Truseq exome capture kit

DNA was isolated from brain tissues using Omniprep Genomic DNA isolation kit (G-Biosciences, USA) as per manufacturer’s protocol. Exome capture was done using Illumina TruSeq Exome capture kit. 100 base pair paired end sequencing was done using Illumina HiSeq 2000 (Ilumina, USA). The exome sequence data is publicly available at the sequence read archive (SRA ID: SRP045655).

Blood was taken for genetic testing in both patients with informed consent. Sequencing of exons 1 and 9–13 of the MAPT gene was performed initially. Subsequently both samples underwent exome sequencing. Exome enrichment was performed using TruSeq Exome Capture kit (Illumina, San Diego, CA, USA). Sequencing was performed on a HiSeq 2000 (Illumina). Reads were aligned to GRCh37/hg19 using BWA, variants called according to GATK best practice guidelines and annotated with ANNOVAR (Wang et al., 2010 (link)). In silico pathogenicity predictions of nonsynonymous variants were done with the combined annotation-dependent depletion (CADD) (Kircher et al., 2014 (link)). Variants were filtered against population databases (1000Genomes, ESP, and gnomAD) and assessed based on variant type (missense, nonsense, splice site, frameshift, nonframeshift) and predicted pathogenicity.

A multigenerational family with European ancestry was selected for exome sequencing, with DNA samples available on five affected and three unaffected individuals (two of the unaffected individuals are marry-in relatives). This family was selected based on the number of affected family members with severe curves and the relative pedigree-distance between affected family members. Exome capture was performed on 1 µg of genomic DNA from three affected individuals (IV:1, IV:6, and II:4) in this family using the Illumina TruSeq Exome Capture kit. Samples were sequenced with a 2x100bp run at the Illumina HiSeq 2000 at the University of Colorado Denver Genomics and Microarray Core Facility.