Identifying susceptible genes for Parkinson disease employing next-generation sequencer

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To identify susceptible genes for Parkinson disease, comprehensive resequencing analysis of candidate genes (responsible genes for lysosomal storage disease) employing a next-generation sequencer was conducted. We arranged 64 DNA samples of patients with Parkinson disease in an 8x8 rectangular array, and formed the pooled DNA sample sets by each row and column (8 rows and 8 columns). Each pooled DNA sample set was subjected to PCR amplifications of candidate genes (approximately 24 kb in total) and every amplicons for each set were mixed. The mixed amplicons for each set was subjected to an analysis of single lane of Illumina GAIIx, single-end, 100bp, according to manufactures' instructions.Reads were aligned to the reference sequences (hg19) of candidate genes by BWA with default parameters, and approximately 1,000-2,000 coverage per allele was obtained. Only 50 bp of reads with more than 20 of QV were used to screen variants. We specifically focused on rare variants not registered in dbSNP, and searching for such variants employing an integer programming algorithm. The results were that 3 variants not registered in dbSNP were obtained; each variant was identified in three different samples in the heterozygous state. All of them were nonsynonymous single nucleotide substitutions. One was reported to be pathogenic for a lysosomal storage disease, and two were unknown variants. All variants were confirmed by an additional direct nucleotide sequence analysis (Sanger method).Based on these results, it was suggested that the matrix pooling approach is an accurate and cost effective testing algorithm for detection of rare variants.