Sample Selection and Demographics
HEX Version 1.0 includes DNA from 468 individuals categorized as cognitively and neuropathologically healthy. DNA was extracted from brain tissue obtained from several UK and US brain banks, including the Thomas Willis Oxford Brain Collection, the London Neurodegenerative Disease Brain Bank, Brains for Dementia Research, the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona, and the MRC Sudden Death Brain Bank.
As a repository of benign genetic variability, HEX includes only sequences from individuals who met the following criteria:
- Age at death over 60 years.
- Absence of clinical diagnosis of neurodegenerative disease.
- Absence of overt neurodegenerative pathology upon neuropathological examination. Braak stage ≤ II.
- Samples from unrelated individuals.
Of the 468 samples analyzed, the age at death ranged from 60 to 102 years old, with an average of 79.78 years. All individuals were Caucasian. Of the 468 samples, 231 were female (49 percent).
Sample Preparation and Sequencing
DNA was extracted from frozen brain homogenates according to standard practices. Libraries were prepared according to either Illumina’s TruSeq or Agilent SureSelect V4 recommendations and sequenced on the HiSeq2000 using 2x100 paired-end base pair reads.
Raw FASTQ files were processed according to the GATK’s Best Practices for version 3.4. In short, reads were aligned using the Burrows-Wheeler Aligner, optical duplicates were excluded using picard, local realignment around indels and base quality score recalibration were performed using GATK. Variant Calling was performed simultaneously for SNPs and InDEls using GATK’s HaplotypeCaller on the entire cohort. Variants were then separated into SNPs and InDels and variant recalibration was processed for each type of variability independently. Variants were then decomposed and normalized using the tool vt. Passing filter variants were annotated with biological information (transcripts, consequences, etc.) using Ensembl’s VEP v75. ExAC frequency data was extracted from the ExAC VCF version 0.3 released on January 13, 2015.