Crime fighting in the 21st century necessitates improved investigation methods, and increased partnership between crime labs and investigation units. Rapid DNA technology emerged over a decade ago, but continues to evolve to meet the changing needs of the criminal justice community. This webinar will provide viewers with insight into recent improvements to the Applied Biosystems™ RapidHIT™ ID platform for crime scene evidence sample processing.
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In the criminal justice community, there's only one mission, the relentless
0:08
pursuit of
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the truth.
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All it takes is one case that keeps you up at night.
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One persistent investigator who never gives up.
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One dangerous criminal still on the street.
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One precious sample that changes everything, one innocent suspect who's
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counting on justice.
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One tenacious analyst who won't stop without an answer.
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One brave victim who's not afraid to speak their truth.
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All it takes is one.
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One match.
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One community.
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One world made safer.
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And there's one trusted partner who's been there from the beginning.
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For over 30 years, Thermo Fisher Scientific has provided forensic laboratories
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all over
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the world with gold standard forensic tools so you can protect your communities
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Doing justice to those who seek it, identify the missing and help prevent
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tomorrow's victims.
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We never stop because you never stop.
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Individually we're strong.
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But together, together we're unstoppable.
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We find the truth.
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Hi, I'm Peter John McEnany and one of my roles as a technical application
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specialist
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is to bring your voice to our new product developments.
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Today, that brings me here to Pleasanton, California and the site of our Human
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Identification
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Headquarters where our development teams are working tirelessly to bring you
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the next
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generation solutions for rapid DNA.
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Come with me and let's take a peek behind the curtain.
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Hey Rob, nice to see you again.
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It feels like it was just yesterday that we were working on a 3500 software
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improvement
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and GMAP or IDX solution to help DNA analysts streamline processing time.
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And here we are again working to drive system improvements addressing the
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growing crime
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rates and resource limitations.
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Hi Peter John, nice to see you too.
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I know in the past few years, crime has increased significantly in my
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neighborhood.
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I live on a street 10 hours long and I'm seeing multiple incidences of crime
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every month.
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I don't think police departments are equipped to handle this increase or
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frequency in the
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crimes I'm seeing.
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Yeah, forensics labs seem to be overburdened with more samples and to make
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matters worse,
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we keep hearing of staff shortages and this combined with an expanding set of
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challenges
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including natural disasters and other situations that demand focus and
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resources.
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It is more important than ever that we deliver tools that help with efficiency.
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You know, I joined the applied biosystem in 2005.
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We were just starting work on our third, what I call a new generation kit, mini
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filer.
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Profiling co-filer have emerged to become identifiler.
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White filer would just come out.
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I was running a single cap 310 in the 3130 at the time, not even an XL for gen
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otyping.
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I then helped work on the development of the 3500 series instruments and the
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introduction
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of the six-dye system.
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For us, this was a significant improvement of the five-dye system and the 3130
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instrument.
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It allowed us to move from the 16 markers and identifiler to the 24 markers and
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global
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filer as we worked to improve workflow and make a better customer experience.
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And this change was required to meet the expanded codist marker requirements.
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Now, I hear some reasons why even more markers or they want a specific set of
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markers for
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centralized high volume workflows.
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And then again, I'm hearing people want decentralized workflows and small sets
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and screening of samples.
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Well, rapid DNA enables a ladder.
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With rapid DNA, labs can quickly triage their samples.
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They can generate investigative leads early on in search for local databases.
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And that enables them to take action while a suspect is still in custody.
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So I hear that rapid assays are having an impact in the world.
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And you know, it's really great for the R&D folks who work really hard to build
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these
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assays and develop them to hear feedback.
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So I'd love to hear from you some feedback on the rapid assays and their impact
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And the program showing the biggest impact are those developed in partnership.
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The true power of rapid DNA is its ability to bring together key members of the
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criminal
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justice and human identification communities to obtain answers faster and help
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prevent
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or solve crimes.
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So Rob, we call our next big rapid DNA solution, our enhanced investigative
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lead solution.
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But what does that entail?
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The new solution is really a completely redesigned workflow.
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We have updated the three critical components of this system.
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Biggest one is we have a new investigative leads crime scene cartridge.
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We have new software on the instrument and we have a new rapid link software.
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Now all of this is compatible with the current rapid head idea instrument.
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We're not going to change anything on the hardware of the instrument.
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And what did the project team set out to accomplish with the development of our
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enhanced investigative
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lead solution?
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A goal we started the project was to design a new assay that would improve on
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the current
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intel cartridge.
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Basically we wanted more green check marks than the intel system.
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Now so we started the program.
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But soon after a paper was published in SSFIG, but a task group made up of the
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FBI, Srigdam
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and ENFSI with a set of recommendations for rapid casework systems and how to
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upload their
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results into codis.
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So we pivoted and we actually enhanced our system.
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So to meet these new requirements we added indicators for PCR inhibition,
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sample degradation,
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sample load and the possibility of an allele dropout there potential.
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These additions were added to the original goal of improving the p-kite balance
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for low
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input samples.
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So Peter John, what do you think customers are going to think of all these
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changes we're
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making?
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I think those are some major changes and major improvements.
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And I think customers always appreciate improved performance, especially when
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it comes to balance
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or sensitivity and the potential for future access to codis will be a game
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changer.
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I think it is all very exciting.
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And you mentioned adding indicators of DNA quantity, DNA degradation or PCR
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inhibition
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I should say.
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Can you elaborate on how we are achieving this and how those features were
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developed?
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To evaluate PCR inhibition we was the same IQC chemistry that was developed for
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the global
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filer IQC kit.
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We just incorporated the same system into the Intel Plus assay.
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And since the system already functioned well within the global filer's T.R. set
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of targets,
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our work was then to optimize the IQC reaction to work well within a rapid
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cartridge.
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And adding the new human non-STR target systems, well that was way more
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challenging.
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We explored two systems.
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We tried a multi-copy system and a single target copy system.
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We quickly found the single target system was way more accurate in evaluating
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DNA load
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and sample degradation.
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So the challenges in adding these new markers to an existing assay, they were
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manyfold.
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You have to have no cross reactivity with any existing markers.
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You have to have consistent target amplification to do the job they are
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supposed to do with
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quantification.
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And they have to be human specific.
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So the target of primer locations, you can't have any SNPs or polymorphisms in
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the targets
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of the primers.
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And the target amplicon itself has to be free of indels or any SNPs are going
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to change
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the mobility of the amplicon.
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So I think we went through over 20 target candidates that failed just to repeat
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morphology in the
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design.
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The process for getting into the assay is quite rigorous.
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In the end, we added two targets to the system for quantification degradation.
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One at the front of the assay, around 65 nucleotides.
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We found this is an ideal size for estimating the amount of sample loaded into
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an assay.
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And we have one near the end of the STR read regions at 408 nucleotides.
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So here we are, we span the assay with targets that are immune to any PCR type
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of amplification
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issues.
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No allele size changes.
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They're one size.
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So this allows us to use the same slope concept we use with the IQC peaks for
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inhibition to
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predict how degraded the sample is.
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And in addition to indicating the level of degradation of the sample, having
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that large
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target, which is out there at the end, to estimate how much DNA is present in
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the reaction
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at the larger STRs.
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This is also a good indicator for how much impact inhibition has on the
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amplification
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of larger markers.
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Because inhibition will also impact the large STRs.
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And this is a good indicator for how severe that impact is.
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Absolutely.
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And I have to challenge in a good way.
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You said that the multi-target quant markers, we quickly identified that the
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single copy
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was going to be better.
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I have to give the R&D team credit for spending actually a lot of time trying
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to optimize
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that multi-copy target and really reap the benefits of that before having to
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move to
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the single copy target for the performance of the assay.
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So props to you and the team.
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But you also mentioned improved performance.
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So as far as performance, can you expand on that for our audience today?
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Yeah, I remember I mentioned we started the project before the SwigDAM
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recommendations
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were announced and published.
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We had already begun to look at ways to improve the performance of the Intel
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assay, which
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is extremely sensitive, but it's subject to stochastic effects.
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So you could say our goal of the project at the beginning was to change the
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yellow check
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marks of Intel to green with Intel Plus.
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So to accomplish this, we needed the system to have better peak height balances
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and be
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more immune to the low sample inputs, which caused these amplification
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artifacts.
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So how did we do this?
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We did it by increasing the amount of DNA available in the PCR by reducing the
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lysis solution volume
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and capturing twice as much lysate, which is where we get the DNA, with an
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additional
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punch in the PCR chamber of the cartridge.
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Of course, this changed until optimizing all our primer mix and a master mix in
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the cartridge
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to have the best performance.
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So with the additional capture DNA, we were able to reduce the number of PCR
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cycles further
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reducing the amplification artifacts while keeping what our target sensitivity
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of the
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system was and what we wanted.
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So while the GFB in ACE and Intel cartridges had made use of the GFB CE
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chemistry kit formulation,
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the Intel Plus cartridge, we re-optimized everything to work specifically with
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the rapid
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hit ID platform.
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Just to try and summarize what you just said there and emphasize to the
12:48
audience how challenging
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it is to make a change to SCR kit chemistry, it's no easy feat to just tweak a
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single
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marker, much less add new markers or add markers that are in flanking regions
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outside of the
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typical SCR read region within a dye channel or within the host of dye channels
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within an
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entire kit.
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So all of these changes are going to benefit the customer.
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Are there any other changes that are going to benefit the customer?
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I hear that the user will be able to use a new swap to maximize performance.
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I think that might have to do something with the lysis buffer that you
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mentioned earlier.
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Is that part of the new cartridge?
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Yeah, that's right Peter, John.
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The Intel Plus cartridge will support two protocols.
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So we have a general protocol which has the same lysis volume as the ACE
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cartridge and
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we wanted to keep this so you can use standard size swabs.
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But in addition, we've added a specialized protocol for these low input samples
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So it does have certain requirements to use the protocol.
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To use that specialized protocol, the user needs to use a micro or a mini fl
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ocked swap
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as a specialized protocol uses that reduced lysis volume to deliver more
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concentrated
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DNA to the PCR chamber.
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So the sensitivity of the specialized protocol also enables the user to save
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the primary
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swap if they want for other processes.
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As the user can, what we call sub sample, the primary swap.
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This is done by transferring a small amount of sample to the micro flocked swap
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And you can do this by simply putting the micro flocked on a table and pressing
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the
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primary swap against it to transfer some small amount of material.
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So now you can use the micro flocked swap to run the Intel Plus system as
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either a screening
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method or as a primary method for taking a genotype.
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And then the result can help inform the lab that the samples may be challenging
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, degraded,
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inhibited before initiating their downstream workflow.
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You touched on something there that I do want to elaborate on a little bit.
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There's not necessarily a difference in the amount of transfer from a
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traditional or general
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cotton swab that you mentioned to the micro flocked swap with regards to
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whether you're
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transferring from one to the other.
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But when it comes to ease of use, it's actually easier to put the blank micro
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flock or mini
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flock or what a hydro flocked swab on the bench on some paper and rub the maybe
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wooden handled
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swab, the stiffer swab that has the sample on it, rub that onto the micro fl
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ocked swap.
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Instead of trying to rub the micro flocked swab onto the general cotton swab,
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right?
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It might not change the amount of transfer, but it's much easier to use.
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Is that true?
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Because of the flexible handle on that micro flocked swab, we found out the
16:07
best way to
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do transfer was to put the micro flocked down and hold the primary swab with
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the stiffer
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handle against it to transfer the DNA.
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All you need is a small amount.
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This protocol is highly sensitive.
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Anywhere between 0.2 to 0.4 microlears of blood is usually enough to pull a
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full profile.
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That's why I said you can save that swab that has a decent amount of material
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on it for
16:32
further use and not use it up in the rapid instrument by doing the sub sampling
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Absolutely amazing.
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In the sensitivity there is wonderful.
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We talked mainly about the new chemistry and the cartridge and a little bit on
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the software
16:48
with regards to the two instrument protocols.
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Are there any other changes that the customer should expect with our new
16:55
solution?
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There's going to be quite a few.
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The good news is for a lot of our early customers in the GMapper world and
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using the chemistry
17:04
kits that are on the 3100 and 3500 instruments, is this system will go back to
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using GMapper
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ID extra genotyping.
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Moving to GMapper on the instrument itself for its software update.
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This effect will have some changes for the user and how they do their work.
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Now the system will require the running of a positive control swab after
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insertion of
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a new primary cartridge followed by the latter.
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This requirement is really a kit level requirement.
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So each kit that's going to be run on the primary cartridge will need to have
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its positive
17:45
control run before running samples.
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However, it does only need to be done once per kit per primary cartridge.
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So the reason for this is the new system on the new system.
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The positive control is going to establish the spectral die matrix which is
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needed for
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reduction of pull-up events.
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So another thing, this new software will only support GFEA's and Intel Plus
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cartridges along
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with the 150 run primary cartridge.
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The current HID, Rapid Hit HID software will not support the Intel Plus kit and
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neither
18:26
will the current RapidLink software.
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So you're going to have to upgrade your software versions for both RapidLink
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and the instrument
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to version 2 to run the new Intel Plus cartridges.
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Now just so people know the GFEA's cartridges will still run under the same
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protocols as
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the previous version.
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All we are changing is new signal processing software and the data analysis
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again is migrated
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to GMIDX for sizing and genotyping.
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So RapidLink.
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RapidLink version 2 is a complete rewrite of the software.
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It's a web-based browser interface now and it has features to manage what the
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instruments,
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user profiles, workflows and sample data.
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In addition, a lot of things had to be added to RapidLink to support the new
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Intel Plus
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cartridges new features.
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So we've introduced, I'm going to call them notifications or flags that were
19:22
added to
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the profiles display screen in RapidLink.
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So for instance, for the things we've been talking about for inhibition and
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degradation.
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Based on the peak height differences of the smaller IQC and amplification and
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quantification
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markers, their respective larger counterparts.
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The ratio difference will be displayed on the system as if you have inhibition
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as a yellow
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triangle next to the letters INH or if you have degradation, a yellow triangle
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next to
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the letters DEG.
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If it is determined to be inhibited or degraded.
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If no flags are generated, we detect no inhibition or degradation, you'll see
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nothing.
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There'll be no flags.
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So based on the results of a measurement system analysis study we did, we can
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now estimate
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the amount of DNA present at both the small quantification target and then
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again at the
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larger quantification target, all based on their peak heights.
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And when we do this, again on the display screen, we will show you the picogram
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estimate display for the small target next to letters QTS.
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And then the estimate for the amount of DNA at the larger target next to the
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letters QTL.
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And the final SWIG DAM recommendation for our systems meet was what is the
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likelihood of
20:49
having a Leel dropout in a sample.
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And this was really interesting and challenging for us.
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So through lots of evaluation of the assays response to very low level inputs
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that induced
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dropouts, we determined actually the best way to predict a dropout was to use
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the peak
21:07
heights of a select set of the large STRs themselves, one from each dive
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channel.
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This proved to be much more accurate than looking at the quantification markers
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, which
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surprised us.
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But we ran hundreds of samples and this had the best method, was the best
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method.
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So how this gets reflected to the user is on the profile screen of RapidLink.
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The user is going to see the letters STO, followed by either a green, yellow,
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or red flag.
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A green flag indicates there should be no dropouts due to sample load.
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A yellow flag that the sample may display various like peak height imbalances
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and other
21:52
artifacts a little bit in the profile itself, but it should see no dropouts due
21:57
to sample
21:57
load.
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The red flag indicates we have a very high likelihood that this sample will
22:02
have a dropout.
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And in our studies, we saw a 75% positive rate call for dropout events when the
22:11
flag
22:11
went yet red.
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So basically 75% of the samples that had a red flag also contained a dropout.
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So Peter John, I have to ask, you know, we've done R&D, has done a lot of work
22:25
redesigning
22:26
this cartridge to satisfy all the requests in this position recommendation
22:31
paper.
22:32
You have been the primary part of contact for the FBI during this work on
22:35
developing
22:36
the Intel Plus project.
22:39
Can you explain a little bit about the FBI's visions for these changes?
22:44
Maybe the future where they see rapid DNA going?
22:47
And since really this is to access CODIS with crime scene samples, can you give
22:51
us a little
22:52
more on that?
22:53
So the position paper was the first step in the FBI's vision for how we as a
22:59
community
23:01
will get to the point where rapid DNA users can upload DNA profiles generated
23:06
from crime
23:07
scene samples on rapid DNA instruments.
23:11
The second step is the multi-lab study in which the FBI along with its SWIG DAM
23:18
counterpart
23:19
will evaluate the suitability of these rapid DNA solutions for future CODIS
23:26
access with
23:27
crime scene samples.
23:29
Then steps three and four are involving the publication of the MLS data or the
23:35
multi-lab
23:36
study data and developing some new SWIG DAM guidelines for CODIS access via
23:43
crime scene
23:44
evidence from a rapid hit instrument.
23:48
So there's a lot that went into it and there's actually a lot that you
23:52
mentioned in that
23:53
last segment about those enhancements.
23:56
And we tried to partner and work together with the various groups to try and
24:02
meet all
24:02
of those requirements.
24:05
Can you tell us a little bit about or do you have any questions about what this
24:10
might
24:10
mean for the community, whether it's forensics or law enforcement?
24:15
Yeah, so we know that booking stations now have approval to upload directly
24:20
into CODIS.
24:21
And that's great.
24:23
But now we're going to investigate of leads.
24:26
And I'm really wondering when the bar for investigative leads will we reach
24:29
where they
24:30
can upload those data into CODIS.
24:32
I think they use now local databases, but they can't go to CODIS.
24:36
So can you talk a little bit about that and the ability and what you see and
24:39
when will
24:40
that be available for the law enforcement?
24:42
At this point, labs will be able to upload crime scene samples to CODIS using
24:48
only the
24:48
modified rapid DNA analysis method, meaning all profiles will need to be
24:54
reviewed by a
24:55
qualified DNA analyst, even when the new crime scene evidence processing
25:01
solutions are going
25:02
to be available.
25:04
And that is after the study results have been published and the SWIGDAM
25:09
requirements have
25:10
been posted and everything has been enabled for any kind of crime scene sample
25:17
profile
25:18
upload to a CODIS index.
25:21
So this doesn't preclude the sample from being run at a law enforcement agency
25:27
or location,
25:29
but the data will still need to be transferred to a crime lab for review and
25:34
potential upload.
25:36
Any questions about that?
25:37
No, I'm just thinking this is so great.
25:38
I mean, it's great for the friends of community.
25:41
We've come along with rapid and honestly as a developer of these assays, it
25:46
really is
25:47
rewarding to hear that what we're doing is having an impact, that our work is
25:51
getting
25:52
traction and that just makes us want to do this more and do the next step for
25:56
the community.
25:57
Yeah, it's been a long road.
25:59
The journey is not over yet.
26:02
Our partnership with the FBI has been invaluable for the criminal justice
26:06
community and our
26:07
close partnership and cooperation brought testing the arrestee samples in the
26:13
booking
26:14
station to a reality and upload into CODIS for those specific samples and that
26:20
specific
26:21
sample type.
26:23
Now we are on a similar path with crime scene samples and I couldn't be more
26:27
excited about
26:28
our solution and what it's going to mean for the criminal justice and
26:33
investigative communities.
26:34
I really look forward to the future.
26:37
Thank you so much for joining me today Rob to discuss this with our Forensic
26:41
Magazine
26:42
community and I look forward to working with you in the future.
26:45
Thanks again Peter John for having me.
26:47
This has been great and very informative.
26:49
Thank you.
26:50
I hope you're as excited as I am about all the new developments and
26:54
enhancements in rapid
26:55
DNA technology at Thermo Fisher Scientific.
26:59
Standby, after this, there's a live Q&A session.
27:03
Thank you.
27:03
(upbeat music)
27:06
(upbeat music)