Abstract
Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive. Recently, novel genome partitioning methods have been developed that allow to target re-sequencing to specific genomic compartments, but practical experience with these methods is still limited. In this study, we have combined a novel droplet-based multiplex PCR method and next generation sequencing to screen patients with X-linked mental retardation (XLMR) for mutations in 86 previously identified XLMR genes. In total, affected males from 24 large XLMR families were analyzed, including three in whom the mutations were already known. Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform. Highly specific and uniform enrichment was achieved: on average, 67.9% unambiguously mapped reads were derived from amplicons, and for 88.5% of the targeted bases, the sequencing depth was sufficient to reliably detect variations. Potentially disease-causing sequence variants were identified in 10 out of 24 patients, including the three mutations that were already known, and all of these could be confirmed by Sanger sequencing. The robust performance of this approach demonstrates the general utility of droplet-based multiplex PCR for parallel mutation screening in hundreds of genes, which is a prerequisite for the diagnosis of mental retardation and other disorders that may be due to defects of a wide variety of genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11568-010-9137-y) contains supplementary material, which is available to authorized users.
Highlights
The core problem of human and medical genetics is to identify genetic variants underlying specific phenotypes.This has been traditionally achieved by Sanger sequencing of PCR products, which is a tedious process and often economically formidable if a large set of genes needs to be studied
Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive
In order to search for mutations in 86 known X-linked mental retardation (XLMR) genes, we applied a novel droplet-based multiplex PCR method to simultaneously amplify their coding regions and splice sites
Summary
The core problem of human and medical genetics is to identify genetic variants underlying specific phenotypes This has been traditionally achieved by Sanger sequencing of PCR products, which is a tedious process and often economically formidable if a large set of genes needs to be studied. Exploitation of the full potential of the current platforms requires a subset of the genome of medical interest to be isolated for targeted sequencing To meet this need, a variety of methods have been developed in the last few years to carry out genome partitioning. With different strengths and weaknesses, most of these strategies can be used to enrich megabase-scale target regions These can be continuous genomic intervals or a full complement of protein-coding exons (Albert et al 2007; Hodges et al.2007; Okou et al 2007; Bau et al 2009; Gnirke et al 2009; Ng et al 2009; Turner et al 2009)
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