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.