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      Accuracy and quality of massively parallel DNA pyrosequencing

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          Abstract

          Error rates were estimated for the Roche GS20 massively parallel pyrosequencing system, and several factors were identified that can be used to remove low-quality reads, improving the accuracy to 99.75% or better.

          Abstract

          Background

          Massively parallel pyrosequencing systems have increased the efficiency of DNA sequencing, although the published per-base accuracy of a Roche GS20 is only 96%. In genome projects, highly redundant consensus assemblies can compensate for sequencing errors. In contrast, studies of microbial diversity that catalogue differences between PCR amplicons of ribosomal RNA genes (rDNA) or other conserved gene families cannot take advantage of consensus assemblies to detect and minimize incorrect base calls.

          Results

          We performed an empirical study of the per-base error rate for the Roche GS20 system using sequences of the V6 hypervariable region from cloned microbial ribosomal DNA (tag sequencing). We calculated a 99.5% accuracy rate in unassembled sequences, and identified several factors that can be used to remove a small percentage of low-quality reads, improving the accuracy to 99.75% or better.

          Conclusion

          By using objective criteria to eliminate low quality data, the quality of individual GS20 sequence reads in molecular ecological applications can surpass the accuracy of traditional capillary methods.

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          Most cited references 19

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          MUSCLE: multiple sequence alignment with high accuracy and high throughput.

           Robert Edgar (2004)
          We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.
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            Genome sequencing in microfabricated high-density picolitre reactors.

            The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
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              ARB: a software environment for sequence data.

              The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.
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                Author and article information

                Journal
                Genome Biol
                Genome Biology
                BioMed Central
                1465-6906
                1465-6914
                2007
                20 July 2007
                : 8
                : 7
                : R143
                Affiliations
                [1 ]Josephine Bay Paul Center, Marine Biological Laboratory at Woods Hole, MBL Street, Woods Hole, MA 02543, USA
                Article
                gb-2007-8-7-r143
                10.1186/gb-2007-8-7-r143
                2323236
                17659080
                Copyright © 2007 Huse et al.; licensee BioMed Central Ltd.

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research

                Genetics

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