic Club
(412) 281-7703
Located in the Cultural District, Downtown Pittsburgh, an easy walk from the Convention Center, Stadiums, Hotels, & Restaurants.
135 9th Street
Pittsburgh, PA 15222
Mon – Sat : 12pm – 2am
Sun : 3pm – 2am
Monday-Friday 4:00-6:00 | Saturday Noon-2:00
Happy Hour
Couples Nights
Couples Get In For Free.
Amateur Nights
Think you have what it takes!? Amateurs take the stage, first prize is $150.00 cash! Second prize is $50.00 cash!
College Nights
Each Wednesday and Sunday night is college night. FREE Admission for college students! Must present student ID at the door. Must be at least 21 years or older to drink alcohol. Those ages 18-20 may be admitted when accompanies by someone at least 25 years old.
135 9th Street
Downtown Pittsburgh


text:
What will atomic energy do for me? (1954)
(Gefunden bei gh2u.tumblr.com)
"Mass is power, says Dr. Albert Einstein. The arguments he presents are a little too hard for the public to follow; but here are some of his conclusions about the power that lies in the atom, if and when we can ever make use of it. But Einstein also says he feels sure that it will not be possible to convert our familiar matter into energy in the manners shown. This will relieve coal miners and oil well operators."
(Gefunden bei blog.modernmechanix.com)
Aus: Tout l'univers n° 9 (Éditions Hachette, 1961). Illustration von A. Feddini
(Gefunden bei mondorama2000)
TES-3, soviet mobile nuclear power plant. In: 50 years of the soviet science and technology exhibition. KGM, Budapest, 1967.
(Gefunden bei scanzen.tumblr.com)
Technicians in plastic protective suits and face masks repair pressure valve at atomic energy plant. LIFE Magazine, 5. April 1954
(Gefunden bei scanzen.tumblr.com)
Paul Franz Brandwein - You and science: Science for better living.
New York, Harcourt Brace & Company, 1955.
(Gefunden bei atompunk.tumblr.com)
Carlyle B. Haynes, When God Splits the Atom. An explanation of the meaning of the discovery and the principles of nuclear fission in the light of the ancient prophecies of the Bible. The Southern Publishing Association (1946)
R. A. Knox, God and the Atom. A Catholic theologian explores the moral dilemma of Hiroshima and atomic weapons in general. Sheed & Ward, 1945
T. V. Smith, Atomic Power and Moral Faith. Foreword by Robert J. Bernard. Claremont College, 1946
(Gefunden bei osulibrary.oregonstate.edu)
On the banks of the Pacific Northwest's greatest river lies the Hanford nuclear reservation, an industrial site that appears to be at odds with the surrounding vineyards and desert. The 586-square mile compound on the Columbia in eastern Washington is known both for its origins as part of the Manhattan Project, which made the first atomic bombs, and for the monumental effort now under way to clean up forty-five years' of waste from manufacturing plutonium for the U.S. nuclear weapons complex. Atomic Frontier Days tells a complex story of production, community-building, politics, and environmental sensibilities
Atomic Frontier Days parade in Richland, Washington during the early days of the Hanford site.
(Gefunden bei zestyblog.tumblr.com)
View of a worker holding reprocessed plutonium recovered from retired nuclear weapons. (1973). - Rocky Flats Plant, Golden, Jefferson County, CO.
(Gefunden bei scanzen.tumblr.com)
These complex beauties are from a collection of Nuclear slide rules and dials gathered together by ORAU (Oak Ridge Associated Universities). They've got dials to calculate radiation dosage, Nuclear bomb effects, Nuclear weapon effects, x-ray exposure, decay rates, treatment times and Radium exposure. A much more useful collection than mine, which mainly consists of vintage promotional pieces!
See the full collection of Nuclear slide rules here and see some of mine here.
(Gefunden bei deliciousindustries.blogspot.de)
G. Peyton Wertenbaker; "The Man From the Atom" (Amazing Stories, May 1926)
(Gefunden im Flickr-Photostream von finsbry | und bei isfdb.org)


text:
Go for it, you will regret going back to cigs. Good luck and whatever you use, you can get lots of help. Just stay away from cheap knock offs. I had them given to me and they suck.
Go for it, you will regret going back to cigs. Good luck and whatever you use, you can get lots of help. Just stay away from cheap knock offs. I had them given to me and they suck.
Started with an ego, went to an innokin cool fire, svd, nzone clone, and now on an Origin mod with a nimbus rda and loving it◀
OK, I went with the Provari and Kayfun , researched the hell out of it, took all of you guys advice into consideration, and I'm just waiting for it to come in Today is day 32 with no cigs, and I am so happy about that! Yay me Thank you all for your help and support, you rock
If you bought the Kayfun, make sure you have Kanthal wire, some wicking material and an ohm meter. It does come with a little but you will need more.
Plus, some of the coil builds you see on Youtube will not work on the Provari...........if the ohms are too low, the Provari will not fire.
Tip of the Week: Don't lick the mixer beater with it running
In response to the OP, don't buy a provari based on mysticism that seems to surround them; some seem to imply there is something special about them, and it would make them appear magical, as if powered by pixie dust and unicorn farts.
From what I have read, it appears to be a durable device with an easy to grasp user interface. It also seems to have a rather nice way of regulating power.
However, the provari (or any battery by itself) doesn't seem to be addressing the OP's concern in simulating the experience in smoking.
I personally do not like pg at all. The route I took is staying with top coils for a warmer vape and going with higher voltages/wattages.
My recommendation to the OP would be to find a decent top coil head, such as the vivi nova or iclear30... And to be careful to make sure they are not the bottom coil variants of those tanks. The only reason I'm not suggesting cartos is they don't seem to like the 100% vg that much, and we don't want to create extra hassles for a new vaper. In fact, I suggest two tanks.
Next I would suggest getting a couple c-twists or spinners, to play with variable voltage (and see if this gives the desired results). Mods are fun, but not always practical for out and about. Even if the provari ends up being the chosen path, these vv egos will be good for out and about.
Next I would suggest finding at least two e-juices that are palatable, to prevent the malady of "vapers tongue". If you vape the same flavor consistently, and without variation, you will stop being able to taste it. This is also why I suggested two tanks earlier.
Happy vaping!


text:
Harriet Wilson |
CHAPTER V, DEPARTURES Life
is a strange avenue of various trees and flowers; JAMES'
visit concluded. Frado had become 53 a
portion of her time. In the absence of the 54 none
of the family around to be merry with, 55 into
the river, and swimming across, remained But
to return. James married a Baltimorean Two
young men seemed desirous of possess- 56 wants,
and watch with interest her improvement In this dilemma, a visitor came to Aunt 57 Abby's;
one of her boy-favorites, George Means, George
returned home, and Jane endeavored 58 George
could not rest after his return, he "What
would mother say?" queried Jane, as "Not much to comfort you." "Now,
aunt, George is just such a man as I "Then
don't marry him," interrupted Aunt "Mother will make me." "Your father won't." "Well,
aunt, what can I do? Would you "Yes, answer it. Tell him your situation." "I
shall not tell him all my feelings." 59 his
manner or appearance; that she was under "Have
you no wish to know the real state of "No!
Go, I say! go!" and Henry opened He
retired to Aunt Abby's. Henry soon fol- 60 upon
Nab, and her satanic influence, told her "Go to your room, Miss Jane," she continued. "Don't
let me know of your being in Nab's for The
storm was now visible to all beholders. Jane
sent Henry a letter of dismission; he her To brave her mother's fury, nearly overcame 61 her,
but the consolation of a kind father and |


text:
- Be the first to Review
Your rating for Museo del Baile Flamenco
Expert Review of Museo del Baile Flamenco
Romance Review of Museo del Baile Flamenco
RomanceExpert Tips & Helpful Hints
- Stop here before visiting one of Sevillas's many tablaos (flamenco bars), and then watch a performance with greater understanding.
Arguments remain as to the exact birthplace of Spain's iconic art form, flamenco, but it was certainly in western Andalucia and most likely Sevilla. The soulful, pulsing fusion of song, music, and dance that is flamenco is interpreted in this highly
(+) More
C/ Manuel Rojas Marcos 3
C/ Manuel Rojas Marcos 3
Visitor Information
Telephone Number: 00 34 954 34 03 11
The details, dates, and prices mentioned here were accurate at the time of publication.
Review by Away Copyright © 2010 Away.com all rights reserved.
- Sevilla, Spain Hotel Prices Made Easy
Orbitz.com - Just Orbitz and go! - Hotel Deals in Sevilla, Spain
Expedia.com - Don't just travel. Travel right! - Book Now with Cheap Tickets and Save on Sevilla, Spain Hotels!
CheapTickets.com - Just click. You're there. - Find the best rates for hotels in Sevilla, Spain
Kayak.com - Life's a trip.


text:
Options and Accessories (View All)
Marketing Information
The HP ProLiant DL380 G7 Server continues to deliver on its heritage of engineering excellence with increased flexibility and performance, enterprise-class uptime and HP Insight Control manageability, 2 socket Intel Xeon performance, and 2U density for a variety of applications.
General Information
Product Type | Server |
Manufacturer Part Number | 639829-005 |
Manufacturer Website Address | www.hp.com |
Manufacturer | Hewlett-Packard |
Product Model | 639829-005 |
Product Name | ProLiant DL380 G7 639829-005 Server |
Product Line | ProLiant |
Product Series | DL380 G7 |
Brand Name | HP |
Processor & Chipset
Processor Speed | 2.53 GHz |
Processor Type | Xeon |
Processor Model | E5649 |
Processor Core | Hexa-core (6 Core) |
64-bit Processing | Yes |
Processor Manufacturer | Intel |
Chipset Manufacturer | Intel |
Chipset Model | 5520 |
QuickPath Interconnect | 5.86 GT/s |
Number of Processors Supported | 2 |
Number of Processors Installed | 2 |
Hyper-Threading | Yes |
Standard Memory | 8 GB |
Memory Technology | DDR3 SDRAM |
Memory Standard | DDR3-1333/PC3-10600 |
Memory Card Reader | Yes |
Optical Drive Type | DVD-Writer |
Controller Type | Serial Attached SCSI (SAS) |
RAID Supported | Yes |
I/O Expansions
Number of Total Expansion Slots | 3 |
Display & Graphics
Graphics Controller Manufacturer | ATI |
Graphics Controller Model | ES1000 |
Graphics Memory Capacity | 64 MB |
Network & Communication
Ethernet Technology | Gigabit Ethernet |
Total Number of USB Ports | 5 |
Package Contents | |
Green Compliant | Yes |
Green Compliance Certificate/Authority | WEEE |
80 Plus |
Physical Characteristics
Form Factor | Rack |
Rack Height | 2U |
Height | 3.4" |
Width | 17.3" |
Depth | 27.3" |
Standard Warranty | 3 Year Limited |
This product is shipped only within Canada and Warranty valid for Canada only.
Computer Valley makes every effort to provide accurate specifications, descriptions, images and price on all products that are found on www.computervalley.ca
However for complete and updated descriptions, compatibilities, specifications and actual photo we recommend our clients to consult with manufacturer's websites or simply call us for proper clarifications. Computer Valley is not responsible and will not accept returns for items purchased that are not compatible with your system. Please ensure that you have verified compatibility and actual photo with your system by visiting the manufacturer's web site prior to purchase or give us a call to make sure what you are purchasing.
For more information, visit our Shipping and Return policy.
Computer Valley wants to provide you with the best and fastest possible service. We try to ship in stock orders placed before 2:00pm EST the same day. For in stock orders placed after 2:00pm EST they are usually shipped the next business day. However there are always exceptions. Please contact us prior to placing your order if you need your purchase by a certain date.
We ship by UPS, FEDEX, USPS, CANADA POST , PUROLATOR and LOOMIS from the nearest locations possible based on stock in hand.
Please note: Computer Valley reserves the rights to accept and cancel any order at anytime due to pricing error or any other reason. An email will be sent as a confirmation of the order status.
Options and Accessories


text:
New Yosemite Falls Webcam Provides a Spectacular View of North America's Highest Waterfall
Working in partnership with the National Park Service and Delaware North Companies Parks & Resorts at Yosemite, the Yosemite Falls webcam is the fourth supported by the Conservancy and the first to offer a view of a Yosemite waterfall. The other Yosemite Conservancy webcams feature spectacular views of Yosemite landscapes, including Half Dome, El Capitan and the Sierra Nevada mountain range. |