A Robust Protocol to Increase NimbleGen SeqCap EZ Multiplexing Capacity to 96 Samples

Contemporary genetic studies frequently involve sequencing of a targeted gene panel, for instance consisting of a set of genes associated with a specific disease. The NimbleGen SeqCap EZ Choice kit is commonly used for the targeted enrichment of sequencing libraries comprising a target size up to 7 Mb. A major drawback of this commercially available method is the exclusive use of single-indexing, meaning that at most 24 samples can be multiplexed in a single reaction. In case of relatively small target sizes, this will lead to excessive amounts of data per sample. We present an extended version of the NimbleGen SeqCap EZ protocol which allows to robustly multiplex up to 96 samples. We achieved this by incorporating Illumina dual-indexing based custom adapters into the original protocol. To further extend the optimization of cost-efficient sequencing of custom target panels, we studied the effect of higher pre-enrichment pooling factors and show that pre-enrichment pooling of up to 12 samples does not affect the quality of the data. To facilitate evaluation of capture efficiency in custom design panels, we also provide a detailed reporting tool.当代遗传学研究常涉及靶向基因测序面板（targeted gene panel）测序，例如由与特定疾病相关的一组基因构成的检测组合。NimbleGen SeqCap EZ Choice 试剂盒常用于靶向富集测序文库，其可覆盖的靶区域最大尺寸可达7 Mb。该商用方法的主要缺陷在于仅支持单索引（single-indexing）建库，即单次反应最多可对24个样本进行多重混样测序。当靶区域尺寸较小时，该方法会导致单样本数据量过剩。本研究提出了一种改良版NimbleGen SeqCap EZ实验流程，可稳定实现最多96个样本的多重测序。我们通过在原始流程中引入基于Illumina双索引（dual-indexing）的定制接头完成了这一优化。为进一步优化定制靶向基因组合的低成本测序方案，我们探究了更高富集前混合倍数的影响，结果表明单次富集前混合最多12个样本不会对测序数据质量产生负面影响。为便于评估定制设计基因组合的捕获效率，我们还提供了一款详细的数据分析报告工具。