3.3. Primer Design and Sanger-based Sequencing
Primers were designed to flank the relevant exon of the SACS gene, including 50 bp of the flanking intronic regions. The UCSC genome browser was used to obtain the reference transcript of the SACS gene (NM_014363.4) and its protein product (NP_055178.3). This website provides a direct link to ExonPrimer for the design of primers flanking coding exons. The primers were checked for underlying SNPs using the online software tool available from the National Genetic Reference Laboratory, Manchester [9]. After passing in silico tests, the primers were tailed with M13 sequences and synthesised by Integrated DNA Technologies (details available upon request).
Sanger-based sequencing was performed to confirm the research-based WES results and the traces were analysed using Variant Reporter™ Software v1.0 (Thermo Fisher Scientific, Cleveland, OH, USA) as described previously [10]. GenBank NM_014363.4 was used as the reference sequence, with cDNA number +1 corresponding to the A of the translation initiation codon (codon 1). Each sequence trace had a minimum trace score of 35, which corresponds to an average false base call frequency of 0.031%.