Accuracy and quality of massively parallel DNA pyrosequencing.
Accuracy and quality of massively parallel DNA pyrosequencing.
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DOI：
10.1186/gb-2007-8-7-r143
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发表时间：
2007
期刊：
影响因子：
12.3
通讯作者：
Welch DM
中科院分区：
文献类型：
--
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
估计了Roche GS 20大规模并行焦磷酸测序系统的错误率，并确定了几个可用于去除低质量读数的因素，将准确度提高到99.75%或更高。大规模并行焦磷酸测序系统提高了DNA测序的效率，尽管Roche GS 20的每碱基准确度仅为96%。在基因组计划中，高度冗余的共有序列可以弥补测序错误。相比之下，微生物多样性的研究，目录之间的差异核糖体RNA基因（rDNA）或其他保守的基因家族的PCR扩增子不能利用的共识大会，以检测和最大限度地减少不正确的碱基调用。我们使用来自克隆的微生物核糖体DNA的V6高变区序列（标签测序）对Roche GS 20系统的每个碱基错误率进行了实证研究。我们计算出未组装序列的准确率为99.5%，并确定了几个可用于去除一小部分低质量读数的因素，将准确率提高到99.75%或更高。通过使用客观标准来消除低质量数据，在分子生态学应用中单个GS 20序列读数的质量可以超过传统毛细管方法的准确性。
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. 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. 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. 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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