Forensic Bioinformatics (www.bioforensics.com) Genophiler® and GenoStat®: Tools for objective evaluation of DNA testing results Jason R. Gilder August 17, 2007 Forensic Bioinformatics (www.bioforensics.com)

Automation of GeneScan and Genotyper Consistent electronic analysis of all samples Organizes all output – can be read on any computer Summary table of all labeled peaks Draws your attention to potential issues

The * indicates that this peak may be involved in pullup… Summary Sheet The * indicates that this peak may be involved in pullup…

Clicking here will show us the GenoTyper annotated electropherogram… Pullup Clicking here will show us the GenoTyper annotated electropherogram…

Simplified table Overall view of samples Single line display Clicking sample displays electropherogram

Project Screen Shot

Analysis Parameters: Size Standards:

Standard electropherogram Similar to laboratory output Limited view Hard to evaluate low-level results

Genotyper zoom Close view of baseline and potential low-level alleles View from 0 to 150 RFU Helps expert make an informed decision

Another example of Genophiler zoom Genophiler also provides a zoomed graph to 150 RFUs Shows smaller peaks more clearly Here, the peaks that were not reported by the lab may be evidence of an unknown, minor contributor

Sample results window All detected peaks Size Peak height Peak area Data collection point

Sample information window Time and date of analysis Analysis instrument Machine conditions Analysis parameters

Raw data window Pre-separated spectral data

Poor raw data

Other Issues: EPT Data Shows current, voltage, laser power, and temperature For good analysis, should be constant (flat)

Problematic EPT data

Side-by-side interface

Consistency report We perform two analyses for every case GeneScan default RFU threshold (50) Reproduce the testing lab’s analysis Compares analysis results between testing laboratory and ABI defaults Highlight peaks that are labeled in one analysis and not the other

Genophiler® LOD/LOQ Table Sample Run Sample Name Sample Nickname Limit of Detection (LOD) Quantitation (LOQ) Average baseline signal Baseline std. dev. Run 2_17_06 C7P16 9947a 6K11_3ul 05-2 Pos Control (2-17)-2 19 49 5.86 4.34 6K11_3ul (2-17)-1 5.72 G7C16 9947a (2-17)-C 18 48 5.82 4.18 D7P16 RB Reagent Blank (2-17) 5.75 4.20    Notes:    - LOD/LOQ derived from the methodology validated in J. Gilder, T. Doom, K. Inman, and D. Krane. "Run-specific limits of detection and quantitation for STR-based DNA testing." Journal of Forensic Sciences. 2007;52(1):97-101.    - Limit of Detection (LOD) is the average baseline signal (in RFUs) plus three standard deviations (μb + 3σ).    - Limit of Quantitation (LOQ) is the average baseline signal (in RFUs) plus ten standard deviations (μb + 10σ).    - Positive controls not exhibiting the full 9947A profile are not included in the above table.

Removing labels in GenoTyper

Default view (as found in printout) Genotyper: Show peaks that were manually removed

Genophiler CD Full electronic record of the case All results in electronic format on CD-ROM All data files, analysis parameters, projects, macros, etc. Works on any computer with a web browser Can make printouts of everything

Potential issues that are flagged

Potential issues that are flagged Mixtures Peak height imbalance Missing genotype information High peaks Low average peak height Indications of degradation Pull-up Inconsistent PP/CO results

Mixture Locus or loci containing three or more labeled peaks

Peak height imbalance Any heterozygous loci differing by more than 30% 65% Defendant

Missing genotype information At least one locus with no labeled peaks

High peak Any peak above the point of saturation (4000 RFUs) Can lead to pull-up Defendant

Low average peak height An average peak height less than 400 RFUs

Degradation Potential breakdown of DNA sample Slope calculated through best-fit linear regression Slope = -17 Defendant

Pull-up Two peaks in two different dyes occurring at approximately the same data collection point Evidence Q5

Inconsistent PP/CO results Different results in Profiler Plus and COfiler Defendant

Potential issues that are flagged Mixtures Peak height imbalance Missing genotype information High peaks Low average peak height Indications of degradation Pull-up Inconsistent PP/CO results

Genophiler report “An expert will be able to explain the significance and implications of these findings in your particular case.” “All of the statements listed below about the data in your case can be verified by any competent expert who has access to GeneScan and Genotyper software and to the data you provided to us.”

Genophiler does not Perform mixture interpretation Make allele calls Assess the quality of a profile Make any judgment calls

GenoStat DNA statistics package Random match probabilities Related random match probabilities Mixture statistics (CPI) 167 population allele frequency databases Mixture resolution Separate mixtures into contributor components Free

Standard stats mode

Random match probability Standard stats mode Random match probability

Standard stats mode Unrelated Sibling Half-Sibling Parent/Child Uncle/Nephew Cousins Standard stats mode

Standard stats mode Mixture stats

Theta substructure factor Standard stats mode Theta substructure factor

FBI population databases Standard stats mode FBI population databases

Load any of 167 population databases Standard stats mode Load any of 167 population databases

Mixture resolution mode

Mixture resolution mode One hypothesis = fully resolved

Mixture resolution mode Set peak height ratio

Mixture resolution mode Minimum peak height threshold

Mixture resolution mode Inferred mixture ratio

Partially-resolved mixture

Partially-resolved mixture Manually exclude mixture hypotheses

Partially-resolved mixture Manually exclude loci from statistics

Partially-resolved mixture Constrained CPI uses only plausible mixture hypotheses

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