Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.
Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.
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DOI：
10.1186/s12864-016-2757-4
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发表时间：
2016-06-13
期刊：
影响因子：
4.4
通讯作者：
Quail MA
中科院分区：
文献类型：
--
作者：
Aigrain L;Gu Y;Quail MA
在过去的十年中，下一代测序（NGS）技术的出现使得DNA测序在每个测序碱基的价格和易于产生DNA文库方面都实现了民主化。当涉及到为目前的市场领导者Illumina准备DNA测序文库时，有过多的试剂盒可用，用户很难确定哪种试剂盒最适合和最有效地用于他们的应用;主要的问题不仅是成本，而且是最小的偏差，产量和时间效率。我们使用相同的DNA样品，通过使用新的液滴数字PCR（ddPCR）测定法探测每个方案步骤后剩余的DNA量，以系统的方式比较了9种市售文库制备试剂盒。该方法允许在连接或PCR富集后精确定量两端带有衔接子或P5/P7序列的片段。我们还研究了DNA输入和DNA片段大小对最终文库制备效率的潜在影响。文库的总体文库制备效率显示出不同试剂盒之间的重要差异，其中将几个步骤组合成单个步骤的试剂盒表现出比其他试剂盒高4至7倍的最终产率。详细的ddPCR数据还揭示了衔接子连接产率本身在试剂盒之间的变化超过10倍，某些连接效率如此之低，以至于它可能损害原始文库的复杂性并破坏测序结果。当需要PCR富集步骤时，较低的接头连接的DNA输入导致更大的扩增产率，隐藏了试剂盒之间的潜在差异。我们描述了一种ddPCR测定，使我们能够探测文库制备中最关键步骤的效率，连接，并得出结论，哪些试剂盒更有可能保留样品的异质性，并减少扩增的需要。本文的在线版本（doi：10.1186/s12864-016-2757-4）包含补充材料，可供授权用户使用。
The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification. The online version of this article (doi:10.1186/s12864-016-2757-4) contains supplementary material, which is available to authorized users.