Bioinformatic Challanges in clinical diagnostic application of targeted next generation sequencing. Experience from Pheochromocytoma.. Bioinformatic Challanges in clinical diagnostic application of targeted next generation sequencing. Experience from Pheochromocytoma.

Background: Recent studies have demonstrated equal quality of targeted next generation sequencing (NGS) compared to Sanger Sequencing. Whereas these novel sequencing processes have a validated robust performance, genomic enrichment and bioinformatic software needs to be further investigated in a diagnostic setting. Methods: DNA from 21 patients with genetic variants in SDHB, VHL, EPAS1, RET, (n=17) or clinical criteria of NF1 syndrome (n=4) were included. Targeted NGS was performed using Truseq custom amplicon enrichment sequenced on an Illumina MiSEQ instrument. Results were analysed in parallel using three bioinformatics pipelines; (1) MiSEQ Reporter, fully automatized and integrated software, (2) CLC Genomics Workbench, graphical interface based software, and ICP (3) an in-house scripted custom pipeline. Results: A tenfold read coverage was achieved in between 95-98% of targeted bases. All workflows had alignment of reads to SDHA and NF1 pseudogenes. Compared to Sanger sequencing, variant calling revealed a sensitivity ranging from 83 to 100% and a specificity of 99.9-100%. Only MiSEQ reporter identified all pathogenic variants in both sequencing runs. Conclusions: We conclude that targeted next generation sequencing have equal quality compared to Sanger sequencing. Enrichment specificity and the bioinformatic performance need to be carefully assessed in a diagnostic setting. As acceptable accuracy was noted for a fully automated bioinformatic workflow, we suggest that processing of NGS data could be performed without expert bioinformatics skills.

背景：近期研究表明，靶向二代测序（targeted next generation sequencing, NGS）与桑格测序（Sanger Sequencing）的检测质量相当。尽管这类新型测序技术已被验证具备稳定可靠的性能，但在诊断场景中，基因组富集策略与生物信息学分析软件仍需进一步探索。

方法：本研究纳入21例患者的DNA样本，其中17例携带SDHB、VHL、EPAS1、RET基因的遗传变异，另外4例符合NF1综合征临床诊断标准。采用Truseq定制扩增子富集方案进行建库，并在Illumina MiSEQ测序仪上完成靶向NGS测序。采用三种生物信息学分析流程并行处理测序结果：(1) MiSEQ Reporter，全自动化集成分析软件；(2) CLC Genomics Workbench，基于图形界面的分析软件；(3) 自研脚本定制分析流程。

结果：95%~98%的靶向碱基可实现10倍测序深度覆盖。所有分析流程均能将测序读段比对至SDHA及NF1假基因。与桑格测序相比，变异检出的灵敏度介于83%~100%之间，特异度为99.9%~100%。仅MiSEQ Reporter可在两次测序运行中检出全部致病变异。

结论：本研究证实，靶向二代测序与桑格测序的检测质量相当。富集特异性与生物信息学分析性能仍需在诊断场景下进行严谨评估。鉴于全自动化生物信息学分析流程已具备可接受的准确性，我们认为无需具备专业生物信息学技能即可完成NGS数据的分析处理。