SNV detection and classification.

<p>(A) The flow diagram depicts the experimental strategy for SNV discovery and confirmation. For the AGRE and control cohorts, orthogonal multiplexing was performed to prepare two distinct sets of sample pools (15 pools of 20 samples each, or 20 pools of 15 samples each). Following enrichment of exonic target regions for all 18 mGluR pathway genes, SNVs were identified and confirmed by deep resequencing of orthogonal pools on two independent NGS platforms (Illumina GAII and the Helicos HeliScope). SNVs concordantly detected on both platforms were then analyzed as shown in panel B. (B) The flow diagram depicts the procedure used to classify the presumptive functional effects of identified variants. SNVs concordantly detected on both NGS platforms were classified as common or rare using a minor allele frequency (maf) threshold of 1%. Common SNVs, rare SNVs occurring in both autism and control populations, and rare synonymous (silent) SNVs were considered likely to be benign. Rare SNVs in intronic sequences flanking exons that did not affect conserved splice donor or acceptor sites or in 5′ untranslated regions were classified as not benign but of unknown significance. Rare SNVs causing missense substitutions or occurring in mRNA 3′ untranslated regions (and therefore possibly affecting mRNA stability or translation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Chen1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Conne1" target="_blank">[29]</a>) were considered possibly deleterious, and rare SNVs causing nonsense mutations or affecting conserved splice donor or acceptor sequences were considered probably deleterious. These latter two categories of SNVs were together considered potentially deleterious.