Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.
Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.
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DOI：
10.1186/s12864-017-4428-5
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发表时间：
2018-01-08
期刊：
影响因子：
4.4
通讯作者：
Thorner AR
中科院分区：
文献类型：
--
作者：
MacConaill LE;Burns RT;Nag A;Coleman HA;Slevin MK;Giorda K;Light M;Lai K;Jarosz M;McNeill MS;Ducar MD;Meyerson M;Thorner AR
当大规模平行测序（MPS）用于敏感应用时，如低频体细胞变异发现、古DNA研究、人类样本中的微生物检测或循环无细胞肿瘤DNA（ctDNA）变异检测，样本索引串扰可能导致假阳性结果。因此，MPS检测试剂盒的检测限与指数串扰的程度直接相关。当使用标准组合衔接子时，观察到高达0.29%的串扰率，在非图案化和图案化的Illumina流动池中，每个泳道分别产生110，180（0.1%串扰率）或1，121，074（0.29%串扰率）个错误分配的读数。在这里，我们证明了使用独特的双匹配索引适配器可以将索引串扰显著降低到每个流动池通道≤1个错误分配的读数。虽然目前的研究是使用双重匹配的指数，使用独特的，双重不相关的指数也将是一个有效的替代方案。对于敏感的下游分析，使用组合索引进行多重杂交捕获和测序是不合适的，因为它导致不可接受数量的错配读段。使用双重匹配的索引适配器，几乎可以消除串扰。这些结果表明，使用这样的衔接子对于降低旨在鉴定低等位基因频率事件的测定中的假阳性率是至关重要的，并且强烈表明双重匹配的衔接子可用于所有敏感的MPS应用。本文的在线版本（10.1186/s12864-017-4428-5）包含补充材料，可供授权用户使用。
Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications. The online version of this article (10.1186/s12864-017-4428-5) contains supplementary material, which is available to authorized users.
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