Laboratory Medicine and Quality Assurance

Table. 1.

Expected and measured VAFs of 21 variants in different dilution ratios using amplicon-based next-generation sequencing

Variants Dilution ratio (%) Equation R2
Undiluted 1:1 1:2 1:4 1:8
ABL1 T315I y=1.3059x–0.3469 0.9445
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 6.61 2.20 0.70 0.80 0.61
Discrepancy* 32.27 –12.05 –44.24 28.31 93.66
ASXL1 W796C y=1.0839x–0.0400 0.9948
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 5.45 2.58 1.11 0.82 0.34
Discrepancy 9.00 3.20 –11.20 31.20 8.80
CBL S403F y=0.7530x+0.2850 0.9919
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 4.02 2.30 1.11 0.63 0.66
Discrepancy –19.60 –8.00 –11.20 0.80 111.20
DNMT3A R882C y=0.4017x–0.3717 0.8243
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 2.42 1.05 1.43 0.64 0.21
Discrepancy –51.60 –58.00 14.40 2.40 –32.80
EZH2 R418Q y=1.0351x+0.1804 0.9545
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 5.64 2.02 1.77 0.92 0.58
Discrepancy 12.80 –19.20 41.60 47.20 85.60
FLT3 D835Y y=0.9507x–0.0779 0.9873
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 4.77 2.22 0.80 0.58 0.45
Discrepancy –4.60 –11.20 –36.00 –7.20 44.00
IDH1 R132C y=0.6163x+0.6779 0.8493
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 3.52 2.44 2.18 0.91 0.31
Discrepancy –29.60 –2.40 74.40 45.60 –0.80
IDH2 R172K y=1.2016x+0.3079 0.9230
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 5.89 4.45 1.31 0.98 0.55
Discrepancy 17.80 78.00 4.80 56.80 76.00
JAK2 V617F y=0.9561x–0.0504 0.9833
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 4.58 2.57 1.43 0.33 0.10
Discrepancy –8.40 2.80 14.40 –47.20 –68.00
JAK2 F537-K539 y=0.7071x–0.4121 0.8533
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 3.53 0.37 0.68 0.21 0.00
Discrepancy –29.40 –85.20 –45.60 –66.40 –100.00
NPM1 W288fs*12 y=0.4414x+0.4988 0.6824
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 2.34 2.23 1.63 0.13 0.44
Discrepancy –53.20 –10.80 30.40 –79.20 40.80
SF3B1 G740E y=0.8877x+0.0521 0.9967
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 4.48 2.36 1.01 0.60 0.41
Discrepancy –10.40 –5.60 –19.20 –4.00 31.20
TET2 R1261H y=1.2071x–0.2808 0.8669
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 6.42 1.16 1.53 0.63 0.55
Discrepancy 28.40 –53.60 22.40 0.80 76.00
TP53 S241F y=1.000x+0.4704 0.9603
Expected VAF 5.00 2.50 1.25 0.63 0.31
Measured VAF 5.38 2.92 2.39 0.80 0.55
Discrepancy 7.60 16.80 91.20 28.00 76.00
NRAS Q61L y=0.8621x+0.8854 0.9116
Expected VAF 10.00 5.00 2.50 1.25 0.63
Measured VAF 9.82 4.15 3.47 3.29 0.40
Discrepancy –1.80 –17.00 38.80 163.20 –36.00
GATA2 G200fs*18 y=1.0731x+5.0021 0.9729
Expected VAF 35.00 17.50 8.75 4.38 2.19
Measured VAF 40.74 27.78 14.71 8.06 6.49
Discrepancy 16.40 58.74 68.11 84.23 196.69
RUNX1 M267I y=1.043x–1.0113 0.9801
Expected VAF 35.00 17.50 8.75 4.38 2.19
Measured VAF 34.31 20.56 6.60 2.60 1.60
Discrepancy –1.97 17.49 –24.57 –40.57 –26.86
ASXL1 G646fs*12 y=0.4391x+0.8642 0.9846
Expected VAF 40.00 20.00 10.00 5.00 2.50
Measured VAF 18.65 9.09 5.82 1.92 2.87
Discrepancy –53.38 –54.55 –41.80 –61.60 14.80
KRAS G13D y=0.9612x+0.9617 0.9944
Expected VAF 40.00 20.00 10.00 5.00 2.50
Measured VAF 38.57 21.75 11.20 5.52 2.26
Discrepancy –3.58 8.75 12.00 10.40 –9.60
BCOR Q1174fs*8 y=0.9654x–0.6417 0.9942
Expected VAF 70.00 35.00 17.50 8.75 4.38
Measured VAF 68.06 30.21 17.88 6.69 4.89
Discrepancy –2.77 –13.69 2.17 –23.54 11.77

Abbreviation: VAF, variant allele frequency

*Discrepancy: (measured VAF–expected VAF)/expected VAF×100
Lab Med Qual Assur 2022;44:76~81 https://doi.org/10.15263/jlmqa.2022.44.2.76
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