High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing
High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing
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DOI：
10.1136/jmedgenet-2012-100973
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发表时间：
2012-12-01
影响因子：
4
通讯作者：
Otto, Edgar A.
中科院分区：
文献类型：
--
作者：
Halbritter, Jan;Diaz, Katrina;Otto, Edgar A.
目的以低成本检测192例肾单位结核相关性纤毛病变患者的11个已知NPHP基因（NPHP 1-NPHP 11）编码区的致病突变。我们应用了10倍引物多重方法，允许同时对48个DNA样品的475个扩增子（251个外显子）进行基于PCR的扩增。在四轮扩增之后，随后在随后的PCR中用不同的条形码索引所有192个患者来源的产物，在HiSeq 2000仪器（Illumina）的一个泳道上进行2x 100个配对末端测序。使用“CLC Genomics”软件进行生物信息学分析。结果生物信息学分析显示168/192（87.5%）份DNA样本的30 x覆盖率（中位数449 x）和251个靶向编码外显子中的234个（灵敏度：93.2%）。为了证明原理，我们分析了20个已知的突变，并确定了其中18个处于正确的接合性状态（90%）。同样，我们在34/192例患者（18%）中发现了致病性突变，并在基因NPHP 3（7），NPHP 4（3），IQCB 1（4），CEP 290（7），RPGRIP 1 L（1）和TMEM 67（1）中发现了23个新突变。此外，我们发现了40个不同的单一杂合错义变异的未知significant.Conclusions我们得出结论，组合的方法，基于阵列的多重PCR扩增的Fluidigm访问阵列平台，其次是下一代测序是非常具有成本效益的，并大大有利于诊断突变分析广泛异质性孟德尔疾病。
Objective To identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost.Methods Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2x100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using 'CLC Genomics Workbench' software. Potential mutations were confirmed by Sanger sequencing and shown to segregate.Results Bioinformatics analysis revealed sufficient coverage of 30xfor 168/192 (87.5%) DNA samples (median 449x) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance.Conclusions We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.