Peterjon McAnany & Robert Legace 27 min

Expanding Your Toolbox with Rapid DNA to Drive Actionable Investigative Intelligence


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.



0:00

In the criminal justice community, there's only one mission, the relentless

0:08

pursuit of

0:09

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

1:09

all over

1:09

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.

2:35

Hey Rob, nice to see you again.

2:37

It feels like it was just yesterday that we were working on a 3500 software

2:41

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.

2:55

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

4:10

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

4:16

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

4:40

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

5:08

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.

10:31

Absolutely.

10:32

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

10:42

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.

11:02

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?

11:11

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

11:40

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

12:24

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

12:43

hit ID platform.

12:45

Just to try and summarize what you just said there and emphasize to the

12:48

audience how challenging

12:50

it is to make a change to SCR kit chemistry, it's no easy feat to just tweak a

12:56

single

12:57

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.

13:14

So all of these changes are going to benefit the customer.

13:19

Are there any other changes that are going to benefit the customer?

13:22

I hear that the user will be able to use a new swap to maximize performance.

13:28

I think that might have to do something with the lysis buffer that you

13:32

mentioned earlier.

13:33

Is that part of the new cartridge?

13:34

Yeah, that's right Peter, John.

13:37

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

13:44

cartridge and

13:45

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.

13:59

To use that specialized protocol, the user needs to use a micro or a mini fl

14:05

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

14:19

the primary

14:20

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

14:39

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

14:49

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.

15:03

You touched on something there that I do want to elaborate on a little bit.

15:09

There's not necessarily a difference in the amount of transfer from a

15:13

traditional or general

15:15

cotton swab that you mentioned to the micro flocked swap with regards to

15:20

whether you're

15:21

transferring from one to the other.

15:24

But when it comes to ease of use, it's actually easier to put the blank micro

15:32

flock or mini

15:33

flock or what a hydro flocked swab on the bench on some paper and rub the maybe

15:41

wooden handled

15:42

swab, the stiffer swab that has the sample on it, rub that onto the micro fl

15:48

ocked swap.

15:49

Instead of trying to rub the micro flocked swab onto the general cotton swab,

15:55

right?

15:55

It might not change the amount of transfer, but it's much easier to use.

16:00

Is that true?

16:01

Because of the flexible handle on that micro flocked swab, we found out the

16:07

best way to

16:08

do transfer was to put the micro flocked down and hold the primary swab with

16:13

the stiffer

16:14

handle against it to transfer the DNA.

16:17

All you need is a small amount.

16:19

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

16:26

full profile.

16:28

That's why I said you can save that swab that has a decent amount of material

16:32

on it for

16:32

further use and not use it up in the rapid instrument by doing the sub sampling

16:37

Absolutely amazing.

16:38

In the sensitivity there is wonderful.

16:41

We talked mainly about the new chemistry and the cartridge and a little bit on

16:47

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?

16:56

There's going to be quite a few.

16:58

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

17:10

using GMapper

17:11

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

17:31

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

18:04

reduction of pull-up events.

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So another thing, this new software will only support GFEA's and Intel Plus

18:13

cartridges along

18:14

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

18:42

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.

19:00

It's a web-based browser interface now and it has features to manage what the

19:05

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

19:15

cartridges new features.

19:17

So we've introduced, I'm going to call them notifications or flags that were

19:22

added to

19:22

the profiles display screen in RapidLink.

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So for instance, for the things we've been talking about for inhibition and

19:30

degradation.

19:31

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

19:48

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

20:21

again at the

20:22

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.

20:37

And then the estimate for the amount of DNA at the larger target next to the

20:41

letters QTL.

20:44

And the final SWIG DAM recommendation for our systems meet was what is the

20:48

likelihood of

20:49

having a Leel dropout in a sample.

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And this was really interesting and challenging for us.

20:55

So through lots of evaluation of the assays response to very low level inputs

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that induced

21:00

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

21:12

channel.

21:13

This proved to be much more accurate than looking at the quantification markers

21:16

, which

21:17

surprised us.

21:19

But we ran hundreds of samples and this had the best method, was the best

21:25

method.

21:26

So how this gets reflected to the user is on the profile screen of RapidLink.

21:33

The user is going to see the letters STO, followed by either a green, yellow,

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or red flag.

21:41

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

21:51

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.

21:59

The red flag indicates we have a very high likelihood that this sample will

22:02

have a dropout.

22:04

And in our studies, we saw a 75% positive rate call for dropout events when the

22:11

flag

22:11

went yet red.

22:12

So basically 75% of the samples that had a red flag also contained a dropout.

22:20

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)