Sanger Sequencing
Confirmation of NGS findings by Sanger sequencing was first suggested in guidelines as a result of low coverage and high false positive error rate of the early NGS instruments (Consortium et al. 2010; Fox et al. 2014; Harismendy et al. 2009; Quail et al. 2008). With the advancement of NGS technology, particularly targeted panel sequencing with higher depth of coverage, the sensitivity and specificity of NGS variant calling has improved. Therefore, validation of variants detected by targeted-capture panels that usually have sufficient coverage, could be waived, while it can be helpful in reducing false positives for variants with depressed allele fraction (<30% of alternate alleles) or located in poorly covered regions. Also, performing manual inspection of the NGS data using IGV obviates the need to carry out further validation studies. However, we employed a hybrid approach utilising Sanger sequencing and IGV review for a subset of variants on a case-by-case basis.