Powerful Probabilistic Genotyping Tools

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 MCMC software solutions, Bullet performs calculations in seconds.  The eDNA Probabilistic Genotyping tool has been designated Bullet for its speed, accuracy, and ability to cut through the fog of uncertainty.
  • The perception that results are not reproducible
    • Unlike MCMC software solutions, Bullet produces identical results each time the same scenario is calculated. When using Bullet 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?

Recently, 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.”

In the example below the system would be unresolvable using CPI/CPE or other binary LR methods. However Bullet incorporates uncertainty and allows the analyst to use all the data and weigh the evidence accordingly.

Drop-out (and Masking) example on a Two Person Mixture

Bullet allows a User to optionally investigate the Evidence Profile “system by system” to further visualize or investigate. Bullet incorporates each allele’s RFU values with associated Base Pair Size in an explicit visual manner allowing the analyst to calculate/verify “on the fly”  the system’s probability of drop-out. 

Optional Contributor Isolation Workspace

Ultimately, by using all data and automated assignment of an appropriate level of uncertainty (based on Signal Strength / Base Pair sizes) in conjunction with a Degradation Curve, all data from the Crime Scene Profile can be used. The resulting Likelihood Ratio is calculated on competing hypotheses used to explain the presence or coincidental match of a suspected contributor’s profile within a Crime Scene profile.

Example of a two-person mixture conditioned on the stochastic minor contributor

Bullet is further enhanced to easily accommodate mixed samples exhibiting differential degradation. 

Bullet will derive and display the Degradation Curve allowing the analyst to gain valuable insight into the Crime Scene Profile.  The optional curve can then be used to predict the allele’s anticipated Signal Strength based on Amplicon length, further normalizing and allowing the analyst to condition the level of uncertainty in systems where the contributor’s alleles are masked or may have dropped out altogether.    

Degradation Curve Visualization

 

Bullet – eDNA’s Probabilistic Genotyping Tool

The eDNA Probabilistic Genotyping tool has been designated Bullet for its speed, accuracy, and ability to cut through the fog of uncertainty.

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.

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