Genophiler® and GenoStat®:Tools for objective evaluation of

DNA testing results

Forensic Bioinformatics (www.bioforensics.com)

Jason R. GilderAugust 17, 2007

• 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

Summary Sheet

The * indicates that this peak

may be involved in pullup…

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)

Limit of Quantitation

(LOQ)

Average baseline signal

Baseline signal

std. dev.

Run 2_17_06C7P16 9947a

6K11_3ul 05-2Pos Control

(2-17)-2 19 49 5.86 4.34

Run 2_17_06C7P16 9947a

6K11_3ulPos Control

(2-17)-1 19 49 5.72 4.34

Run 2_17_06 G7C16 9947a

6K11_3ulPos Control (2-17)-C 18 48 5.82 4.18

Run 2_17_06 D7P16 RBReagent

Blank (2-17)

18 48 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

Standard stats mode

Random match

probability

Standard stats modeUnrelated

Sibling

Half-Sibling

Parent/Child

Uncle/Nephew

Cousins

Standard stats modeMixture

stats

Standard stats mode

Theta substructure

factor

Standard stats mode

FBI 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

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

Questions?

www.bioforensics.com