Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence.
Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence.
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DOI：
10.1093/nar/gku607
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发表时间：
2014
影响因子：
14.9
通讯作者：
Hallwirth CV
中科院分区：
文献类型：
--
作者：
Deakin CT;Deakin JJ;Ginn SL;Young P;Humphreys D;Suter CM;Alexander IE;Hallwirth CV
条形码载体是研究造血基因治疗中克隆多样性和动态的有前途的工具。当确切数量、序列同一性和丰度未知时，对标记有条形码载体的克隆进行分析需要准确识别潜在大量的单独稀有条形码。这是一个具有内在挑战性的应用，使用当代下一代测序技术的可行性尚未解决。为了凭经验探索这种潜在的应用，在没有事先假设的情况下，我们对已知复杂性的条形码文库进行了测序。使用Illumina平台对含有1个、10个和100个Sanger测序的条形码的文库进行测序，其中100个条形码文库也使用SOLiD平台进行测序。含有1和10个条形码的文库通过丰度的几个对数倍差异与由测序错误产生的假条形码区分开。然而，在100个条形码库中，预期条形码和假条形码重叠，并且不能通过生物信息学过滤和聚类策略来解决。在独立测序运行中，多个假阳性条形码似乎以比已知条形码更高的丰度表示，尽管它们确认不存在于原始文库中。这些错误可能以应用依赖的方式影响条形码研究，与随机和系统错误的存在一致，其机制尚未完全解决。
Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved.
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