Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Collaborative Innovation Center for Brain Science, Shanghai Jiao Tong University, Shanghai, 200030, People's Republic of China.
Hereditas. 2021 Feb 17;158(1):10. doi: 10.1186/s41065-021-00171-3.
Next-generation sequencing technology is developing rapidly and target capture sequencing has become an important technique. Several different platforms for library preparation and target capture with different bait types respectively are commercially available. Here we compare the performance of the four platforms with different bait types to find out their advantages and limitations. The purpose of this study is to help investigators and clinicians select the appropriate platform for their particular application and lay the foundation for the development of a better target capture platform for next-generation sequencing.
We formulate capture efficiency as a novel parameter that can be used to better evaluations of specificity and coverage depth among the different capture platforms. Target coverage, capture efficiency, GC bias, AT Dropout, sensitivity in single nucleotide polymorphisms, small insertions and deletions detection, and the feature of each platform were compared for low input samples. In general, all platforms perform well and small differences among them are revealed. In our results, RNA baits have stronger binding power than DNA baits, and with ultra deep sequencing, double stranded RNA baits perform better than single stranded RNA baits in all aspects. DNA baits got better performance in the region with high GC content and RNA baits got lower AT dropout suggesting that the binding power is different between DNA and RNA baits to genome regions with different characteristics.
The platforms with double stranded RNA baits have the most balanced capture performance. Our results show the key differences in performance among the four updated platforms with four different bait types. The better performance of double stranded RNA bait with ultra deep sequencing suggests that it may improve the sensitivity of ultra low frequent mutation detection. In addition, we further propose that the mixed baits of double stranded RNA and single stranded DNA may improve target capture performance.
下一代测序技术发展迅速，目标捕获测序已成为一项重要技术。目前市场上有几种不同的文库制备和目标捕获平台，分别采用不同类型的探针。本研究比较了不同探针类型的四种平台的性能，以了解它们的优缺点。本研究旨在帮助研究人员和临床医生为其特定应用选择合适的平台，并为下一代测序的更好的目标捕获平台的发展奠定基础。
我们提出了捕获效率作为一个新的参数，可以更好地评估不同捕获平台的特异性和覆盖深度。比较了低输入样本中各平台的目标覆盖度、捕获效率、GC 偏倚、AT 缺失、单核苷酸多态性检测灵敏度、小插入和缺失检测灵敏度以及各平台的特点。总体而言，所有平台的性能都很好，并且显示出它们之间的微小差异。在我们的结果中，RNA 探针比 DNA 探针具有更强的结合能力，并且在超深度测序中，双链 RNA 探针在各个方面的表现均优于单链 RNA 探针。在 GC 含量高的区域，DNA 探针的性能更好，而在 AT 缺失率方面，RNA 探针的性能更低，这表明 DNA 和 RNA 探针与具有不同特征的基因组区域的结合能力不同。
双链 RNA 探针平台具有最平衡的捕获性能。我们的结果显示了四种不同探针类型的四个最新平台在性能方面的关键差异。超深度测序中双链 RNA 探针的更好性能表明，它可能提高超低频率突变检测的灵敏度。此外，我们进一步提出双链 RNA 和单链 DNA 的混合探针可能会改善目标捕获性能。
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