In total, selleck compound 43,142 unique baits were used for these experiments covering a total of 2.58 MB in the chromosome 9p FTD/ALS

locus (c9FTD/ALS). For subsequent mutational screening of the GGGGCC hexanucleotide repeat expansion, we used DNA from 402 Finnish ALS cases and 478 Finnish neurologically normal individuals that had previously been used to identify the chromosome 9p21 association signal (Laaksovirta et al., 2010). An additional 268 DNA samples were obtained from affected probands in unrelated ALS families (198 U.S. cases, 41 German cases, and 29 Italian cases) and from 75 Finnish individuals who had presented with isolated FTD. Control samples consisted of 262 neurologically normal US individuals obtained from the NINDS repository at Coriell, 64 neurologically normal German individuals, and 83 neurologically normal Italian individuals. An additional series of 300 anonymous African and Asian samples that are part of the Human Gene Diversity Panel (Cann et al., 2002) were included in the mutational analysis as controls to evaluate the genetic variability of the repeat expansion in non-Caucasian populations. Demographics and clinical features of these samples are summarized in Table 1. Appropriate institutional

review boards approved the study. Paired-end sequencing was performed on a next-generation HiSeq2000 sequencer according to the manufacturer’s BMS-907351 research buy protocol (Illumina, San Diego, CA, USA). This generated 56.7 gigabases of alignable sequence data for the control sample ND11463 (mean read depth for the chromosome 9 region 27,367,278 to 27,599,746 bp = 42.2) and 114.4 gigabases for the case sample ND06769 (mean read depth = 170.4). Sequence alignment and variant calling were performed against the reference human genome (UCSC hg 18). Sequencing

reads were aligned using BWA (Li and Durbin, 2009). Sorting, indexing, read duplicate removal, and merging of BAM files were preformed with Picard (http://picard.sourceforge.net). The Rutecarpine Genome Analysis Toolkit was used to perform base quality score recalibration and to call variants (McKenna et al., 2010). SNPs identified within CEU individuals from the 1000 Genomes project (April 2009 release, http://www.1000genomes.org) or in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/, Build 132) were excluded. The remaining variants were annotated to RefSeq transcripts and protein coding variants prioritized for examination. Repeat-primed PCR was performed as follows: 100 ng of genomic DNA were used as template in a final volume of 28 μl containing 14 μl of FastStart PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA), and a final concentration of 0.18 mM 7-deaza-dGTP (New England Biolabs, Ipswich, MA, USA), 1× Q-Solution (QIAGEN, Valencia, CA, USA), 7% DMSO (Sigma-Aldrich), 0.9 mM MgCl2 (QIAGEN), 0.7 μM reverse primer consisting of ∼four GGGGCC repeats with an anchor tail, 1.