Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and Next-Generation Sequencing

Table 2

Validation of several variants and comparison of NGS and HRM determined variant allele frequencies.

Pool	No. of samples^a	117^b in intron 9		156 in intron 9		174 in intron 9		5153 in exon 13		6943 in exon 15
		NGS^c	HRM^c	NGS	HRM	NGS	HRM	NGS	HRM	NGS	HRM
Ethnic	23	6.2%	6.5%	0.01%	0.00%	6.7%	8.7%	62%	63%	12%	13%
P1	27	5.6%	5.6%	0.01%	0.00%	0.0%	0.0%	74%	74%	13%	19%
P2	29	8.1%	8.6%	1.12%^d	1.70%	0.0%	0.0%	69%	72%	14%	16%

^aA total of 79 samples were individually tested for variants by HRM analysis for three regions of the RET gene (which analyzed the 5 NGS-detected variant positions shown in this table).
^bRET amplicon position shown, see Table 1 for more information on each variant change.
^cNGS: illumina genome analyzer determined allele frequency (variant read percentage) from a pooled sample set. HRM: high-resolution melting analysis determined allele frequency, where each individual in the pool was tested separately for variation.
^dLowest NGS variant read percentage for all pools. This suspected variant was verified as a singleton allele within Caucasian pool P2 by HRM and Sanger sequencing.