Background noise in STR testing

1. Background noise in STR testing Travis Doom Dept. of Computer Science/Eng. Wright State University, Dayton, OH Forensic Bioinformatics (www.bioforensics.com)

2. Motivation • How is an electropherogram peak classified as a “true allele peak”, “technical artifact”, or “noise”? • What are the sources of variation in lab RFU thresholds? • How might we determine these thresholds objectively?

3. What is “Signal?” • Analytical methods rely on a measured signal for the analysis. • This signal is often a change in electronic voltage from an instrument. • The instrument measurement output (total/output/measurement signal) is the sum of the analyte signal and the nominal baseline/background signal. • St = Sx +Sb • The net signal indicates the concentration of the analyte. • Sx = St – Sb (the net signal) • Sx = gCx

4. What is “Noise”? • For real instruments and samples, there are always some errors in determining baseline signal: • Drift: slow change in the baseline level over time • Noise: unwanted random fluctuations in the nominal baseline signal • Baseline = nominal +/- noise (~Sb) • St = Sx + ~Sb • If the baseline signal was exactly constant, any amount of analyte, no matter how small (within the resolution of the instrument) would be detectible. • Perfect correction for baseline could be made without error. • Perfectly precise results could always be obtained. • Noiseless experiments involving chemical concentrations and light detection instruments are not possible.

5. How is analyte signal distinguished from noise? • Sx = St – ~Sb • Precision and accuracy of experimental results depend on: • The magnitude of the analyte signal • The magnitude of the noise • The limit to measuring/detection the presence of an analyte signal is not the size of the signal, nor the size of the noise alone, but the relative magnitude of the two. • Signal-to-Noise Ratio (S/N) • Noise can be characterized if reproducible • Assumption #1: Noise magnitude is independent of analyte signal magnitude • Assumption #2: Noise variance is Gaussian in nature • Thus, baseline signal is characterized by a mean μb and standard deviation σb • Role of the reagent blank

6. Limit of Detection • Limit of detection: the lowest concentration of analyte [or analyte signal] that the analytical process can reliably detect (American Chemical Society). • The detection limit is based on a statistical calculation • Detection limit is closely related to the noise characterization of a series of measurements, including blanks under the same experimental conditions • Statistical certainty is expressed as a confidence limit for the noise error, generally 99.7% (3-SIGMA) • Sx = St - μb • Precisely correct only when Sb is μb. • As Sb varies due to noise, error varies into the reported net signal Sx. • Analyte “reliably detected” if: St – Sb >= 3 σb • Relative Magnitude (S/N): Sx >= 3 σb

7. Thresholds for Detection/Quantization • How much output signal do we need to be reliably certain that we observe an analyte? • Detection threshold: μb + 3σb • Analyte signal at limit of detection = St - threshold • How much output signal do we need to be reliably certain that we can quantize the amount of analyte observed? • It is more difficult to determine the concentration of an analyte than merely to detect its presence or absence. • This is generally accepted as a 10-SIGMA decision • Quantization threshold: μb + 10σb

8. Signal Measure Saturation Quantization limit μb + 10σb Measured signal (In Volts/RFUS/etc) μb + 3σb Detection limit Mean background Signal μb 0

9. Objective threshold determination • The limit of detection is an extrapolated value. • While easy to use, carte blanche thresholds make assumptions that may not be valid for a particular experiment/run. • FBS study (currently unpublished) • Study characterizes noise signal in 42 runs taken from 7 cases analyzed by the FBI. • Each run contains a regent blank, a positive control, and a negative control. • Output signal data was collected only from regions of the electropherogram free of analyte signal (positive control peaks, ROX peaks, +/-4 stutter) in all channels. • In-line regent blanks/controls

10. Study Results

11. Positive Control (Average) Saturation 47 Quantization limit Measured signal (RFUs) 19 Detection limit Mean background Signal 4 0

12. Negative Control (Maximum) Saturation 262 Quantization limit Measured signal (RFUs) 90 Detection limit Mean background Signal 25 0

13. Contributions • Rubinson and Rubinson. Contemporary Instrumental Analysis. Prentice Hall, 2000. • Formal definitions in Section 5.5: Signals, Noise, and Detection limits • National Center for Biotechnology Information (NCBI) • BatchExtract • Forensic Bioinformatics • Jason Gilder