DNA Sequence Capture and Enrichment by Microarray Followed by Next-Generation Sequencing for Targeted Resequencing: Neurofibromatosis Type 1 Gene as a Model

BACKGROUND: The introduction and use of next-generation sequencing (NGS) techniques have taken genomic research into a new era; however, implementing such powerful techniques in diagnostics laboratories for applications such as resequencing of targeted disease genes requires attention to technical issues, including sequencing template enrichment, management of massive data, and high interference by homologous sequences. METHODS: In this study, we investigated a process for enriching DNA samples that uses a customized high-density oligonucleotide microarray to enrich a targeted 280-kb region of the NF1 (neurofibromin 1) gene. The captured DNA was sequenced with the Roche/454 GS FLX system. Two NF1 samples (CN1 and CN2) with known genotypes were tested with this protocol. RESULTS: Targeted microarray capture may also capture sequences from nontargeted regions in the genome. The capture specificity estimated for the targeted NF1 region was approximately 60%. The de novo Alu insertion was partially detected in sample CN1 by additional de novo assembly with 50% base-match stringency; the single-base deletion in sample CN2 was successfully detected by reference mapping. Interferences by pseudogene sequences were removed by means of dual-mode reference-mapping analysis, which reduced the risk of generating false-positive data. The risk of generating false-negative data was minimized with higher sequence coverage (>30X). CONCLUSIONS: We. used a clinically relevant complex genomic target to evaluate a microarray-based sample-enrichment process and an NGS instrument for clinical resequencing purposes. The results allowed us to develop a systematic data-analysis strategy and algorithm to fit potential clinical applications. (C) 2009 American Association for Clinical Chemistry