Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes.
Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes.
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DOI：
10.1186/s12864-015-1806-8
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发表时间：
2015-08-07
期刊：
影响因子：
4.4
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Peng Q;Vijaya Satya R;Lewis M;Randad P;Wang Y
PCR扩增子测序已广泛用作DNA和RNA序列分析的靶向方法。高度多重PCR进一步使得能够在一个简单的反应中富集数百个扩增子。同时，PCR扩增子测序的性能可能受到诸如高重复读数、聚合酶伪影和PCR扩增偏倚等问题的负面影响。最近，研究人员通过将分子条形码引入PCR引物设计中，在解决这些缺点方面取得了一些良好的进展。到目前为止，大多数工作都是使用一到几对引物进行证明的，这限制了可以分析的区域的大小。我们开发了一种简单的方案，其使得能够在具有数百个扩增子的高度多重PCR中使用分子条形码。使用该方案和参考材料，我们展示了在大区域内以非常低的分数进行精确变体调用和靶向RNA定量的应用。我们还评估了该方案在分析FFPE样品中的实用性。我们证明了分子条形码在高度多重PCR中的成功实施，多重规模比早期工作高出许多倍。我们表明，新方案结合了高度多重PCR和分子条形码的优点，即分析非常大的区域、低DNA输入要求、非常好的再现性和以最小的假阳性（FP）检测低至1%突变的能力。本文的在线版本（doi：10.1186/s12864-015-1806-8）包含补充材料，可供授权用户使用。
PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. High multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently researchers have made some good progress in addressing these shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze. We developed a simple protocol, which enables the use of molecular barcodes in high multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we demonstrated the applications in accurate variant calling at very low fraction over a large region and in targeted RNA quantification. We also evaluated the protocol’s utility in profiling FFPE samples. We demonstrated the successful implementation of molecular barcodes in high multiplex PCR, with multiplex scale many times higher than earlier work. We showed that the new protocol combines the benefits of both high multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1 % mutations with minimal false positives (FP). The online version of this article (doi:10.1186/s12864-015-1806-8) contains supplementary material, which is available to authorized users.