Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

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 material: The online version of this article (doi:10.1007/s11568-010-9137-y) contains supplementary material, which is available to authorized users.

Keywords: Droplet-based multiplex PCR; Massive parallel sequencing; Mutation screening; X-linked mental retardation.

Fig. 1

Normalised sequencing coverage distribution. a Normalised coverage cumulative distribution of the sequenced bases within all amplicons. b Normalised coverage cumulative distribution of all amplicons. Normalised coverage is the absolute coverage divided by the mean coverage. Amplicon coverage is the median coverage of all the bases within the amplicon. Solid lines represent the average among all the 24 samples and the dashed lines represent 25th and 75th percentiles

Fig. 2

The correlation of sequencing coverage between 2 samples. a The sequence coverage of each base from sample 1 was plotted against sample 3. b. The sequence coverage of each amplicon from the same 2 samples was plotted. The sequence coverage of an amplicon is the median sequence coverage of all bases within the amplicon. The data shown here are representative for all 24 patients

Fig. 3

The dependence of sequencing coverage of amplicons on their GC content (a) or amplicon length (b). The amplicons were grouped depending on their GC content (a) or length (b), the distribution of sequencing coverage within each group is shown as boxplot