text:
Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes
Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like
We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.
We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.
Among the eukaryotic pathogens whose genomes have so far been sequenced, the malaria parasite,
Attempts have been made to overcome coverage problems associated with PCR amplification by omitting the PCR step in library preparation
Here we report alternative library-amplification procedures optimized for AT-rich templates and suitable for low mass DNA starting material especially from malaria clinical isolates. We have tested various library amplification methods including linear and isothermal amplification strategies. We have also explored various PCR amplification conditions by testing different polymerases, and their tolerance to AT-rich templates, in the absence or presence of tetramethylammonium chloride (TMAC), a PCR additive that increases thermostability of AT base pairs
Conventional PCR amplification of an AT-rich locus
In order to identify polymerases and PCR conditions that are efficient and tolerant to high AT content templates, we performed conventional PCR using commercial enzymes. The PCR was targeted at a 1217 bp locus (Pf3D7_01:55900-57116) with a high AT-content (84%) and a 540 bp locus (Pf3D7_11:1294982-1295521) with a relatively balanced base composition (positive control) from the
Additional file 1
Figure S1. Bioanalyzer quantification and analysis of libraries. Aliquots of the PE-adapter-ligated library (shown) was amplified using enzymes/conditions indicated. After purification with Agencourt Ampure XP beads, library products were analyzed using Bioanlyzer. Bioanalyzer traces show various yields obtained by different conditions and amplification methods used. T7 and PCR-free produced the lowest yield. Phusion and AccuPrime amplifications were undetectable in the presence of TMAC (Tetramethylammonium chloride). Additional file
Click here for file
Figure 1
Screening for tolerance to an AT-rich template using conventional PCR amplification
Screening for tolerance to an AT-rich template using conventional PCR amplification. Top panel: PCR amplification of a 540 bp locus (Pf3D7_11:1294982-1295521) with a relatively balanced (70% AT) base composition (positive control) in the presence or absence of TMAC. Bottom panel: PCR amplification of a 1217 bp locus (Pf3D7_01:55900-57116) with extreme AT content (84%) in the presence or absence of TMAC. M, 100 bp DNA ladder (NEB); (1) PWO master; (2) PWO master + TMAC; (3) PfuULTRA; (4) PfuULTRA + TMAC; (5) Kapa HiFi; (6) Kapa HiFi + TMAC; (7) AccuPrime Taq HiFi; (8) AccuPrime Taq HiFi + TMAC; (9) AccuPrime pfx SuperMix; (10) Phusion; (11) Phusion +TMAC; (12) Platinum HiFi; (13) Platinum HiFi + TMAC; (14) Platinum pfx; (15) Platinum pfx + TMAC, (16) Ex Taq; (17) Ex Taq + TMAC; (18) Kapa2G Robust; (19) Kapa2G Robust + TMAC.
PCR amplification of Illumina genomic libraries
Although Phusion polymerase is the standard enzyme for Illumina sequencing library amplification, it produces libraries with poor representation of loci with extreme base composition particularly the AT-rich regions
Using these metrics, we show that libraries prepared under standard conditions (using Phusion polymerase) produced less even coverage compared to the optimized conditions in both 3D7 and the clinical isolate. As shown in Figure
Figure 2
A plot of genome coverage against normalized average depth
A plot of genome coverage against normalized average depth. Duplicate data sets were normalized and pooled. Variance in coverage above and below the normalized average depth (red vertical line) across the genome is shown. Deviation of sample curves from the average depth indicates level of evenness in coverage depth distribution across the genome. The closer the sample curve is to the vertical line, the more even the coverage. The theoretical curve represents average normalized depth at 100% genome coverage. A) Coverage by libraries made from
Figure 3
GC profile analysis of sequenced data
GC profile analysis of sequenced data. The GC content distribution for different library preparation methods are shown alongside theoretical data for comparison. A) GC content analysis on libraries prepared from
Table 1
Average GC content
Kapa HiFi
Av. %GC content
Av. %GC content
Calculated values of average GC content corresponding to each library preparation. Deviation from the 19.4% (value of the unamplified genome) indicates the effect of amplification bias. See Figure 3 A & B for a global graphical presentation of the GC content distribution. 3D7,
We also assessed coverage across chromosome 11 and then focused on specific regions of balanced GC content, high AT content and variable base compositions. A box plot showing variation in depth coverage for both
Figure 4
Box plots showing coverage analysis of
Box plots showing coverage analysis of
Isothermal exponential amplification of Illumina genomic libraries using RPA technology
High extension temperatures in conventional PCR procedures have been associated with bias and other negative effects on high AT content libraries. We therefore assessed the suitability of an isothermal exponential amplification method provided by the recombinase polymerase amplification (RPA) technology
Linear amplification of Illumina genomic libraries using T7 RNA polymerase
Exponential amplification of sequencing libraries is thought to be the source of biases and artifacts. To avoid this, we explored a recently described linear amplification for deep sequencing (LADS) protocol
Effect of TMAC on Illumina genomic library amplification
TMAC has been shown to improve thermostability of AT-base pairs
Fidelity and coverage ranking
We analyzed the accuracy of each enzyme in terms of base incorporation by counting mismatches and indels at regions of low complexity (TATA repeats). At these regions of low complexity, RPA generated the least number of errors (scoring best) whereas T7 polymerase had the highest (Table
Table 2
Ranking of
Coverage score
MM score
Rank ALL
PCR -Free
Kapa HiFi
Platinum pfx
AccuPrime Taq HiFi
Kapa2G Robust
Ranking was based on genome coverage and accuracy. Coverage score: PCR-free and Kapa HiFi had the lowest percentage of genome covered at 0 or <5× thereby scoring the highest. Mismatch (MM) score: Loci of low complexity were used to generate mismatch ranking (see Material and Methods under Fidelity section). The number of True positive mismatches, False positive mismatches, Deletion and Insertions obtained for each library were used to generate a score before combining all scores to generate an overall mismatch rank. Using these criteria, RPA produced the highest score and T7 scored the least. See additional file
Library amplification is an important step in preparing samples for sequencing with the current NGS platforms, especially when the starting material is severely restricted in quantity. PCR amplification, however, has been shown to be the major source of artifacts and base-composition bias in library fragments. Although alternative library-preparation approaches that omit the PCR amplification show a significant reduction in base-composition bias, this strategy requires large quantities of starting material. Therefore, PCR-free library preparation is unsuitable where amounts of DNA starting material are limited, as is the case with most clinical isolates.
We demonstrate that problems associated with PCR can be ameliorated through optimization, thereby allowing amplification to be used in generating sequencing libraries even from extremely AT-biased genomes. Through careful selection of polymerases and the use of a PCR-additive that normalizes melting temperatures of the different library fragments, we have optimized Illumina library amplification. These alternative PCR conditions generate library fragments with increased coverage of extreme AT-rich regions.
Standard Illumina amplification procedures that use Phusion polymerase generate libraries with biased base-composition and are depleted of AT-rich loci. The sequence data generated under these standard conditions are of reduced complexity and maybe unsuitable for resequencing to identify relatively rare genetic variants of important biological function. We set out to solve this problem by focusing on the library amplification step, which is the primary source of biases in almost all NGS platforms. We started by screening commercially available polymerases for tolerance to an extremely AT-rich template. This initial screening procedure identified a preferred polymerase and amplification conditions that showed improved tolerance to AT-rich templates. We demonstrated that most commercial polymerases are inhibited by TMAC. However, this chemical can have a tremendous positive effect in amplifying AT-rich templates for the TMAC-tolerant enzymes. With TMAC additive we have developed Illumina library-amplification protocol suitable for extreme AT-rich genomic DNA. Quality analyses of sequence data from both
To date, no single polymerase or amplification method has been shown to efficiently amplify both extremes of base composition with equal and optimum efficiency. The two isothermal amplification methods (RPA and T7) tested here showed some level of improved coverage relative to standard amplifications, but they were both associated with high levels of chimeric and duplicate reads. Under the optimized conditions we show that the Kapa series of polymerases in the presence of TMAC produced the best coverage that was relatively consistent across regions of extreme base composition. Worthy of note is the low template-dependent fidelity score for Kapa HiFi (Table
Despite the bias problem associated with it, PCR still remains an essential step in many NGS library preparation procedures, especially when the starting DNA material is limited. We describe robust alternative PCR conditions optimized for extremely AT-biased genomes. We show that the optimized conditions significantly reduce bias and also retain the complexity of either extremes of base composition. This observation was consistent in both the lab-cultured strain (3D7) and the clinical isolate. The optimized procedures described here will greatly benefit the malaria genomic research and control initiatives that have faced serious challenges with the parasite's extreme AT-rich genome.
Genomic DNA
Screening for an AT-rich tolerant enzyme
Various commercial enzymes were used to perform a conventional PCR (50 µl total volume) targeted at a 1217 bp locus (Pf3D7_01:55900-57116) with a high AT-content (84%) and a 540 bp locus (Pf3D7_11:1294982-1295521) with a relatively balanced base composition (positive control). Ten ng of
Illumina sequencing libraries
Genomic DNA (0.05 - 1 μg in 75 µl TE buffer) from
Illumina PCR library amplification
All PCR amplifications were performed with an MJ Research Thermo Cycler PTC-225. Illumina PE 1.0 and 2.0 primers or PE 1.0- and 2.0-derived indexing primers were used to amplify 10 ng adapter-ligated library fragments by PCR. From a single pool of adapter-ligated library, aliquots were amplified using standard Illumina PCR reagents or using various alternative polymerases under optimized PCR conditions. Standard Illumina PCR (50 μl) with Phusion polymerase (Thermo) contained 1× Phusion DNA polymerase master mix and 0.4 μM of each primer pair and was amplified with thermocycling conditions of 1 min at 98°C for the initial denaturation, followed by 12 cycles of [10 s at 98°C, 30 s at 65°C, 30 s at 72°C] and a final extension for 5 min at 72°C. PCR (50 μl) with Kapa HiFi (KAPA Biosystems, South Africa) contained 1× Kapa HiFi buffer (containing TMAC) or 1× Kapa buffer B, 0.3 mM of each dNTP, 0.4 μM of each primer pair, 1 unit of Kapa HiFi and was amplified with thermocycling conditions of 1 min at 98°C for the initial denaturation followed by 12 cycles of [10 s at 98°C, 1 min at 65°C] and a final extension for 5 min at 65°C. PCR (50 μl) with Kapa2G Robust (KAPA Biosystems, South Africa) contained 1× Kapa2G buffer B, 0.3 mM dNTPs mix, 0.4 μM of each primer pair, 0.4 units of Kapa2G Robust and was amplified in the presence or absence of 60 mM TMAC with the same thermocycling conditions as Phusion above. PCR (50 μl) with Platinum pfx (Invitrogen) contained 1× Platinum pfx buffer, 0.3 mM dNTPs mix, 0.4 μM of each primer pair, 1 mM MgCl2, 0.8 units of Platinum pfx and was amplified in the presence or absence of 60 mM TMAC with thermocycling conditions of 2 min at 94°C for the initial denaturation followed by 12 cycles of [15 s at 94°C, 30 s at 60°C, 30 s at 65°C] and a final extension for 5 min at 65°C. PCR (50 μl) with AccuPrime Taq HiFi (Invitrogen) contained 1× AccuPrime buffer II, 0.4 μM of each primer pair, 1 unit of AccuPrime Taq HiFi and was amplified in the presence or absence of 60 mM TMAC with the thermocycling conditions used for Platinum pfx. PCR products were purified with 1× Agencourt Ampure XP beads (Beckman Coulter) and eluted in EB buffer (Qiagen). PCR products were analysed by running on an Agilent 2100 Bioanalyzer (Agilent Technologies) to assess amplification. Only successful amplifications were processed for sequencing.
Illumina isothermal library amplification using RPA technology
We used recombinase polymerase amplification (RPA) technology as provided in the TwistAmp™ DNA-amplification kit
Illumina linear library amplification using T7 RNA polymerase
Linear amplification for deep sequencing (LADS)
PCR-free libraries
Genomic DNA (1-2 μg) was sheared, end-repaired and A-tailed as described under "Illumina sequencing libraries". Pre-annealed paired-end Illumina No-PCR adapters were ligated using the same ligation procedure described under "Illumina sequencing libraries". Resulting adapter-ligated fragments were loaded alongside a low molecular weight ladder (NEB) on a TBE 2% agarose gel. Fragment bands of 350 to 500 bp were cut and purified using a Qiaquick gel extraction kit. Gel purification was performed at room temperate and DNA eluted in EB buffer.
Library quantification by qPCR
Pre-PCR libraries were quantified by qPCR using a previously quantified and sequenced library in dilutions containing 100 ng, 10 ng and 1 ng as standard. The qPCR reaction (25 μl) contained 12.5 μl of 2× Sybr Green PCR Master Mix (Applied Biosystems), 0.75 μl of 10 μM Pre-PCR qPCR top and bottom primers (see additional file
Data processing
Replicate data sets were analyzed independently by mapping sequence reads to the
Mapped reads for each replicate data sets were normalized by average genome coverage to 11× and 21× for clinical and non-clinical samples respectively, and then pooled or analyzed independently depending on analysis metrics used. For the clinical samples, reads originating from host contamination were filtered out (by aligning all reads to
The value lci (0.5) that gives a measurement of the coverage below half of the average depth in the distribution was used to compare evenness of coverage for each data set.
We counted the number of bases in the genome with zero coverage (Cov = 0) and less than 5× coverage (Cov<5×). We used SAMtools to generate coverage plots and bash/awk scripts for coverage counting. Tolerance to regions of extreme base composition was analyzed after normalizing data sets by overall average coverage depth. Coverage distribution on selected regions (on chromosome 11) are presented as a boxplot generated in R
Most polymerases introduce errors during base incorporation in regions of low complexity. To assess these forced errors (or template-dependent errors), we compared variant calls by each amplification method at sites of low complexity (mainly TATA repeats). We also assessed the ability of each amplification condition to find a set of known consensus errors in the reference, and used data obtained to score true and false positives. The known consensus errors in the reference genome were previously identified and published
We ranked the results by assigning a score of 1 to the best and a score of 0 to the worst. For each amplification condition, we calculated the average score value obtained in both coverage and fidelity metrics. Conditions with the highest average score was ranked highest while that with the least assigned the lowest rank (see Table
Chimeric and duplicate reads analysis
We used the BWA paired-end alignment algorithm to generate mapped data sets. Picard software
All datasets used in this study have been deposited in the ENA read archive under accession number [ERP000832].
TMAC: Tetramethyammonium chloride; PCR: Polymerase Chain Reaction; NGS: Next-Generation Sequencing; PE: Paired-end; qPCR: quantitative PCR; RPA: Recombinase Polymerase Amplification; LADS: Linear amplification for deep sequencing; SNP: Single Nucleotide Polymorphism.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Research in the lab was carried out by SOO, MAQ and SC. Bioinformatic analyses were carried out by TDO, YG, GM and MM. The research was coordinated by BM and HPS. DPK, DJT, HPS and MAQ conceived the project. SOO wrote the paper. All authors read and approved the final manuscript.
This work was supported by the Wellcome Trust [grant number 079355/Z/06/Z].
The grand assault
Genome sequence of the human malaria parasite Plasmodium falciparum
Substantial biases in ultra-short read data sets from high-throughput DNA sequencing
Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries
Optimization of protocol for sequencing of difficult templates
Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes
Low concentrations of tetramethylammonium chloride increase yield and specificity of PCR
PlasmoDB: a functional genomic database for malaria parasites
BamView: viewing mapped read alignment data in the context of the reference sequence
Artemis: sequence visualization and annotation
DNA detection using recombination proteins
Linear amplification for deep sequencing
Fast and accurate short read alignment with Burrows-Wheeler transform
The Sequence Alignment/Map format and SAMtools
A language and environment for statistical computing
Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology


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Combining psychedelic chamber pop with Midwestern Americana is The Republic Tigers, the latest band likely to flood the blogosphere in the upcoming months. The reason for that will be the Kansas City, Missouri quintet's upcoming debut, Keep Color. But before dropping that bomb, the group has released a solid four-song EP, named EP, to give us all a taste.
As soon as "Buildings and Mountains" starts up, you cannot help but be drawn in by the soaring vocals that haunt the track. And the slightly brooding keys and synths just add more layers to an already lush soundscape. Just as psychedelic, but slightly more experimental, are the aptly-titled "The Drums" and "Rollercoaster". The former features plucked acoustic guitar strings that overlap and give a feel of organized chaos. "Rollercoaster", on the other hand, is completely instrumental, except for even more choir-esque "aaahs," until the last minute. It plays a lot like post-rock on valium, though the change one-third of the way through gives it a slight boost. The only song that doesn't immediately grab you is "Sinkin' Annie, Down, Down, Down". Although the '60s pop vocal harmonies are a nice touch, their and the track's appeal are fleeting.
The Republic Tigers
(Chop Shop)
US: 19 Feb 2008
UK: Available as import
If this EP is any indication, The Republic Tigers are sure to garner some attention. Just make sure you're on the look-out for Keep Color, which sounds just as good as EP but just with eight more spaced-out tracks.
- "Buildings and Mountains" MP3
- Multiple songs MySpace
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Vulture Whale "Can't Help It" (video) (premiere) // Music
Paul McCartney: Pure McCartney (take 2)
// Sound Affects
"Pure McCartney really is a compilation for those wanted to dip their toes in wading pool instead of jumping waterfalls.READ the article

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Are copay coupons making drugs more expensive?
By Charles Ornstein ProPublica A few months back, after returning from a family vacation that involved lots of pool time,Read more
By Charles Ornstein ProPublica A few months back, after returning from a family vacation that involved lots of pool time,Read more
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By Lawrence Hurley Reuters WASHINGTON – The U.S. Supreme Court on Monday struck down a Texas abortion law imposing strictRead more
An Education Department advisory committee took the unprecedented step of calling on the government to revoke powers of for-profit collegeRead more
By Ben Hirschler & Martinne Geller Reuters LONDON – Chief executives from Tokyo to Denver prepared for long-term disruption, job cutsRead more
The U.K. has voted to leave the European Union, 51.9 to 48.1 percent. Prime Minister David Cameron announced early FridayRead more
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I`ll have a Bengali wedding: Bipasha Basu
Mumbai: Actress Bipasha Basu, who is reportedly dating Harman Baweja, refused to speak on her relationship status but said that she will have a typical Bengali wedding.
"Whenever I get married it will be a Bengali wedding. If I won`t have a Bengali wedding my mother won`t come. She has warned me. So, I am going to have a Bengali wedding for sure," Bipasha, known for films like `Jism`, `Corporate` and `No Entry`, told reporters.
"But the only condition is that nobody will dance at my wedding, everybody will only exercise," added the 35-year-old, a fitness freak who has launched the third volume of her fitness DVD titled `Unleash`.
Her ex-beau John Abraham recently got married to Priya Runchal, an investment banker.
When asked about her views on John`s wedding, she said: "I am not answering that."
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Rangers Expect To Score $44M Float At London's Alternative Investment Market
Published December 17, 2012
SORRY SEEMS TO BE...: The SCOTSMAN reported "furious Rangers fans have demanded an apology from Montrose FC" after Saturday's match-day program notes "poked fun at the Ibrox club's financial troubles earlier this year." The introduction to the Rangers team read: "Playing their first season in Division Three, The Rangers are a newco of the now defunct Glasgow Rangers. Currently top of the table, they will be hoping to go on to seal the title and clinch their first silverware" (SCOTSMAN, 12/16).


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Home > News > Men, War, and Narcissism
June 30th, 2006
Mike Treder: Scientists have provided objective evidence to back suspicions that men who are over-confident and narcissistic are the likeliest to start wars -- but also are the likeliest to lose.
A study like this is prime material for easy jokes. But it's also sobering, especially when you consider, first, that narcissism and self-confidence are two traits that drive individuals to become leaders who have the power to start wars, and second, that wars in the Nano Era could easily result in worldwide devastation, global enslavement, or both.
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SWEDEN: Muller wanted to drop Saab Griffin logo before Scania objections
By Simon Warburton | 5 October 2012
Former Saab and current Spyker, CEO, Victor Muller, says he wanted to end the association with the Swedish automaker and its Griffin logo long before current owner, National Electric Vehicle Sweden (NEVS) was forced to drop the iconic symbol.
NEVS has been allowed to continue using the Saab name, but truckmaker, Scania, which also uses the Griffin logo, objected to the the automaker featuring the symbol because of fears around potential Chinese piracy.
"I wanted to abolish the Griffin logo - I wanted to go back to the aeroplane logo," Muller told just-auto. It [Griffin] was a logo of Scania and Saab AB - it did not say anything.
"It [aircraft symbol] was beautiful - it was going to replace the Griffin everywhere."
Saab used to trade heavily on its aerospace association before Saab AB assumed responsibility for manufacturing fighter aircraft, often featuring aviation imagery in its television advertising.
Muller said he wished NEVS "all the luck in the world" although added cryptically: "I don't understand their business model but who am I?"
The Spyker CEO said Chinese manufacturer, Youngman, with whom Saab attempted to form a partnership before bankruptcy, had come on board with the Dutch automaker on the basis of its licence of the Phoenix platform "where Saab left off."
"The fact they [Youngman] chose to work with us again shows they had no hard feeling about losing the money they put into Saab," said Muller. "They knew we did everything."
NEVS is playing down the impact of not being able to use the Griffin logo, preferring to concentrate on its continued permission to feature the Saab name for its electric vehicle programme.
"The important thing for us is to have the Saab name," a NEVS spokesman recently told just-auto from Sweden. "The Griffin logo is not that important...because we want to build our own identity. "We won't use the Griffin logo at all - the logo has to be something we develop."
Scania scuppered hopes NEVS could use the Griffin symbol by raising concerns about the the prevalence of copies in China - the Saab buyer has a Chinese connection - although it stressed there was no link between the two.
Sectors: Vehicle manufacturers, Vehicle manufacturing
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|By PR Newswire
Article Rating:
|December 10, 2012 02:11 PM EST
BOSTON, Dec. 10, 2012 /PRNewswire-iReach/ -- axle Video, a startup focused on making media management simple and cost effective for small-to-midsize production teams, today introduced axle™ 2012, the next generation of its award winning media management solution. axle lets video producers, small postproduction teams and owners of media libraries organize, search and comment on their media through a simple web interface, without complexity or headaches.
(Photo: http://photos.prnewswire.com/prnh/20121210/CG26655)
"We were impressed with how quickly and simply axle could be set up and implemented into the video workflow for our online school, Berkleemusic.com." said Craig Reed, video producer at Boston's Berklee School of Music media department. "We began using it almost immediately to track content, support review and approval processes, and mark up raw content for rough cut decisions. The browser-based interface makes it easy for different teams to quickly find the content they need, and for our remote clients to easily review media and provide feedback"
Like the earlier versions of the software, axle 2012 is radically simple, with a browser-based interface that's equally easy to understand and use on an iPad, Windows PC or Mac. axle 2012 adds a number of key benefits to creative production teams, many of whom have not been able to adopt media asset management technology until now.
- Scalable transcoding - accelerates management, review, and approval of large amounts of media: file-based cameras help producers generate many terabytes of material, often faster than production teams can manage and review it. In response, many production / post-production workflows involve remote editorial assistants, editors, producers, directors, or clients. axle 2012 integrates Telestream's industry-leading Episode Pro so that up to 10 transcode software licenses can be harnessed for the fastest possible processing pipeline of incoming media – up to 20x realtime throughput. The whole production team can then review and select from this media, whether connected locally, or remotely from anywhere in the world.
- Finished output delivery – axle 2012 enables the creation and selection of Telestream Episode Pro processing presets from within axle 2012's browser-based interface. Local and remote axle 2012 users can initiate transcoding workflows and the delivery of finished output. Any video asset stored in axle 2012 can be sent for transcoding. Episode Pro supports a very wide range of output formats, including Apple's ProRes and Avid's DNxHD.
- Tiered storage – axle 2012 supports up to three tiers of storage: Tier 1 (Editing) for high-speed devices such as SANs and optimized NASs for direct video editing from connected workstations; Tier 2 (Parking) for nearline media file systems for migration to and from editing storage at short notice; and Tier 3 (Library) for tape and cloud-based storage options with longer retrieval times. axle 2012 lets users easily migrate their high-res media among these 3 tiers of storage, without worrying about details of the migration. Most network storage systems are compatible; axle 2012 has been successfully qualified with Active Storage, Facilis and Small Tree Tier 1 storage; Active Storage mVault, Object Matrix, and QNAP Tier 2 storage; and Cache-A and XenData Tier 3 storage.
- Support for audio, photo and print assets – axle 2012 adds support for a wide range of media formats besides video. Types supported include most mainstream audio files (MP3 and AAC); TIFF, EPS and JPEG image files; Camera RAW files from most DSLR manufacturers including Canon and Nikon; and PDFs, both single-page and multi-page. axle's simple web browser interface gives users a way to browse, review and annotate these file types.
The axle 2012 server software runs on a Mac mini, can monitor standard network file systems and is compatible with files from major content creation tool vendors such as Apple and Adobe. axle lets users view, log, comment, upload, download and review and approve material from wherever they are located and on whatever device they choose, including Macs, PCs, and IPads. Axle has also concluded an OEM agreement with Telestream under which it is distributing Episode Pro software coupled with axle. Each Episode Pro license can run on a separate Mac mini, Mac Pro or Windows system networked to the axle server, and is priced at $995.
"We are thrilled to expand our partnership with axle Video and to offer its users the encoding flexibility and scalability they need in today's multiformat production environments," said Barbara DeHart, vice president of marketing at Telestream. "Direct integration of our products provides axle users with easy access to Episode's vast range of file formats and enables centralization of powerful encoding functions,"
The network equipment vendor Cisco will be holding a webinar with axle on Wednesday Dec. 12th ( www.tinyurl.com/ciscoaxlewebinar ) describing the axle 2012 solution, and axle will be holding further, technical webinars on Wednesday Dec. 19th ( www.tinyurl.com/axle9amEST and www.tinyurl.com/axle3pmEST ). Participation in these webinars is free and open to the public.
axle 2012 is priced at $1295 per 5 users for the software, and $295/year for code updates and support; combined solutions start at $2290 including one Telestream Episode Pro license. Shipments begin December 19th. Axle has begun recruiting a worldwide network of certified resellers who are able to configure systems with the axle and Telestream components, as well as accompanying Apple hardware. Creative professionals and resellers can learn more at http://www.axlevideo.com.
Trademarked company and product names are the property of their respective companies.
About axle
axle video, LLC is a Boston-based company aiming to bring radical simplicity to media management for small and midsize workgroups. Its founders, Sam Bogoch, Steven Ryan and Patrice Gouttebel, all have extensive experience in the fields of media asset management, digital asset management and workflows for creative applications. The company's products take an "appliance" approach, to solve what has previously been the domain of highly complex software configurations and deployments. axle's first product release, version 1.0, received a prestigious Best of IBC award from TVB Europe magazine.
To view this video on YouTube, please visit: http://www.youtube.com/watch?feature=player_detailpage&v=7OKsNOaMQr8#t=22s
Media Contact: Stephen Beals axle Video, LLC, 315-569-9150, [email protected]
News distributed by PR Newswire iReach: https://ireach.prnewswire.com
SOURCE axle Video, LLC
The Internet of Things will challenge the status quo of how IT and development organizations operate. Or will it? Certainly the fog layer of IoT requires special insights about data ontology, security and transactional integrity. But the developmental challenges are the same: People, Process and Platform and how we integrate our thinking to solve complicated problems. In his session at 19th Cloud Expo, Craig Sproule, CEO of Metavine, will demonstrate how to move beyond today's coding paradigm ...
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Kettering Peking Chinese Restaurant
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Tres Señoritas Travel To Spain – Beautiful Barcelona
By A Baby Boomer Woman's Life After 50 on June 12, 2014
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Last week I shared the first half of my trip to Spain, where the tres señoritas – my daughter A, my sister N, and I – explored Madrid. Are you ready to travel around another Spanish city? Hold on tight, as I take you on a fast-paced tour of beautiful Barcelona. . . .
Read more from Tres Señoritas Travel To Spain – Beautiful Barcelona at A Baby Boomer Woman's Life After 50
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Christmas Tree Growers Introduce Green Standards
Christmas tree growers, members of the Coalition of Environmentally Conscious Growers in Oregon, have created a set of standards that will identify trees grown under certain environmental conditions, AP reports.
This is the first year the program will be seen in the market. More than 200,000 tags will hang on trees, indicating the trees were farmed by the coalition's standards. The group hopes to take the tag system nationwide.
To use the tag, a farm must meet certain standards for managing wetlands, nutrients and pests. More than a dozen tree growers are on a waiting list to be inspected.
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Energy Manager News
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Government ( tax money ) does have a legitimate and necessary place in society. Hope the Florida taxpayer, research institutions and the citrus industry together can find a viable and timely solution to the greening disease.
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9183 Broadview Road
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For on-line donations (e-donations) go to: www.parishpay.com
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As we celebrate Independence Day this weekend, let us remember that all freedom comes as a result of great sacrifice and that we must be willing to sacrifice if we are to attain and preserve this freedom. Many people have the mistaken impression that freedom mean that I can do whatever I want, when I want, and how I want. This is why there is so much trouble and chaos in the world.
God given freedom, which is the freedom envisioned by the Declaration of Independence, is our empowerment to respect one another's rights and to assist each other in our task of fulfilling God's plan for our lives. When this mutual respect and assistance become a reality, the life, liberty, and pursuit of happiness follow as a natural result.
Picnics, fireworks, and games will be our chief occupations on this holiday.
But at Mass today and for a few moments today, let's stop and thank God for the freedoms and rights we have as citizens of the United States of America. Let's not forget the men and women fighting for freedom in trouble spots of the world but ask God to shield them under the protection of his mighty arm that shatters the weapons of war and lead us all to accept the gentle rule of the King of Peace.
A Blessed and Happy Independence Day to All!!


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What is an 'Inferior Good'
An inferior good is a type of good for which demand declines as the level of income or real GDP in the economy increases. This occurs when a good has more costly substitutes that see an increase in demand as the society's economy improves. An inferior good is the opposite of a normal good, which experiences an increase in demand along with increases in the income level.
BREAKING DOWN 'Inferior Good'
Inferior goods can be viewed as anything a consumer would demand less of if they had a higher level of real income. An example of an inferior good is public transportation. When consumers have less wealth, they may forgo using their own forms of private transportation in order to cut down costs (car insurance, gas and other car upkeep costs) and instead opt to use a less expensive form of transportation (bus pass).
Normal GoodAn economic term used to describe the quantity demanded for a ...
Substitution EffectThe idea that as prices rise (or incomes decrease) consumers ...
Income EffectIn the context of economic theory, the income effect is the change ...
Income Elasticity Of DemandA measure of the relationship between a change in the quantity ...
DemandAn economic principle that describes a consumer's desire and ...
SubstituteA product or service that a consumer sees as comparable. If prices ...
What Does Inferior Good Mean?The term "inferior good" does not describe a lack of quality, but rather, is an economic term used when discussing elasticity of demand for a good.
What is a Normal Good?A normal good is any good or service that sees an increase in demand due to an increase in income.
Calculating Income Elasticity of DemandIncome elasticity of demand is a measure of how consumer demand changes when income changes.
What is Demand?Demand is the economic term for the cumulative wants and desires of consumers as they relate to a particular good or service. Generally speaking, if all other factors remain constant, as demand ...
Understanding the Substitution EffectThe substitution effect is an economic term used to describe consumer behavior relative to price or income changes.
What's a Substitute?A substitute is a good that satisfies the same needs as another.
Explaining Quantity DemandedQuantity demanded describes the total amount of goods or services that consumers demand at any given point in time.
Law of DemandThe law of demand is one of the most fundamental principles in microeconomics. It's all about how price affects demand. According to the law of demand, for all other things remaining constant, ...
What's Aggregate Demand?Aggregate demand is a macroeconomic term describing the total demand in an economy for all goods and services at any given price level in a given time period.
Do Deflationary Shocks Help Or Hurt The Economy?Find out how deflationary shocks can both benefit and hurt consumers and businesses.
What's the difference between the income effect and the substitution effect?Learn more about the income effect and substitution effect in economics. Find out how these two principles impact consumer ... Read Answer >>
How are industrial goods different from consumer goods?Understand the difference between industrial goods and consumer goods, and learn the different types of industrial goods ... Read Answer >>
Which factors are more important in determining the demand elasticity of a good or ...Learn about demand elasticity of goods and services and the main factors that influence the elasticity of demand. Read Answer >>
What is the difference between a capital good and a consumer good?Learn to differentiate between capital goods and consumer goods, and see why capital goods require savings and investment ... Read Answer >>
How does aggregate demand affect price level?Read about the relationship between aggregate demand and the general price level, and learn why it is difficult to determine ... Read Answer >>
What are some examples of demand elasticity other than price elasticity of demand?Learn about income elasticity of demand and cross elasticity of demand and how to interpret these two measures of demand ... Read Answer >>


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8698 S Main StEden, NY 14057
22. VinoIntellio
379 Jewett Holmwood RdEast Aurora, NY 14052
From Business: We bring an affordable means to serve wine by the glass, market products and services, and entertain guests.
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Researchers Have 3D Printed A Solution To One Of The Most Common Sleeping Disorders In The US
Published 10:35 am, Wednesday, May 7, 2014
Researchers in Australia have 3D printed a mouthpiece designed to combat one of the most common sleeping disorders in the United States—sleep apnea.
Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO) and Australian dental company Oventus have discovered a way to 3D print a mouthpiece that can prevent dangerous pauses in breath during sleep.
The scientists used a 3D scanner to create a map of the patient's mouth, according to Medical Xpress, and used the results to print titanium-built mouthpiece coated with medical-grade plastic.
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The device features a duckbill that extends from the wearer's mouth like a whistle. This creates two separate airways, which allows air to avoid obstructions in the nose, back of the mouth, and tongue by traveling through the back of the wearer's throat.
Sleep apnea is an involuntary cessation of breathing that occurs during sleep. There are three types of sleep apnea: obstructive, central, and mixed. Obstructive sleep apnea, called OSA for short, is the most common type according to the American Sleep Apnea Association.
All three types of sleep apnea can cause one to stop breathing repeatedly during sleep, sometimes hundreds of times during the night. These pauses are often longer than one minute, the ASAA notes. The sleeper is usually unaware of these pauses because they do not trigger a full awakening.
If left untreated, sleep apnea can have long term health affects such as high blood pressure, heart disease, diabetes, and depression.
According to the American Association for Respiratory Care, sleep apnea affects up to 18 million Americans, and officials estimate that 10 million Americans with the condition haven't been diagnosed.
The CPAP mask is the most common, non-invasive method for treating sleep apnea. The new 3D printed mouthpiece, however, is a more comfortable solution for those affected by the disorder. Oventus CEO Neil Anderson said the following to Medical Xpress:
This new device is tailored to an individual's mouth using a 3D scan and is used only on the top teeth which make it more compact and far more comfortable.
The 3D printed mouthpiece is expected to be available for patients next year, but it's unclear whether or not it will come to markets outside of Australia.
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iHerb Customer Reviews
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Great product, 5 stars
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Excellent for dry hands


text:
Flight crew crash
Posted in the Keavy Forum
Since: Jan 11
Location hidden
#1 Jun 7, 2013
This is so sad.
#2 Jun 7, 2013
Was the Eddie Sizemore killed in the crash the Sizemore that use to be a deputy sheriff here in Laurel?
#3 Jun 7, 2013
Yes it was Eddie Sizemore from London
#4 Jun 7, 2013
"The company identified the crewmembers as pilot Eddie Sizemore, flight paramedic Herman "Lee" Dobbs and flight nurse Jesse Jones. They were on their back to base in Manchester after transporting a patient to a hospital in London, Ky.
Laurel County Sheriff's spokesman Gilbert Acciardo told the Lexington Herald-Leader that Sizemore had retired as a deputy from the sheriff's office, but returned in 2011 before leaving the position to become a pilot for Air Evac Lifeteam.
"He enjoyed flying," Acciardo said. "That was his passion."
Air Evac Lifeteam President and CEO Seth Myers said the company is devastated by the loss of the crew.
Federal air safety officials planned to investigate the accident."
#5 Jun 7, 2013
To the family's who have lost all these great hero's.
You will all be in our thought's and prayers.They gave their lives doing what they loved and will always be remembered as HERO'S!!!!!!!!
#6 Jun 7, 2013
#7 Jun 7, 2013
What a tragedy.So sorry to read about this today.Prayers for all the family and friends of these wonderful men.They will be missed greatly.
#8 Jun 7, 2013
grew up with eddie went to school with him and his brother they were always good to me
#9 Jun 7, 2013
yeah and sometimes he worked over in the courtroom years ago if this is the same sizemore
#10 Jun 7, 2013
Our hearts are so saddened for the families of all three men. What a shame that they died so tragically. I'm sure each was doing a job he loved. Our prayers and thoughts are sent to their families and their communities. Their jobs, like that of law enforcement officers, is so needed and often unappreciated until a tragedy occurs. I'm sure the patient transported to St. Joe London was a life and death transport and can realize that they may have saved his/her life but gave theirs. I'm sure the men and women who worked with Eddie at LCSO are hurt beyond words. He worked there so much longer than at his air flight job. They band together and I am sure they will extend every protocol possible to him and his family. It seems that he may have been given the opportunity to return to his first love--flying. I understand he was trained as a flight officer during the Vietnam War. How sad. God bless them.
Since: Jun 11
Location hidden
#11 Jun 7, 2013
Tragic. My thoughts are with the family & friends of these men.
#12 Jun 7, 2013
how in the world could something like this happen to such a knowledgable pilot?
#13 Jun 7, 2013
what a terrible loss.
fog is oh so dangerous even driving in a car.
#14 Jun 8, 2013
This company has had 16 accidents since 1998. And this is the fourth set of three crew members to die from crashes since 2007. Unbelievable!!! If you would like to read the story it's in the Lexington Hearld Leader!
#15 Jun 8, 2013
And here we go with the dirt slinging. How sad I feel sometimes to live among such negative and callous people. Can't you just be polite and allow one string to be left alone so everyone can honor these men for a change? Why is everything always a conspiracy? You're opening a can of worms that will bring out every hateful and lying troll on these boards now. Just wait and see what you've started. But you already know that don't you? How very very sad to want to turn this string into an accusation throwing fest. You should be ashamed. I hope you're satisfied with yourself once you get your thrills at the expense of these men and their memory.
#16 Jun 8, 2013
You've got it all wrong so sick. You are the one thats trying to start crap. I read all of these post and they say nothing but sympathy except for the one just above yours and thats public knowledge. Where is the dirt slinging except in your quote? You are the start of dirt slinging. I'd be ashamed.
#17 Jun 8, 2013
Exactly WHERE in the post by 'nurse' was there anything but placing of blame ? Sometimes there is just a thing called an 'accident!' This one may have absolutely nothing to do with the others. You both are starting something. It is what it is.
#18 Jun 8, 2013
No one is slinging mud at these people this was a tragedy and we are devastated as a community.
However, just to ignore the circumstances in which these people died would just be ignorant! Do want to just stick your head in the sand and let it happen again? Do you want to put more great people in danger? I would hope not.
This company has had several deadly accidents and it bears looking into.
Also, why were they flying people to London? Are you serious? There is a reason our medical bills are skyrocketing. I think we should get ready for some more problems when Medicare/Medicaid looks into how many of these transports they have been paying for!
Let me make this clear...I mean no disrespect for these great men that lost their lives! On the contrary, It is because I value heroes like them that I feel we must get to the bottom of why this happened and try to prevent it from happening again!!!
United States
#19 Jun 8, 2013
This is a tragedy. I am confident their will be a through Investigation. It is a sad thing when people loose their lives to such tragedy.
However, things from the past often come back to the fore front and the lawyers will come out of the wood work with law suits.
When the Laurel County Sheriff's Department had a Helicopter they got from the Government, Military Surplus, I believe Eddie Sizemore was the Pilot of that aircraft when it crashed. Luckily he walked away from that crash. However, the Lawyers will bring this up, should a wrongful death suit be filed.
Right now it is Mourn those who were Killed in this tragedy then wait and see the Headline assessing blame for the crash. It is sad but most likely will happen.
United States
#23 Jun 8, 2013
id blame the person they had to fly to london that night, if not for whomever it was these three men would still be alive..was one life worth the three that were lost? so in my opinion whomever they flew out that night can personally hold themself responsible for these three mens lives
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Tom Smart, Deseret News
SALT LAKE CITY — The University of Utah has named Alberta Davis Comer as dean and director of the J. Willard Marriott Library and university librarian effective Aug. 19.
Comer has served as dean and associate dean of library services at Cunningham Memorial Library, Indiana State University since 2004. She holds a Master of Library Science degree from Indiana University-Bloomington.
"She brings to the U. an expansive knowledge of libraries, as well as a strong focus on students, faculty collaboration, and the cultivation of positive community and donor relations,″ said Michael Hardman, interim senior vice president of academic affairs at the U.
"The Marriott Library is one of the nation's premier academic libraries, and I am looking forward to leading its impressive team," Comer said.
Comer replaces Joyce Ogburn, who served as dean until 2012 and recently accepted a position with the Appalachian State University libraries.
The J. Willard Marriott Library is the flagship academic library for the Utah State System of Higher Education and the largest state-funded academic library in the six-state region of Idaho, Montana, Nevada, New Mexico, Utah and Wyoming.
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