Development and Analytical Validation of a Targeted Next-Generation Sequencing Panel to Detect Actionable Mutations for Targeted Therapy

Figures & data

Table 1 Five HapMap Normal Samples and Their Dilution Ratios in Five Diluted Samples for SNV Validation

	SNVM1	SNVM2	SNVM3	SNVM4	SNVM5
HG00119	30%	35%	5%	10%	20%
HG00556	5%	10%	20%	30%	35%
HG01028	10%	20%	30%	35%	5%
NA18548	20%	30%	35%	5%	10%
NA19058	35%	5%	10%	20%	30%

Table 2 Performance of SNV Calling at AF ≥5% for Five Diluted Samples at Different Sequencing Depths

Mean Depth	Total TP	Total FN	Total FP	Sensitivity	Specificity
500X	318	2	0	99.38%	100%
700X	320	0	0	100%	100%
900X	320	0	0	100%	100%
1000X	320	0	0	100%	100%
Total	1278	2	0	99.84%	100%

Figure 1 Detected AF vs expected AF for five SNV diluted samples (A–E) and the reference standard sample HD701 (F).

Table 3 Performance of INDEL Calling at AF ≥2% for Five Diluted Samples at Different Sequencing Depths

Mean Depth	Total TP	Total FN	Total FP	Sensitivity	Specificity
500X	64	1	0	98.46%	100%
700X	64	1	0	98.46%	100%
900X	64	1	0	98.46%	100%
1000X	64	1	0	98.46%	100%
Total	256	4	0	98.46%	100%

Figure 2 Detected AF vs expected AF for five INDEL diluted samples. Please note that the INDELs in these five diluted samples are plotted together.

Table 4 The Validation Results of Reference Standard Sample HD701 at the Sequencing Depth of 500X for Confirmed Hotspot SNVs and INDELs

Gene	Variant	Variant Type	Confirmed AF	Detected AF
ALK	P1543S	SNV	33%	28.33%
APC	R2714C	SNV	33%	31.22%
BRAF	V600E	SNV	10.50%	8.88%
BRCA2	A1689fs	INDEL	33%	32.60%
EGFR	E746_A750del	INDEL	2.00%	2.12%
EGFR	L858R	SNV	3.00%	1.75%
EGFR	T790M	SNV	1.00%	0.64%
EGFR	G719S	SNV	24.50%	21.42%
FBXW7	G667fs	INDEL	33.50%	34.08%
FGFR1	P150L	SNV	8.50%	8.51%
FLT3	V197A	SNV	11.50%	7.80%
IDH1	S261L	SNV	10%	12.65%
KRAS	G13D	SNV	15.00%	13.93%
KRAS	G12D	SNV	6.00%	8.54%
MET	V237fs	INDEL	6.50%	5.49%
MLH1	L323M	SNV	8.50%	6.77%
NF1	L626fs	INDEL	7.50%	7.42%
NOTCH1	P668S	SNV	31.50%	26.15%
NRAS	Q61K	SNV	12.50%	10.66%
PDGFRA	G426D	SNV	33.50%	29.83%

Figure 3 The normalized mean depths for all the targeted regions for different validation samples.