The Experts below are selected from a list of 43353 Experts worldwide ranked by ideXlab platform
Baback Gharizadeh - One of the best experts on this subject based on the ideXlab platform.
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viral population estimation using Pyrosequencing
PLOS Computational Biology, 2008Co-Authors: Nicholas Eriksson, Mostafa Ronaghi, Lior Pachter, Yumi Mitsuya, Sooyon Rhee, Chunlin Wang, Baback Gharizadeh, Robert W Shafer, Niko BeerenwinkelAbstract:The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (Pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to Pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the Pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation–maximization (EM) algorithm. We demonstrate that Pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, Pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.
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viral population estimation using Pyrosequencing
PLOS Computational Biology, 2008Co-Authors: Nicholas Eriksson, Mostafa Ronaghi, Lior Pachter, Yumi Mitsuya, Sooyon Rhee, Chunlin Wang, Baback Gharizadeh, Robert W Shafer, Niko BeerenwinkelAbstract:The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (Pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to Pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the Pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation–maximization (EM) algorithm. We demonstrate that Pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, Pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.
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characterization of mutation spectra with ultra deep Pyrosequencing application to hiv 1 drug resistance
Genome Research, 2007Co-Authors: Chunlin Wang, Mostafa Ronaghi, Yumi Mitsuya, Baback Gharizadeh, Robert W ShaferAbstract:The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep Pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep Pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in ≥3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep Pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.
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large scale Pyrosequencing of synthetic dna a comparison with results from sanger dideoxy sequencing
Electrophoresis, 2006Co-Authors: Baback Gharizadeh, Zelek S Herman, Robert G Eason, Olufisayo Jejelowo, Nader PourmandAbstract:Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PCR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25-30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.
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long read Pyrosequencing using pure 2 deoxyadenosine 5 o 1 thiotriphosphate sp isomer
Analytical Biochemistry, 2002Co-Authors: Baback Gharizadeh, Tommy Nordstrom, Mostafa Ronaghi, Afshin Ahmadian, Pal NyrenAbstract:Abstract Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in Pyrosequencing the natural dATP was replaced by dATPαS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84–89). The applied dATPαS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2′-deoxyadenosine-5′- O ′-(1-thiotriphosphate) Sp-isomer in Pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.
Fabrice Armougom - One of the best experts on this subject based on the ideXlab platform.
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microbial diversity in the sputum of a cystic fibrosis patient studied with 16s rdna Pyrosequencing
European Journal of Clinical Microbiology & Infectious Diseases, 2009Co-Authors: Fabrice Armougom, Fadi Bittar, Nathalie Stremler, J M Rolain, C Robert, J C Dubus, J Sarles, Didier Raoult, B La ScolaAbstract:Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare Pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and Pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, Pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S Pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of Pyrosequencing apparatus.
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use of Pyrosequencing and dna barcodes to monitor variations in firmicutes and bacteroidetes communities in the gut microbiota of obese humans
BMC Genomics, 2008Co-Authors: Fabrice ArmougomAbstract:Background Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput Pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA Pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans.
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use of Pyrosequencing and dna barcodes to monitor variations in firmicutes and bacteroidetes communities in the gut microbiota of obese humans
BMC Genomics, 2008Co-Authors: Fabrice Armougom, Didier RaoultAbstract:Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput Pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA Pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans. The main result was the identification of DNA barcodes targeting the Firmicutes and Bacteroidetes phyla. These barcodes were validated using previously published 16S rRNA gut microbiota clone libraries. In addition, an accurate F/B ratio was found when the DNA barcodes were applied to short Pyrosequencing reads of published gut metagenomes. Finally, the barcodes were utilized to define the F/B ratio of 16S rRNA Pyrosequencing data generated from brain abscess pus and cystic fibrosis sputum. Using DNA barcodes of Bacteroidetes and Firmicutes 16S rRNA genes combined with Pyrosequencing is a cost-effective method for monitoring relevant changes in the relative abundance of Firmicutes and Bacteroidetes bacterial communities in microbial ecosystems.
Craig S Cary - One of the best experts on this subject based on the ideXlab platform.
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groundtruthing next gen sequencing for microbial ecology biases and errors in community structure estimates from pcr amplicon Pyrosequencing
PLOS ONE, 2012Co-Authors: C K Lee, Craig W Herbold, Shawn W Polson, Eric K Wommack, Shannon J Williamson, Ian R Mcdonald, Craig S CaryAbstract:Analysis of microbial communities by high-throughput Pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of Pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on Pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The Pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.
Pal Nyren - One of the best experts on this subject based on the ideXlab platform.
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long read Pyrosequencing using pure 2 deoxyadenosine 5 o 1 thiotriphosphate sp isomer
Analytical Biochemistry, 2002Co-Authors: Baback Gharizadeh, Tommy Nordstrom, Mostafa Ronaghi, Afshin Ahmadian, Pal NyrenAbstract:Abstract Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in Pyrosequencing the natural dATP was replaced by dATPαS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84–89). The applied dATPαS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2′-deoxyadenosine-5′- O ′-(1-thiotriphosphate) Sp-isomer in Pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.
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single nucleotide polymorphism analysis by Pyrosequencing
Analytical Biochemistry, 2000Co-Authors: Afshin Ahmadian, Pal Nyren, Baback Gharizadeh, Mathias Uhlen, Anna C Gustafsson, Fredrik Sterky, Joakim LundebergAbstract:There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, Pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with Pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the Pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by Pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.
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direct analysis of single nucleotide polymorphism on double stranded dna by Pyrosequencing
Biotechnology and Applied Biochemistry, 2000Co-Authors: Tommy Nordstrom, Mostafa Ronaghi, Lena Forsberg, Ulf De Faire, Ralf Morgenstern, Pal NyrenAbstract:Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of Pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPXI) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.
Benjamin N Bimber - One of the best experts on this subject based on the ideXlab platform.
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whole genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep Pyrosequencing
Journal of Virology, 2010Co-Authors: Benjamin N Bimber, Michael Lauck, Dawn M Dudley, Ericka A Becker, Emily N Chin, Haiying Li Grunenwald, Nicholas Caruccio, Simon M Lank, Mark Maffitt, Nancy A. WilsonAbstract:Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep Pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies Pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
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ultradeep Pyrosequencing detects complex patterns of cd8 t lymphocyte escape in simian immunodeficiency virus infected macaques
Journal of Virology, 2009Co-Authors: Benjamin N Bimber, Benjamin J Burwitz, Shelby L Oconnor, Ann M Detmer, Emma Gostick, Simon M Lank, David A Price, Austin L Hughes, David H OconnorAbstract:Human and simian immunodeficiency viruses (HIV/SIV) exhibit enormous sequence heterogeneity within each infected host. Here, we use ultradeep Pyrosequencing to create a comprehensive picture of CD8+ T-lymphocyte (CD8-TL) escape in SIV-infected macaques, revealing a previously undetected complex pattern of viral variants. This increased sensitivity enabled the detection of acute CD8-TL escape as early as 17 days postinfection, representing the earliest published example of CD8-TL escape in intrarectally infected macaques. These data demonstrate that Pyrosequencing can be used to study the evolution of CD8-TL escape during immunodeficiency virus infection with an unprecedented degree of sensitivity.
