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Massive update: We are ready for NGS analysis!



We have had some very, very busy few weeks working through validation and implementation of the analysis components of our Next Generation Sequencing casework operation. It's been a lot of blood, sweat and tears to get it all done, but as of this week (the sign off of the analysis standard operating procedures and final competencies).. we are complete and ready to begin "normal" operations!


I'd like to share some milestones since our last update...

Chemistry validation of NGS was completed and implemented on November 18, 2020.

-This allowed us to start running samples that were "on hold" for NGS processing through the MiSeq FGX instrument. It is a longer and more difficult process than traditional DNA processing and we needed to be absolutely confident in the chemistry and our ability to utilize it.


It was at that point that we realized we needed a better system to concentrate samples. The Forenseq NGS Identity kit only requires 5 microliters of DNA extract volume and our process had 40ul. Thus, we had to get our samples concentrated from 40 to 10 (we never consume our extracts) in a manner that preserved the DNA. This was a more difficult prospect than we had hoped. We found that drying down the sample without some stabilizing agent did not work well as we lost DNA in the process. Quality is the most important thing to us, so we needed to find a better way.


We evaluated a product: GenTegra DNA stabilizing tubes

https://gentegra.com/gentegra-dna-2/


These tubes have a matrix imbedded on the plastic that protects and preserves DNA through the dry down process. Our evaulation was a success and GenTegra tubes worked as expected, allowing us to do a formal validation of the product. We would also like to thank James Nelson and the Gentegra team for partnering with us on this endeavor. We believe this will be an essential product in the Forensic DNA community and we are excited to use it in our casework.


Concentration: GenTegra Dry Down validated and implemented on November 30, 2020

-This allows us to 1) dry down our extracts to a lower volume for NGS processing and 2) send our extracts back to our clients "dry" and stable. This will allow for inexpensive shipping and easier storage for clients.


The next step in this process is to analyze and interpret the NGS samples as the results are obtained from the MiSeq FGX instrument. That initial interpretation includes evaluating the samples for quality and/or rework, assessing controls and initial interpretation of single source and mixture profiles using the MiSeq FGX Universal Analysis Software (UAS).


Verogen Forenseq Signature/MiSeq FGx STR Interpretation validated and implemented on December 17, 2020

-Interpretation of single source samples and evaluation of quality and rework. Analytical, Stochastic thresholds established. Read Count Ratios and Major/Minor contributor percentages identified and integrated into interpretation procedures.


A HUGE thank you to the Verogen team for being our essential partners in this validation and implementation. They worked hand in hand with us to help us through the learning curve for forensic NGS. Meredith Turnbough, Warren Robinson, Katherine Johnson, Melissa Kotkin, Anna McMullin and all the others that helped us at Verogen. Thank you so much for your support and partnership. We appreciate you all more than we can express.


Lastly, because NGS samples generate so much more information than traditional Forensic DNA samples, it becomes essential to use software to help with interpretation and statistical analysis. NGS allows analysts to see the actual sequence of a forensic STR, allowing for much greater ability to interpret mixed DNA samples. We will provide more information regarding the power of IsoAlleles in NGS interpretation at a future date, but, for now it's important to know that there are differences in the DNA that the traditional Forensic DNA intepretation cannot identify.


We turned to the pioneers of NGS interpretation, NicheVision. They had the foresight and vision to see this trend in forensics coming and were adapting their software (ArmedXpert) to utilize the DNA sequence information obtained in NGS samples. They have created a plug-in to their ArmedXpert software called MixtureAce. This allows us to utilize the power of DNA Sequencing that is obtained in NGS profiles.


https://nichevision.com/mixtureace/


Partnered with NicheVision we learned, trained, validated and implemented the ArmedXpert MixtureAce software. It took some time to overcome the learning curve and adapt to new ways of thinking about our data, but, we feel confident this will give us more interpretable profiles and much better statistics on our partial profiles and complex mixtures. It turns out you can teach and old dog DNA analyst new tricks!


ArmedXpert with MixtureAce Validated on January 7th, 2021, implementation on January 13, 2021.

-This allows us to interpret NGS data including isoalleles and their corresponding allele frequencies in our analysis. Modified Random Match Probability (restricted, unrestricted, obligate alleles, allele/any) statistical analyses implemented for up to three person mixtures.


We look forward to working with our friends and partners at NicheVision to implement more features specific to NGS samples (PACE software based contributor number interpretation, probabilistic genotyping for NGS samples etc.). The future is really exciting to think about when utilizing the wealth of information derived from NGS profiles.


Thank you so much to the NicheVision team, Luigi Armogida, Brian Young, Vic Meles, Tracy Bauer and the rest of the NicheVision team that helped us. You are all amazing and were such a help to us in getting to this point. We look forward to our continued partnership in the future as you guys blaze the path forward for NGS forensic interpretation.


We've had a really hard couple of weeks pushing hard on completing our validations and implementing this essential science. Thank you to the IMF team as well, Derek Cutler, Alyssa McElreath and Elena Rojas for all the hard work to get to this point. It took a lot longer to get this accomplished than we had planned or hoped, but we are confident in our quality and effectiveness now that we've surpassed some of our biggest milestones. I could not be more proud to be a part of this IMF family.


Let's do this! Let's solve some cases! Let's change the world!!


-Danny


Photo by Ian Stauffer on Unsplash



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