Rare Deleterious Mutations in ALS Genes

Genomic DNA was enriched for selected RNA-binding proteins and known ALS genes using a custom designed Agilent SureSelect in solution kit. Sequencing was performed using an Illumina HiScan platform according to manufacturers instructions.
Rare deleterious mutations were defined by frequency within the Exome Aggregation Consortium data set of <1/10,000 control alleles (Lek et al., 2016 (link)), and a Phred-scaled Combined Annotation Dependent Depletion (CADD) score >10 (Kircher et al., 2014 (link)). Comparison of various pathogenicity prediction tools recently supported the sensitivity and specificity of CADD (Salgado et al., 2016 (link)). Given that we were focused on exonic changes with an effect on protein function, synonymous changes were excluded. We excluded any changes with a read depth <10 and validated by Sanger sequencing any changes with read depth 10–15 or a novel allele frequency less than one third the reference allele frequency (Supplementary Figure 1).
ExAC defines constrained genes based on an observed frequency of loss of function mutations which is much less than predicted by sequence specific mutation probabilities (Lek et al., 2016 (link)). A threshold for “constrained” is set as probability of a gene being loss of function intolerant (PLi) > 0.95.