High-quality DNA sequence capture of 524 disease candidate genes

Abstract

The accurate and complete selection of candidate genomic regions from a DNA sample before sequencing is critical in molecular diagnostics. Several recently developed technologies await substantial improvements in performance, cost, and multiplex sample processing. Here we present the utility of long padlock probes (LPPs) for targeted exon capture followed by array-based sequencing. We found that on average 92% of 5,471 exons from 524 nuclear-encoded mitochondrial genes were successfully amplified from genomic DNA from 63 individuals. Only 144 exons did not amplify in any sample due to high GC content. One LPP was sufficient to capture sequences from <100-500 bp in length and only a single-tube capture reaction and one microarray was required per sample. Our approach was highly reproducible and quick (<8 h) and detected DNA variants at high accuracy (false discovery rate 1%, false negative rate 3%) on the basis of known sample SNPs and Sanger sequence verification. In a patient with clinical and biochemical presentation of ornithine transcarbamylase (OTC) deficiency, we identified copy-number differences in the OTC gene at exon-level resolution. This shows the ability of LPPs to accurately preserve a sample's genome information and provides a cost-effective strategy to identify both single nucleotide changes and structural variants in targeted resequencing.DNA sequencing | mitochondrial disease | statistical analysis | target capture | copy number detection D NA variant discovery is performed in an increasing number of individuals with specific disorders. In addition, thousands of disease candidate regions have been prioritized through linkage mapping and functional approaches. Studying hundreds of candidate genes in hundreds of samples is nontrivial given the high standards of accuracy and completeness in medical resequencing. To select genomic regions at a fraction of the cost of traditional sample preparations, novel DNA sequence capture methods are under development that include hybridization-based target enrichment (e.g., microarrays, beads) and in-solution methods (1-11). Hybrid capture is quickly scalable, shows high levels of uniformity, and has been applied in exome sequencing (5, 12). In-solution methods, and in particular molecular inversion probes (MIPs) (6-8, 13), provide the highest target specificity (>98%) at comparably lower costs and DNA requirement, which has advantages when studying many samples. However, MIPs' target size limitation of 191 bp (8) could lead to amplification failures or overlooked sample variants if an exonic SNP is located in the probes' annealing regions, which become part of the final amplification sequence. To capture most human exons (≤500 bp) using only a single capture probe per exon, we have developed an in-solution method using long padlock probes (LPPs) (14). Here we describe an optimized protocol for LPPs and the application to the capture of 524 candidate genes (5,471 exons) in a simple and robust single-tube assay. The nuclearencoded mitochondrial genes selected a...

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene, gene panel, or exome/genome sequencing

“…Although the contributions of these sorts of changes may be low for most single-gene diseases, they help rule out the gene(s) and may provide a diagnosis. Complementing NGS testing with Sanger sequencing is inevitable, at least with current target capture and sequencing technologies, which consistently fail to amplify certain regions owing to several factors that include sequence homology with pseudogenes, 13,14 high GC-rich content, 15,16 highly repetitive regions, and other sequence complexities. Sanger sequencing can also be used to "fill in" missing data from bases or regions that are supported by an insufficient number of reads to call variants confidently.…”

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

“…The resulting method, termed smMIP (for single molecule Molecular Inversion Probes), combines the MIP strategy for targeted capture (Turner et al 2009;Shen et al 2011;O'Roak et al 2012a) with single molecule tagging (Hiatt et al 2010;Casbon et al 2011;Fu et al 2011;Jabara et al 2011;Kinde et al 2011;Kivioja et al 2012;Shiroguchi et al 2012). MIPs represent an attractive platform for targeted capture because of their very low per-sample cost, workflow simplicity, target-set modularity, and low sample input requirements.…”

Key Principles and Clinical Applications of “Next-Generation” DNA Sequencing

“…Therefore, it is often more cost-effective for clinical researchers to sequence only the exome (the 2% of the genome represented by protein-coding regions or exons), the "Mendelianome" (coding regions of 2,993 known disease genes), or targeted disease gene panels to screen for relevant mutations in the diagnosis and treatment of disease (30,42). In targeted NGS reactions, sequencing reads are intentionally distributed to specific genomic locations, which allows for higher sequencing coverage and therefore ensures accurate detection of sequence variants at these loci, regardless of platform error rates.…”