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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 12

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          Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

          Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.
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            The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis

            The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101 632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.
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              BioEdit: a user friendly biological sequence alignment editor analysis program for Windows 95/98/NT

               TA Hall,  T.A. Hall,  T., HALL (1999)
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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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