Accuracy and quality of massively parallel DNA pyrosequencing

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.