Table 3.
AF Performance
| AF methods | N | MAE, % | Average error, % | Range errors, % | Linear regression | R2 |
|---|---|---|---|---|---|---|
| No duplicated primer sites | ||||||
| AMP base versus CE | 49 | 1.95 | 0.95 | −7.0 to 9.0 | 1.02x + 0.005 | 0.980 |
| AMP raw versus CE | 49 | 2.19 | 1.09 | −7.2 to 9.9 | 1.04x + 0.004 | 0.978 |
| AMP deduplication versus CE | 49 | 8.99 | 8.30 | −16.3 to 23.2 | 0.94x + 0.093 | 0.859 |
| AMP standard versus CE | 34 | 13.99 | −13.61 | −76.6 to 2.8 | 0.20x + 0.025 | 0.296 |
| ITD size < 40 bp | 21 | 0.51x + 0.007 | 0.926 | |||
| ITD size 40–60 bp | 13 | 0.09x + 0.009 | 0.400 | |||
| TSCA versus CE | 74 | 2.53 | 0.83 | −13.7 to 11.1 | 1.04x − 0.001 | 0.983 |
| TSCA versus HC | 5 | 3.26 | −0.05 | −6.3% to 7.1 | 1.05x − 0.010 | 0.889 |
| ITDs with duplicated primer sites | ||||||
| AMP base versus CE | 15 | 9.73 | −9.70 | −24.9 to 0.3 | 0.59x − 0.005 | 0.843 |
| AMP adjusted versus CE | 15 | 6.21 | 1.48 | −10.3 to 16.3 | 0.99x + 0.017 | 0.801 |
AF estimates were compared under various methods within clinical next-generation sequencing panels (AMP and TSCA) versus CE or HC. Only ITDs detected by both AMP/TSCA and CE/HC were included. Not utilizing unique molecular identifiers (AMP raw) had relatively minimal effect for ITDs without duplicated primer sites. A simple AF adjustment for ITDs with duplicated primer sites improved the linear regression slope from considerably <1 (AMP base), indicative of underestimation, to near 1 (AMP adjusted); however R2 remained moderate.
AF, allelic fraction; AMP, anchored multiplex PCR; CE, capillary electrophoresis; HC, hybrid-capture; ITD, internal tandem duplication; MAE, mean absolute error; TSCA, TruSeq Custom Amplicon.