Powerful Probabilistic Genotyping Tools

The eDNA Consortium provides Enhanced Semi Continuous (Bullet) and Continuous Methodologies (BulletProof Sentry) for all your Probabilistic Genotyping and Mixture Deconvolution needs.

Is one method better than the other?  No.  But one or the other may be more suitable for an organization.  The various methodologies are complementary-and facilitate a robust and sensible validation process.  As an eDNA Consortium Member you will have full access to both Bullet and BulletProof.

BulletProof-Sentry is the latest addition to the arsenal of Probabilistic Genotyping tools made available to the eDNA Consortium.

Request full access to BulletProof

Forensic Laboratories in the United States are slow in moving towards Probabilistic Genotyping: Why?

  • The “Commercial” software is too expensive…
    • Join the eDNA Consortium and take advantage of the many free calculation tools
  • The dread of validating a new calculation procedure
    • The eDNA Consortium has developed a comprehensive User Guide, training program, and companion Manual Verification Spreadsheet doing much of the validation work for you.
  • The fear of overly complex calculations—and the ability to adequately testify to the results
    • The eDNA Consortium is cognizant of the “Black Box Fear” when implementing software solutions—Therefore eDNA’s Probabilistic Genotyping (PG) tools include a comprehensive Step by Step calculation manual, numerous Published Papers, and multiple Spreadsheet Solutions demonstrating the PG algorithm in action—and full concordance.
    • Become comfortable using eDNA’s PG Tools with onsite PG training provided by the eDNA Steward.
    • Join the eDNA Probabilistic Genotyping User’s group for sharing information among colleagues.
  • The perception that calculations can take hours or days to perform
    • Unlike Commercial software solutions, BulletProof Sentry performs calculations in seconds.  The eDNA Probabilistic Genotyping tool has been designated BulletProof Sentry for its speed, accuracy, and ability to cut through the fog of uncertainty.
  • The perception that results are not reproducible
    • Unlike some MCMC software solutions, BulletProof Sentry produces identical results each time the same scenario is calculated. When using BulletProof Sentry the expert will never need to face the question, “Is it true your results are not reproducible?”

Why is Probabilistic Genotyping necessary.

The trend in the type of samples Forensic Laboratories are asked to resolve, along with improved extraction methods, amplification chemistries, and improved CE instrumentation have exceeded the ability to adequately resolve mixtures using binary mixture interpretation protocols. When laboratories try to analyze highly complex mixtures, such as “touch” items with more than two contributors and stochastic data, binary methods (CPE, CPI, Modified RMP) fail miserably. With traditional binary methods there exists no way to factor uncertainty.

Current strategies to evaluate low-level mixtures with dropout using the binary methods are insufficient. The existing CPE / CPI methods have no fundamental validity, there exists no unambiguous and rigorous mathematical proofs—it is fundamentally flawed and at best, a “stab” at the weight of the evidence.

What is Probabilistic Genotyping?

Years ago, the ISFG published recommendations for the interpretation of low-level mixtures when dropout is possible (Gill et al. 2012). eDNA’s Probabilistic Genotyping tools provides the ability to utilize all data above the Laboratory’s empirically determined analytical threshold by accounting for “uncertainty” wherein we incorporate a probability of allelic drop-out, drop-in (and if warranted, alternate hypotheses including the ability to account for possible “related suspects”) in the LR.

Quoting the 2016 SWGDAM Guidelines for Validation of Probabilistic Genotyping Systems:

“Probabilistic genotyping refers to the use of biological modeling, statistical theory, computer algorithms, and probability distributions to calculate likelihood ratios (LRs) and/or infer genotypes for the DNA typing results of forensic samples (“forensic DNA typing results”). Human interpretation and review is required for the interpretation of forensic DNA typing results in accordance with the FBI Director’s Quality Assurance Standards for Forensic DNA Testing Laboratories . Probabilistic genotyping is a tool to assist the DNA analyst in the interpretation of forensic DNA typing results. Probabilistic genotyping is not intended to replace the human evaluation of the forensic DNA typing results or the human review of the output prior to reporting.”

BulletProof Sentry

BulletProof Sentry provides a high level of automation. The Maximum Likelihood approach is based on the frequentist inference maximizing the likelihood function with respect to the unknown parameters to obtain the maximum likelihood estimate.

The software optimizes the Likelihood (under each hypothesis) as a function of the unknown parameters in the continuous model:

1)         Mix-Proportion: (mx1,…, mxC): mixture proportion for contributor 1,..,C.

2)        PH Expectation:  mean of a heterozygote peak height allele

3)        PH Variability: coefficient of variance for a heterozygote peak height allele

4)        Degradation: degradation slope

5)         Stutter Prop: (n-1) and (n + 1) stutter proportions

Lab Agnostic

This approach chooses the parameters which provide the best fit to the observed peak heights. Hence the uncertainty of the parameters are not required which makes this a great tool to implement in lab agnostic analysis. The Maximum Likelihood method does not require lab specific “training sets of data”.

This also makes intra-lab data simpler to manage when running multiple chemistries on multiple pieces of equipment. E.g. 3130 vs. 3500 & Fusion 6C vs. GlobalFiler

BulletProof additionally provides the MCMC Bayesian LR, and options to run the Numerical Integration Bayesian methods.

The BulletProof Run Wizard makes it simple to Setup and Queue a Probabilistic Genotyping calculation runs.

New Run Wizard 1

All new runs are populated with the organization’s default run parameters but can be changed based on the sample specifics.

In this example we will ask BulletProof to determine if two or three contributors best explains the evidence.

Multiple Users can simultaneously Queue runs, eliminating the expense for multiple software licenses.  BulletProof supports multiple simultaneous users thus removing PG as a laboratory workflow bottleneck.

During the six year development of BulletProof Sentry we incorporated improved workflows stemming from “lessons learned” and user feedback of other PG software tools.

In the time it required to compose the past few sentences BulletProof Sentry completed the example run (detailed above).  BulletProof correctly assigned the true number of contributors.

Run Results

Request full access to begin using these tools today.

By selecting the Results tab the user will explore a vast array of intuitive yet comprehensive PG run details.

Notice the weight of evidence is calculated using three methods; Maximum Likelihood, Bayesian Numerical, and Bayesian MCMC (Sensitivity is reported as the 5% quantile of Bayesian MCMC).  Each model is based on a different mathematical algorithm being used to calculate the parameters best explaining the evidence.

The Non-contributor analysis substitutes hundreds of known Non-contributors for the Person of Interest to measure the strength of the reported Likelihood ratios.

2500 Run Details

The user can further explore the results by selecting the Hp and Hd tabs.

The Hp tab below shows the mixture proportions of the two contributors with the Peak Height Expectation and Variability used in the gamma distribution.

Parameters under Hp

The Hp tab also includes graphs providing a visual cue as to how well BulletProof modeled the data.  The PP-Plot is a quick way to assess the expected vs. observed probabilities.

2050

The Fitted Model graph is another great tool to assess the models fit to the evidence.

 Another useful graph includes the MCMC Parameters for a good fitting model.

Mixture deconvolution is a great tool for no suspect crimes.  The BulletProof Sentry deconvolution functionality is frequently used in no suspect Sexual Assaults and property crime cases.

Bullet & BulletProof eDNA’s Probabilistic Genotyping Tools

BulletProof, eDNA’s Continuous Probabilistic Genotyping tool provides a Frequentist and Bayesian methodology to resolve complex mixtures and provide mixture deconvolution.  Click below to schedule a free demo and access to the fully operation application.

Take the First Step

Contact the eDNA Steward to arrange for an online primer demo and for login access to the fully operational demo site featuring eDNA 3.2 with eSolve Indexing System, Brutus, and Bullet.

There will be no sales pitches—just a pure tour of the high-level functionality of the eDNA LIMS…then the user will be turned loose to explore the deeper domain functionality at their pleasure.

The demo login credentials provided during the tour remain valid indefinitely so come back whenever and as often as you wish.