Achieving high-sensitivity for clinical applications using augmented exome sequencing
Achieving high-sensitivity for clinical applications using augmented exome sequencing
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DOI：
10.1186/s13073-015-0197-4
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发表时间：
2015-07-16
期刊：
影响因子：
12.3
通讯作者：
Chen, Richard
中科院分区：
文献类型：
--
作者：
Patwardhan, Anil;Harris, Jason;Chen, Richard
背景资料：全外显子组测序越来越多地用于遗传疾病的临床评估，但对基因组的医学相关部分的覆盖率和灵敏度的变化仍然知之甚少。几个测序为基础的检测继续提供覆盖面是不够的clinical assessment.Methods：使用从NA 12878参考样品和预定义的列表中获得的序列数据的医学相关的蛋白质编码和非编码序列，我们比较了四个商业外显子捕获平台和全基因组测序的覆盖范围的广度和深度。此外，我们评估了增强外显子组策略ACE的性能，该策略扩展了医学相关区域的覆盖范围，并增强了序列挑战性区域的覆盖范围。利用参考调用集，我们还研究了改进的覆盖率对变异检测灵敏度的影响。结果：我们观察到覆盖率不足，每个传统的外显子组捕获和全基因组平台在几个医学上可解释的基因。这些空白包括报告最近建立的次要发现（ACMG）和已知疾病相关基因座所需的基因组区域。增强的外显子组策略恢复了许多这些缺口，从而提高了这些领域的覆盖率。在临床相关覆盖水平（100%碱基覆盖>= 20倍），ACE提高了医学可解释基因组中基因的覆盖率（相对于其他平台的10- 78%，覆盖率> 90%），ACMG次级发现基因组（91%覆盖，相对于其他平台的4- 75%）和已知与人类疾病相关的变体子集（99%覆盖，相对于其他平台的52- 95%）。覆盖率的提高转化为灵敏度的提高，ACE变异检测灵敏度（> 97.5%SNVs，> 92.5%InDels）超过了传统的全外显子组和全基因组平台所观察到的灵敏度。结论：临床医生在进行临床评估时应考虑分析性能，因为即使是一些遗漏的变异也可能导致报告假阴性结果。扩增的外显子组策略提供了用其他平台无法实现的覆盖水平，从而解决了关于在临床重要区域中缺乏敏感性的问题。在基因组的医学可解释区域的全面覆盖需要更高的局部测序深度的临床应用中，增强的外显子组方法提供了优于其他基于测序的测试的成本和性能优势。
Background: Whole exome sequencing is increasingly used for the clinical evaluation of genetic disease, yet the variation of coverage and sensitivity over medically relevant parts of the genome remains poorly understood. Several sequencing-based assays continue to provide coverage that is inadequate for clinical assessment.Methods: Using sequence data obtained from the NA12878 reference sample and pre-defined lists of medically-relevant protein-coding and noncoding sequences, we compared the breadth and depth of coverage obtained among four commercial exome capture platforms and whole genome sequencing. In addition, we evaluated the performance of an augmented exome strategy, ACE, that extends coverage in medically relevant regions and enhances coverage in areas that are challenging to sequence. Leveraging reference call-sets, we also examined the effects of improved coverage on variant detection sensitivity.Results: We observed coverage shortfalls with each of the conventional exome-capture and whole-genome platforms across several medically interpretable genes. These gaps included areas of the genome required for reporting recently established secondary findings (ACMG) and known disease-associated loci. The augmented exome strategy recovered many of these gaps, resulting in improved coverage in these areas. At clinically-relevant coverage levels (100 % bases covered at >= 20x), ACE improved coverage among genes in the medically interpretable genome (>90 % covered relative to 10-78 % with other platforms), the set of ACMG secondary finding genes (91 % covered relative to 4-75 % with other platforms) and a subset of variants known to be associated with human disease (99 % covered relative to 52-95 % with other platforms). Improved coverage translated into improvements in sensitivity, with ACE variant detection sensitivities (>97.5 % SNVs, >92.5 % InDels) exceeding that observed with conventional whole-exome and whole-genome platforms.Conclusions: Clinicians should consider analytical performance when making clinical assessments, given that even a few missed variants can lead to reporting false negative results. An augmented exome strategy provides a level of coverage not achievable with other platforms, thus addressing concerns regarding the lack of sensitivity in clinically important regions. In clinical applications where comprehensive coverage of medically interpretable areas of the genome requires higher localized sequencing depth, an augmented exome approach offers both cost and performance advantages over other sequencing-based tests.