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BMC Genomics  2011 

Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing

DOI: 10.1186/1471-2164-12-245

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We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.Scientific strategies and approaches based on next-generation sequencing (NGS) have been revolutionizing genetics over the last few years. Many aspects of basic, applied and clinical research now rely on the generation of enormous amounts of sequence data from various sample sources, to assess polymorphism (mostly SNPs), or expression data (RNA-Seq) at the genome level [1,2]. This shift in the scale of sequence acquisition has been achieved by simultaneous progress in bioinformatics, the availability of genome assemblies and key technical findings in the domains of biochemistry and sequencing device physics [3]. In this context, the 454 GS-FLX (Roche Diagnostics Corporation), Illumina? technology (Illumina, Inc.) and SOLiDTM systems (Applied BiosystemsTM) offer a number of complementary solutions for specific requirements (see Metzker [4] for a review). 454 GS-FLX Titani


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