Finding the Right Tool for the Job: CE Tools that Improve Efficiency and Save Time
Watch this webinar to learn about in-depth evaluations of CE analysis tools designed to minimize hands-on time and time spent on routine data review, enabling more complex sample analysis.
Hello and welcome to Finding the Right tool for the job, CE Tools that improve
efficiency and save time.
This is the third webinar in the sixth part Future Trends in Forensic DNA
Technology series.
My name is Michelle Taylor, Editor-in-Chief of Forensic, and I will be your
moderator throughout.
For today's webinar, you can earn one hour of continuing education credit.
Following the conclusion of the webinar, you will receive an email with
information on how to obtain CE credit documentation.
We have a great line-up scheduled to present to you today, but before we begin,
I'd like to take just a moment to cover a few logistics.
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Today, you will be hearing from Jamie Brockhold, senior technical application
scientist at Thermo Fisher Scientific.
In her current role, she provides trouble shooting and technical support to
friends at laboratories globally
and offers subject matter expertise, as new products are introduced into the
human identification portfolio.
Back to joining Thermo Fisher Scientific, Brockhold earned an MS in Forensic
Science with a concentration in Criminalistics
from the University of New Haven and went on to work in the Forensic Biology
Unit at the Massachusetts State Police Crime Laboratory.
You will also hear from Robert O'Brien, Forensic Biology Section Lead at
Florida International University.
O'Brien evaluates equipment and techniques used in biological collection and
DNA analysis.
His technology evaluations have been published as reports, presentations, and
scientific posters at industry events, and as reference publications.
He also advises operators on tests and techniques available for field use in
biological sample detection and screening.
O'Brien develops curricula for forensic DNA and serology testing programs,
delivering instruction both in person and remotely, something that's very
important right now.
Thank you for joining us for our third session in the 6th part Future Trends in
Forensic DNA Technology webinar series.
After the webinar, please be sure to check your email for more information on
CE Credit Documentation.
We look forward to seeing you on August 20th for part 4, tips, tricks, and best
practices for gaining efficiency in your Forensic DNA Laboratory.
Without further ado, I'm going to hand it off to Jamie to get us started.
Thank you so much for that introduction, Michelle.
My presentation today is focusing on new features in our latest CE Data
Collection software programs that will help improve efficiency and save time.
We're going to be discussing enhancements in the 3500 Data Collection version 4
.0.1, and also new features of the SEEK Studio instrument and SEEK Studio Data
Collection version 1.2.1.
Here's a quick overview of what the presentation will cover.
We'll discuss at a high level the new features of the latest software versions
on each of the CE instruments.
Unfortunately, we don't have time to go into great detail on all of the new
features, but we will get to drill down into the pull-up reduction aspects,
which are similar, but do have slight nuances for each instrument.
After reviewing the technicalities behind pull-up reduction, we'll jump into an
STR Kit comparison where we can get paired pull-up and data analysis across
five STR kits, two from applied biosystems, and three from other manufacturers.
We'll get started talking about the new enhancements in the 3500 Data
Collection software version 4.0.1.
After taking in a lot of customer feedback, since we first released the 3500
with Data Collection, we were able to include some great benefits with this
newest version.
As it relates to improved data interpretation, we did introduce an algorithm
that will reduce pull-up peaks for applied biosystems chemistry, and this is
what's going to be covered in a lot more detail in the upcoming slides.
We also optimized signal across the capillaries, and we did this in two ways.
In 24-cap systems, we introduced a spatial optimization to account for optical
variation across the 24 capillaries, and on both the 24 and 8-cap instruments,
we have a recommendation for a Z offset or a higher position in the well when
the capillaries go into the well for injection, and that can be performed by an
engineer
or a routine PM, or when they first installed the software by doing an auto sam
pler calibration.
We also introduced an algorithm for reducing off-scale data for data-vacing
labs, or those labs running robust single-source reference samples.
Off-scale data recovery, what it does is it lifts the cap off the top end of
the dynamic range, so right now where your data is capped or off-scale, around
32,000, if you're running with off-scale data recovery, that cap is lifted up
to about 65,000.
Also, you will see pull-up related to off-scale peaks reduced. You do see a
little asterisk here that mentions GMapper IDX version 1.6 for full
functionality of the off-scale recovery feature, and that is because it's only
in version 1.6 that you'll see that cap lifted up to 65,000.
If you analyze these HID files in earlier versions of IDX, you will see reduced
pull-up. You will see reduced pull-up also as it relates to the off-scale data,
but you won't see that cap lifted up to 65,000.
You'll still get the off-scale indicator when peaks reach about 32,000.
We also aimed to improve the user experience, and a lot of feature requests
went into these enhancements. The first thing we did was we added a plate
loading flexibility, where you can pause a run, bring the auto sampler forward,
add a second plate, and restart the run.
You also, if you have two plates on, could pause a run after the first plate is
completed, for example, pull the auto sampler forward, take the first plate off
, put a third plate on, and start the same run, and that's continual.
We also added a six-die installation standard, so on earlier versions of
software, the installation standard for the HID performance check was done
running Identifyler Ladder.
You can now have the option to run a five-die with Identifyler Ladder, same as
before, or a new six-die with Global Filer Ladder performance check.
We included, like I mentioned, other feature requests, things like being able
to export the injection list, have a consumable log that is very easy to read
to see when consumables were taken off or put on the instrument, and by whom.
Those are a couple other ones, but there is a full list of all the new features
in the data collection, usable item.
This data collection version, 4.0.1, is Win10 supported.
Talking a little bit about the Seek Studio, which is our newest CE instrument.
For those that don't know, the Seek Studio is a 4-cap CE instrument.
A couple of ways that we are going to save time with this instrument is there
is an automatic optical alignment, meaning that when the capillary is installed
on the instrument, the spatial is automatic.
So no need to run that manually.
There is also an automatic spectral calibration adjustment, or also called in
the literature auto calibration, and we are going to be talking more about that
feature.
But basically, you only need to run a manual spectral.
You have to only set up a spectral one time for each die set on the instrument.
And after that, you don't have to run a manual spectral calibration again.
You can see that this instrument is pretty small.
The dimensions are there.
So a nice easy to lift, easy to move instrument, fit on a small bench top space
, if that is concerned in your lab.
The data collection software is run right on the instrument itself.
There is a touch screen that you do everything on.
You set up your plates.
You can monitor your run.
You view results.
So there is no need to have a computer attached to this instrument at all.
The consumables, we have an all-in-one reagent cartridge, which you can see
sitting in front of the instrument in the picture here.
Everything but your cathode buffer is in this easy to handle cartridge.
Your array is here.
Your polymer is here.
And your polymer sits in a little insulator that when the cartridge is put into
the instrument stays chilled,
which gives the polymer a six-month on instrument storage lifetime.
Your anode buffer reservoir is in the cartridge and your polymer delivery
system.
The cartridge is RFID tagged as is the cathode buffer container, which will sit
on the auto sampler.
There is also no more plate sandwich.
The CE plate sits right on the auto sampler, and then there is just a lid on
the auto sampler that closes down over the septa that is on your plate.
GMIDX version 1.6 is the IDX version that can read the .fsa files coming from
the seek studio data collection software.
So no longer .hid files on this instrument and software.
And then there is also some adjunct software, which is optional to use.
You will get an SAE module that you can use to set up different users and
different permissions within the software and on the instrument.
And then there is also a seek studio plate manager, which can be used to help
set up your plate maps that can be imported into the instrument.
I should also mention it is a single polymer type.
It is a pop one polymer for sequencing general fragment analysis and STR
fragment analysis.
So only pop one. So if you are running different applications on the seek
studio, you don't have to worry about changing your polymer out.
It is a 28 centimeter capillary array. I don't think I mentioned that either.
All right. So let's get into the pull up reduction features that are included
with these data collection versions.
So just to make sure we are all on the same page, we will do a quick overview
of pull up. We will talk about definitions and expectations first.
So generally we expect that pull up peaks are going to be within the range of 1
to 3 percent of the parent peaks that they fall underneath.
With off-scale data, we do know that these values could be slightly higher.
So you may see pull up above that 3 percent more often when you are talking
about an off-scale sample.
When we talk about pull up, there are really two different types of pull up.
There is spectral pull up and there is instrument specific pull up.
Spectral pull up occurs because when we run our manual calibration, we are
using pure dyes.
The matrix for deconvolution that is generated from that pure dye calibration
then gets applied to our samples.
In our samples, our dyes are attached to DNA fragments. That could affect the
spectra differently.
The difference between the spectra from the dyes being attached to the samples
and the dyes being pure dyes, that difference is what causes the error that
causes spectral pull up.
In the raw data, spectral pull up, you can see a pull up peak that sits
directly centered under the parent peak and the same is true in the analyzed
data.
When we talk about instrument specific pull up, it is a little bit different.
Instrument specific pull up all has to do with the way that the fluorescent
molecules move across the detection window.
As the fluorescent tagged fragments move across the detection window, center in
the window and move out of the window, the spectrum shifts on the CCD.
What you end up seeing in the raw data is a sinusoidal shape underneath the
parent peak.
In the analyzed data, rather than seeing the pull up peak centered under the
parent peak, it will be shifted.
If there is instrument specific pull up and spectra pull up in the same dye
channel at the same time, they would be additive.
You would see maybe a single pull up peak, but the percentage might be slightly
higher.
What does the pull up reduction algorithm do on the 3500 in particular? This is
pull up reduction on the 3500 data collection version 4.0.1.
What the algorithm does is it allows the software to use sample specific
spectral data for AB dye sets.
That is chemistry using G5, J6 or J6T.
Rather than using a matrix for deconvolution based on the manual spectral that
was run, that pure dye spectral, the algorithm uses data from the samples
themselves.
It is a more accurate deconvolution for those samples.
If, for some reason, there is limited data in a particular sample.
If you think about a low level sample where maybe there is only a few peaks,
the algorithm is going to step back and say,
"Hey, there is not enough data here for me to generate a spectral that is any
better than the manual spectral that is stored here."
Rather than trying to do something with limited data, just revert back to the
manual calibration.
Even with samples that don't have a lower quality or less data, there still
will be pull up reduction.
It will just be our traditional pull up reduction based off of the manual
spectral.
But when there is good quality data available and the algorithm is able to
generate a sample specific matrix, it will.
All pull up reduction happens prior to the creation of the HID files, and that
includes when a sample specific data is being used.
So any raw data that you see in IDX will already have the reduction applied.
Now when we talk about pull up reduction on the seek studio, it's somewhat
similar and somewhat different.
So there are really two levels of pull up reduction on the seek studio. The
first level is called auto spectral or auto calibration,
and it is similar to what we just talked about with the 3500 pull up reduction
feature.
It is going to use sample specific spectral data, but it's going to do this for
any die set.
So it doesn't just have to be an AB chemistry.
Any chemistry will have the benefit of the auto spectral.
Also similar to the 3500, if there is limited sample specific data, the
algorithm will still apply some level of pull up reduction.
It's a little bit different.
If a previous auto calibration matrix has been run, so you've had at least one
injection in a capillary with a particular die set that has generated an auto
calibration,
the software will revert back to that auto calibration.
If there has never been an auto calibration in that capillary for that die set,
then the software will go back to the manual calibration that was run.
The second level of pull up reduction on the seek studio is called Marketer
Marker Correction, and this is for applied biosystem die sets only, G5, J6, and
J6T.
Here we have taken into account Marketer Marker variation as it relates to pull
up across the read region of the chemistry.
And this software will apply an optimized correction factor for each marker in
each kit.
And the way that you enable Marketer Marker Correction is on plate setup, you
'll select the chemistry that you're running.
It's also similar to the 3500, and that all pull up reduction takes place prior
to the creation of the FSA files, so any data that you view at IDX will already
have pull up reduction applied.
Moving on to the STR Kit comparison.
So this study that we did, we had some general conditions for the study that I
'll cover here.
We also used the same sample types for each chemistry and across the different
instruments that we ran.
So all amplification done on a proflex PCR system.
Analysis, all done in gene mapper IDX version 1.6.
We also analyzed using minimum peak amplitude thresholds, which I'll discuss in
the upcoming slides.
Otherwise we followed manufacturers recommendations and parameters for each kit
as it related to amplification and CE injection.
You can see the kits listed in the blue table.
So Global Filer and NGM, Global Filer IQC and NGM Detect were the two applied
biosystems kits.
Promega Fusion 6C and Promega PowerPlex ESI 17 FAST were two kits from Promega
that we ran, and the KIAJIN investigator 24plex QS kit was the kit from KIAJIN.
So five kits in total.
You can see the different cycles that are recommended, which is how we
performed amplification and the different DNA inputs, either half an anagram or
one anagram, depending on the kit.
We ran what we call NP or non-probative samples.
So we picked four samples that could be commonly encountered by a laboratory.
So the first was a swap from a cell phone, the next a cigarette butt, swap from
a baseball hat and blood on cotton.
We also ran the kit positive control.
We utilized 1.3500 XL with data collection 4.0.1 and two different seek studio
instruments with data collection version 1.2.1.
So I mentioned that we used minimum thresholds for our analysis.
And the way that we calculated a minimum threshold was we analyzed non-template
control data for each of the kits on each instrument at one RFU.
We went into the sample and edited out any pull up.
So that could have been spikes, pull up from the size standard, or any other
artifacts.
So if there was a die blob or any raised baseline areas, that all got edited
out.
Once we were left with all the non-artifactual data, we did some calculations
to get to the limit of quantification.
So we take the average peak height of all those peaks at 1 RFU plus 10 standard
deviations, and that gets us to our LOQ.
And this is the RFU value where you'd expect nearly all your noise to fall
below.
We took that LOQ and rounded it to the nearest 5, and that was the minimum
threshold.
So for each die channel, we had a different minimum threshold.
If your LOQ was 22, we would have rounded down to 20 for the minimum threshold.
On the 3500 instruments, across the 5 different chemistries, the minimum
thresholds ranged from 35 to 85 across the die channels.
That's omitting orange, that's omitting the size standard.
On the seek studio, if you recall, I mentioned we ran two different instruments
, so the range is a little wider.
The range was from 25 to 140 RFU across the die channels, across all the chem
istries, for the two different instruments.
Okay, I just want to -- I use this term "complex spectral artifacts" when I
start talking about the results of the study,
so I just want to make sure everyone's on the same page with what I mean.
So traditional pull-up, that's pull-up that I'm going to be talking about that
is pull-up that falls underneath a parent peak.
It's very easy to calculate a pull-up percentage, so like the top figure that
you see here.
When I talk about complex spectral artifacts, these are pull-up peaks that it's
more difficult to figure out maybe what the parent --
which parent peak is related to the pull-up peak, or it can be like what you
see in this figure here,
a bridging effect, or what some people might call a pull-down effect, where you
have -- we can count it as a spectral artifact if these peaks were called,
these red peaks,
but I didn't calculate any sort of pull-up percentage to a parent peak here.
So looking at the results on our 3500 for the pull-up assessment, applied bios
ystems kits, as a reminder,
we'll have pull-up reduction enabled, so the use of sample-specific spectral
data.
The non-applied biosystems kits have pull-up reduction disabled, so that would
just be using the traditional spectral calibration with those particular die
sets.
We had a positive control and four non-probative samples for this analysis.
So in the chart here, you can see -- there's a left side and a right side. To
the far right, you can see the count of that with those complex pull-up peaks.
So any pull-up peak that was counted but a percentage wasn't calculated for.
And then on the left side, you can see the more traditional pull-up peaks
counted and then the percentages that were calculated.
So overall, you can see if you look at the average percent of the parent peak,
which is in the third column here, this is all below 3%.
So for all five kits, the average pull-up was below 3%.
You can see that in the non-applied biosystems kits, there was some pull-up
above 3% and even some pull-up above 5% in the two promega kits.
And I just have a different way to look at this on the next slide since this is
a lot of numbers to look at quickly.
Here, what you see across the x-axis are the five different kits, across the y
-- up the y-axis, I should say, or the number of pull-up peaks.
So that's traditional and complex counts of pull-up combined.
The different colors in the bars themselves, the red indicates the count of
complex pull-up peaks.
The lightest blue is pull-up that was above 5%.
The next shade of blue, pull-up below 5% and the purple pull-up that was below
3%.
So what you can see overall is that the non-applied biosystems kits have at
least three times the number of pull-up related artifacts
compared to global filer IQC and NGM detect.
I wanted to put this into a relatable -- a relatable way to look at if you were
an analyst in the lab.
So I wanted to consider time-saving.
So what does a three times reduction in the number of pull-up peaks look like
to someone who's taking the time to analyze the data?
So I had to start with an assumption. I said, let's say it takes about 30
seconds per pull-up edit.
I figured that was fair, given that some peaks will be pretty easy to visualize
as pull-up others.
You'll have to go into the raw data, maybe do some calculations, things like
that.
So I averaged about 30 seconds per pull-up edit.
So we can say that if an applied biosystems kit had 70 pull-up peaks in five
samples, which is similar to the data that we saw in this study,
a non-applied biosystems kit would have three times that, or 210 pull-up peaks
in five samples.
So that would mean that it would take someone 35 minutes to analyze the applied
biosystems kit --
with an applied biosystems kit, five samples, just the pull-up peaks, versus an
hour and 45 minutes to analyze pull-up and five samples run with a non-applied
biosystems kit.
If you double that time, considering there has to be a secondary review, we're
talking about an hour and 10 minutes to analyze an applied biosystems kit, five
samples, just talking about pull-up,
versus three hours and 30 minutes for a non-AB kit, five samples, just talking
about pull-up.
So going over to the seek studio instrument, if you remember, we had two
different instruments, so this table is even a little bit busier than the last
one we looked at.
As a reminder, applied biosystems kits will have the auto-spectral enabled and
marker-to-marker correction.
The non-AB kits will just have auto-spectral enabled.
Again, it was a positive control and four non-probative samples that were
analyzed.
So, again, on the far right, you can see those complex pull-up peak counts, and
then on the left, you can see the traditional pull-up counts as well as the
percentages that were calculated.
Again, all kits had an average pull-up percentage of less than 3%.
All of the non-AB kits, in this case, had pull-up greater than 3%, and a few
had pull-up still greater than 5%.
So for those that like to look at things in a different fashion, I much prefer
this fashion.
Very similar, five kits across the X-axis, the number of total pull-up peaks
across up the Y-axis.
You can see broken down the complex pull-up that we saw in the non-AB kits, and
then the different percentage levels in the blue and purple colors.
You can see a difference between Global Filer IQC and NGM detect as far as the
number of pull-up peaks, 6 verse 21 for the two kits.
And so when we compared the applied biosystems kits to the non-AB kits, I did
split it out because there was that discrepancy.
So for Global Filer IQC, the other kits had at least 13 times the number of
pull-up related artifacts, and for NGM detect, at least four times the number
of pull-up related artifacts.
Again, considering time-saving, so how might this impact you if you're the
analyst and/or the reviewer?
Again, I assumed 30 seconds per pull-up edit, but I did split it out based on
the difference we saw with Global Filer IQC and NGM detect.
So for Global Filer IQC, if there were three pull-up peaks in five samples, the
other kits would have 13 times that, or 39 pull-up peaks in those five samples.
So this would mean a time savings from one minute, one and a half minutes,
excuse me, to analyze the three pull-up peaks in Global Filer IQC, verse 19 and
a half minutes in a non-AB kit.
Doubling the time for review, you have it see a 33-minute savings in time.
Sorry, a 36-minute savings in time.
For NGM detect, which would have, let's say, 10 pull-up peaks in five samples,
the other kit would then have four times that, or 40 pull-up peaks in those
samples.
So for initial analysis, we're looking at five minutes, verse 20 minutes,
whether you're running NGM detect or a non-AB kit, and double-a-in that time
for review, you're talking about a 30-minute time savings.
So in conclusion, pull-up reduction on both the 3500 and seek studio with the
latest data collection software versions greatly reduced the number of pull-up
related artifacts for the applied biosystems kits.
On the 3500, it reduced the pull-up artifacts by at least three times on the
seek studio, depending on the kit, four or 13 times the number of edits
reduction in the number of edits for the applied biosystems kits.
And just as a reminder, the seek studio does have that added level of pull-up
reduction, the marker-to-marker correction.
So taking into account the pull-up reduction and also some of the new features
I talked about at the very beginning of the presentation, both the 3500 data
collection version 4.0.1, and the seek studio with data collection version 1.2.
1 can streamline data analysis, especially when used in conjunction with ABSTR
kits.
Thank you so much for your time. That concludes my presentation, and I'm going
to pass the presentation over to Robert.
Okay, so today I'm going to be talking about the right tool for the job. I'm
Robert O'Brien from the Natural Forensic Science Technology Center at Florida
International University.
Before I begin, I just want to thank Michelle and Jamie.
What I'll be talking about is the applied biosystems seek studio genetic analy
zer for HID or the applied biosystems rapid ID system.
So the right tool for the right job, what we'll be looking at is the seek
studio and the rapid ID system. We'll be looking at systems and features, the
instrument operation, the maintenance, and some data that we have run on both
systems.
So first, let's talk about the seek studio versus the 3500.
So dimension wise, the seek studio instrument has a width of about 49.5
centimeters, a depth of 64.8 centimeters and height of 44.2 centimeters.
Whereas the 3500 has a width of 61 centimeters, a depth of 61 centimeters, and
a height of 72 centimeters.
What it is important to note is that when you, for the 3500, you do need to
have a clearance base about 122 centimeters in order to open the door.
So the seek studio has a much smaller imprint traditional CE instruments like
3500.
So it's definitely, it's going to be a shorter instrument and it's not as wide.
With usually makes it the most crucial factor since that dictates a space
needed on a bench top on a laboratory bench.
So if you consider the space that is needed to open the door of the 3500, you
can in fact fit two seek studios in that space.
And the seek studio door actually opens upwards instead of to the right.
So therefore the opening of the door of the seek studio does not take up any
additional space.
The seek studio instrument is a cartridge based instrument.
This puts the majority of the components of the CE system into one easy to
change cartridge.
The cartridge shown contains a capillary with four capillaries.
The palm of the CE system is actually part of the cartridge with the detection
windows sitting behind the optical cover.
The pop one allows the same cartridge to be used for fragment nastles and
sequencing.
The anode buff is also contained inside the cartridge.
The only other consumable that needs to be changed is the cathode buffer.
So the cartridge system makes changing of the capillary array very simple.
This reduces the training time that is needed to ensure that you have a perfect
alignment with the laser every time.
Its spatial does not need to be performed when it's starting a new cartridge as
the system performs an auto optical alignment every time you place it a new
cartridge.
Apart from the main cartridge, the cathode buffer and auto sampler are shown in
diagram as well as you can see from the arrows.
It has a 96 well plate or 8 strip tubes can sit directly on the auto sampler
which has a lid attached.
So you do not need a plate holder as you did for the 3500.
And this system uses the same plates as the 3500 so no spatial consumables will
be needed if you switch it from the 3500 to the C Studio.
A J6 spectral will be performed during the installation along with the AHD
install check which uses global file or ladder.
Since the system has auto spectral calibration as Jamie discussed, there is no
need to run in manual spectral calibration other than a single time.
The touchscreen display on the C Studio allows for full operation of the
instrument including plates set up on the instrument itself.
This means that a separate computer system is not used to operate the
instrument like 3500.
So this once again is reducing the amount of space and the footprint of the
instrument.
However, a desktop or laptop is available to interface with the instrument for
optional SAD control, a plate managed setup software and/or G-mapper IDX
version 1.6.
The C Studio was tested against the 3500.
C Studio's results were 100% concordable with the 3500 in the following data
sets.
We did 20 buckle swabs reference samples.
We did do a sensitivity series based on total input of DNA.
However, these were all normalized to one nanogram.
This actually data was used later on with the rapid ID.
We did a sensitivity based on volume samples with 4 microliters, 2 microliters,
1 microliters and 0.5 microliters.
For saliva, we did 8 microliters, 4 microliters, 2 microliters and 1 microlit
ers.
And for mixture samples, we did a 1 to 1, a 1 to 4, a 1 to 8 and a 1 to 16.
Once again, it's important to note that we ran the same plate that we ran on
the C Studio with us a 3500 and we got 100% concordance between the two
instruments.
The one difference I was noted for the data analysis, the C Studio does require
gene map or IDX version 1.6.
And this also shows that implementation of a C Studio does not in any way
affect the quality of the data that you're going to get from your CE instrument
So now we're going to look at the rapid ID system.
The rapid ID system is once you're going to talk about instrument
specifications.
So the height is about 48 centimeters, the length is 53 centimeters and it has
a width of 27 centimeters.
So therefore it makes it's very small and is very easily able to be placed into
nearly any room,
whether this be in the laboratory or booking station, etc.
The approximate weight is 28.4 kilograms with a primary cartridge installed and
25.4 kilograms with all the primary cartridge.
The only other space requirement that may be needed for this instrument is if
you're going to have a laptop next to it to get the data coming off of the
instrument itself.
However, you can have that laptop in a centralized location and have the data
sent to it. We'll talk about that later.
So based on these dimensions, many of the instruments could be placed alongside
each other.
So you can actually have a bank of these instruments and they can easily expand
the capability without requiring much more space on this larger predecessor,
the rapid ID 200.
The weight of the instrument also makes the instrument itself an easy one
person carry or in a case it could be a two person carry, which makes it ideal
for transporting two crime seats.
The rapid ID system is also a cartridge based system, so it has a primary
cartridge and a sample cartridge.
This allows for easy operation and maintenance of the instrument and because of
the ease of use very little training is required for the user.
The primary cartridge contains the following. It contains all the components
necessary for CE. It contains the Palma, which is shipped separately and stored refrigerated. It is loaded into the primary cartridge before the cartridge is placed into the
instrument.
It contains the capillary and anode and capillary buffers.
The primary cartridge is guaranteed for 100 samples. In tests with continued
daily use, it has been possible to get more than 100 samples out of the primary
cartridge.
The sample cartridges, there are two different sample cartridges. There is the
Ace Global File Express cartridge with the purple label and the Rapid Intel
cartridge with the pink label.
The sample cartridges can be stored up to six months when refrigerated or for
two months at room temperature and both cartridges use a Global File Express
chemistry.
Let's talk a little bit more about the Ace vs. the Intel cartridge.
The Ace Global File Express sample cartridge is intended for use with reference
samples like buckle swabs.
It can also be used for other high-level samples, for example, blood. The
runtime is approximately 90 minutes.
The Rapid Intel sample cartridge is intended for use with single-source crime-
scene type samples, examples to live and blood.
It has improved performance for low-level samples. It could be used for blood,
drinking containers, cigarette bugs and other similar items, and the runtime is
approximately 95 minutes.
The instrument is able to determine which runtime to use based on the cartridge
inserted. Therefore, the user does not have to make adjust runtimes depending
on what cartridge they're using. It's done automatically for you.
The Rapid Hit ID system comes with a laptop or desktop which has loaded onto it
the RapidLink software. Samples are instantly imported and can be used with the
software to generate matches or other investigative leads.
We'll talk a little bit about the RapidLink software now. The RapidLink
software can be used to link several instruments to one main computer.
The map shows the location of each instrument connected to the computer with
the RapidLink software.
If the location is flagged green, it means the instrument or instruments of
that location are all connected and operational.
The RapidLink software is used to determine the following. Location,
functionality, runs perform per day and runs perform per instrument.
So at the bottom left of the screen with the blue and red bars, this shows the
number of runs being performed per day and total across all instruments at that
location.
Blue means successful runs and red means unsuccessful.
At the bottom right, this shows the number of runs performed at each different
instrument. In this way, the operator of the software can track the reagent use
for each instrument.
This allows one central location to be in charge of reordering of reagents and
taking the burden off of the users at crime scenes or booking stations.
So very similar to Gene Mapper, we do have quality flags also on the RapidID
system. So at the end of the run, a quality flag appears on the screen of the
RapidID for the sample.
The colors are green, which basically means a sizing past. The profile can be
used in all functions of the RapidLink software.
Yellow means the sizing also has past but does require review before it can be
used in all functions of the RapidLink software and red means that the sizing
has failed.
These actually are displayed on the instrument itself, so the user is able to
tell instantly whether the sample is going to be a good sample, it's going to
require a view or may need to be run or if there was some other issue.
Regardless of the quality flag displayed on the instrument screen, the run
information is transferred to the RapidLink software and the quality flags are
also seen in the software.
So as you can see from the image on the right there, the quality flags are dupl
icated on the software. So if it's green, you'll see a green checkmark, if it's
yellow, you will see a box with a yellow arrow and if it is red, you'll see a
circle with a red X in between.
So that's how the quality flags are displayed. In this way, someone with access
to the RapidLink software can monitor the quality of the data coming off the
instrument and take any steps necessary in case of a problem taken to burden
off the user.
So one person at central location can see all the data coming off of all the
different instruments and be able to tell at a glance whether it's good quality
data coming off, whether that person perhaps will have to do reviews or if
there's several failures occurring,
they can actually address the problem and probably look into some troubles
hooting. So therefore, once again, the user, whether they're at a booking
station or crime scene, does not have to take on this burden.
Now there's some other features of the RapidLink software. So the RapidLink
software has additional apps or applications that can be purchased separately.
These apps have the following features. So first you've got the one to the far
left is a match app. This can be used to match any DNA profiles that are
imported into that computer with the RapidLink software.
So all the data being fed into this one computer with the RapidLink software
can be, you can use that to generate matches.
The familiar app can be used to do a familiar search of all profiles imported
into that RapidLink software.
The Kinship app can be used to verify a stated relationship between two
profiles and the SED, which stands for the staff elimination database.
The DNA profiles from staff members can actually be imported into the RapidLink
software and can be automatically compared to profiles imported into the
software to check for possible contamination.
It is important to note that since many instruments can be connected to one
computer with the RapidLink software, one advanced operator can perform all
these functions in one central location as opposed to having all the users,
whether the crime scene or booking station is attempting to carry out the
analysis.
The RapidLink software is built to allow users with minimal training to operate
the instrument while in more advanced use of or a DNA analyst, for example,
monitors the instruments and checks the quality of the data generated and
controlling how the data will be used in an investigation.
So let's look at some data generated from the RapidID.
So some of the studies that we ran on the RapidID to measure the performance of
the instrument. We did reference samples which are just simply buckle swabs.
We did a sensitivity series based on total nanogram input of DNA.
We did another sensitivity series based on the volume of DNA placed on the swab
We did a mixture detection where we were looking for the ability of the
instrument to detect a minor.
And of course we had a concordance with the CE 3500 and 6 studio.
So here's an example of 20 buckle swabs that were run in the RapidID system
which had a 100% first pass success rate.
This was done with the ACE cartridge.
These samples were all concordant with the samples run under 3500 and the CE
studio.
This means that all swabs registered a green quality flag.
It is important to note that for a sample to get a green flag, all alleles must
be present and called.
Therefore the first pass success rate means that there were full profiles
generated for all the swabs run and no reruns were necessary.
For males the profiles gave all 24 to 24 nails and for females 22 out of 22.
The image here shows how these were displayed on the RapidID link software
screen.
From left to right you have the quality flag, the date and time of the run, the
sample name, the cartridge being used,
the location, the instrument serial number and then the user.
So a sensitivity series is also done with the total input of DNA.
We basically did a range from 1280 nanograms to 10 nanograms.
So from the first as seen from the heat map from 1280 nanograms to 40 nanograms
all alleles were present.
For 20 nanograms and 10 nanograms there were some samples where dropout was
detected.
Even with dropout the lowest number of low side detected was 17 which is more
than enough for comparison purposes.
This allows the great dynamic range of the Intel cartridge and this is useful
for high and low level samples.
This was done with blood.
This makes it especially useful for crime scene samples where the amount of
input DNA is not known.
So with the RapidID ID Intel cartridge you do not have to worry about possibly
putting in too much blood or too little blood.
It's going to give you a good result either way.
We did a sensitivity series with sample volumes and here we have liquid saliva
was pipedted onto swan.
We also did that with blood samples.
We did various volumes of four microliters to microliters and one microliters.
And you can see at one microliters saliva there was still 85% of alleles
present.
This shows that RapidID is able to handle very low volumes of saliva with still
generating enough data to be used for comparison purposes.
We also did that with blood samples. We did various volumes of four microliters
, two microliters, one microliters and a 0.5 microliters.
And once again from the graph this shows that even at a volume of 0.5 microlit
ers 96% of the alleles were detected using the RapidID.
And this shows that the RapidID is suited for detection of DNA from very small
samples.
For mixture distractions, mixtures were run in the RapidID in the farmed
proportions, one to one, one to four, one to eight and one to 16.
The RapidID consistently detected the presence of the minor at the one to eight
proportion.
On some samples the minor was detected at the one to 16 proportion.
The ability to detect a minor at such low proportions provides utility for
crime scene type samples.
Perhaps indicating the presence of a low level male in a sample with mostly
female DNA.
And this information can be used by the laboratory to decide how to proceed
with further testing.
So, concordance for the 3500 in the seek studio. All samples tested in the
following studies gave them core entry results for the 3500 in the seek studio.
So, in all studies performed, sensitivity with total nanograms and volumes of
blood and saliva, the reference samples and the mixture studies, there was no
discordance observed.
Between the allele calls from the RapidID and those of the 3500 and the seek
studio.
Now it's important to note the RapidID system does not consume the sample.
The ACE and Intel cartridges allow easy removal of the swab.
Once removed, the swabs can be rerun using traditional methods of extraction,
quantitation, amplification and CE.
The sample's tested gave full DNA profiles after they were run on the RapidID.
This allows one swab to be used twice since the RapidID does not consume the
sample.
This becomes very useful if there is a limited number of samples or in case if
two swabs are taken and one fails to give a result.
So, this chart shows samples that will run the RapidID using the ACE cartridge.
The samples were removed from the ACE cartridge and placed in a hood to air dry
The time between when they were run on the RapidID and then run again using
traditional DNA methods range from a week to a month.
The DNA quantities shown are from the swabs after they were run on the RapidID.
In all cases, the samples gave full profiles using traditional CE testing.
Even with as little as 20 microgives of blood or one swab of the energy.
These samples also gave a full profile on the RapidID and the results were
recorded.
So, future testing is planned for lower level samples using the Intel cartridge
to see if we can also get DNA profiles after it's run on the Intel cartridge
with lower level samples.
So, candy systems we use together can they actually be integrated?
Well, both of them have a small footprint. Both require little maintenance and
both are very easy to run.
The RapidID is ideal for the booking station or crime scene unit, while the
seek studio is ideal for smaller full service DNA laptops, but can they exist
together?
One model would be to have, for example, the RapidID used for reference case
work samples.
The ease of use and minimal training required makes it ideal for technicians in
the laboratory to process these samples.
The high first pass success rate ensures good quality results.
In the same laboratory using robotic extraction platforms like the Automate,
with the Quant Studio 5 for quantitation and the Proflex for amplification and
the seek studio for CE, only crime scene type samples would need to be
processed using traditional DNA methods.
This would reduce the number of samples being run through the entire
conventional DNA process, so the full capillary system of the seek studio
should be adequate to meet those needs.
By running the reference samples on RapidID, there will also be a separation of
nodes and unknowns and preventing cross-contamination between the reference and
the question samples.
Let's do a little system comparison summary. The RapidID system has one cap
illary, whereas in the seek studio you have four capillaries.
With the RapidID system, it's very simple. Obviously there's no pipetting
required. The seek studio has easy with one-click universal cartridge.
The user level of experience for RapidID system is a technician or non-
technical operator.
Forensic lab experience is not required for the RapidID system.
For the seek studio trained forensic scientists, all technicians can easily
operate that system.
The runtime is 90 minutes from sample to answer on the RapidID, whereas the
seek studio is 39 minutes, which is the CE run.
But you also have your upstream processing time, that is DNA extraction, quant
ification and SDR amplification.
If you have sample type, the casework, you have the rapid intel cartridge,
whereas for the seek studio, for casework you have purified DNA.
For the database you can have a reference or rapid ACE cartridge. The database
reference, you can have a swab treated or untreated paper for the seek studio.
And testing environments for the RapidID system, you can have the forensic
laboratory, satellite laboratory, a mobile CSI unit, or police booking station.
For a seek studio, you can have obviously a full-service forensic science
laboratory, or you can have a smaller satellite laboratory, since it does not
require some space.
So in conclusion, the question which is the right tool for the job as a sample
answer.
Both the seek studio and RapidID as separate units can do the job they intended
to perform.
The seek studio is a small alternative to the 3500, where the RapidID for user
crime scenes and booking stations.
However, together they do complement each other, increasing efficiency of the
current laboratory, allowing the setup of a smaller satellite laboratory.
They are both the right tool for the right job of processing DNA samples.
So I just wish to thank you and thank Thermo Fisher, and we'll turn this back
over to Michelle.
Thank you, Robert, for that great information and insight.
Audience, it is now time for the Q&A portion of our webinar.
If you have not already, please take just a moment here to ask Robert and Jamie
a question using the Q&A dialog box on your screen.
So Robert, let's start with you.
What demo opportunities are available to try out these instruments?
So for basically FIU and FSC and FIU has a relationship with Thermo Fisher
scientific that we are there, center of excellence for rapid DNA testing.
So what we have in our facility is we actually have two RapidID units where we
can have people come in and see demonstrations of the units.
They can actually run the units themselves.
We have reagents available for testing.
Especially in this time right now where we are having some issues with COVID
and social distancing and so forth, we also do virtual demonstrations where we
assume we've already done five of those internationally for international
laboratories who are wanting to see the units.
And then also we have done some for local agencies also.
Some samples if you want, we have samples here that you can actually prepare
yourself and run and that way you get to actually get some hands on time with
the instrument.
We aren't selling it. We are not going to be able to discuss price, so that you
're going to have to go to Thermo Fisher scientific.
So really we're just a nice environment for people to come get some hands on
time with the instrument and get a feel for it and see how best they think they
can incorporate it into their laboratory.
That sounds awesome. How different is data analysis with the Rapid hit compared
to traditional CE?
Well the data looks the same, so you get especially since the RapidID is using
Global Father Express, so you are going to see your Global Father data.
I have used the 3500 M6 studio and switching over to the RapidID. There's
really no learning curve involved and looking at the data.
You can see all your PKI balances, your LEO calls, your PKI, basically
everything is the same.
So there really is no learning curve involved and the data is comparable.
Gotcha, that sounds easy. So Robert, tell us in your response to COVID-19, have
you had extreme difficulty making your demos virtual or what was the process
like to go ahead and get that back on for people that aren't able to get to the
lab and such?
No, the demo has actually been quite easy. We do them over the Zoom format and
we have cameras set up, so we are actually able to run a sample live and then
take a sample off so that everybody can actually see the beginning and end
process.
Then we move into the software and we basically go over the software with them,
show all the different features of the software.
Since it's all live, you can take questions live or we take questions after. We
can talk about our experiences with the software or any of the testing that we
have done here.
And show them the primary cartridge, the sample cartridge, basically anything
they need. So we haven't really had any issues with transitioning to the Zoom
and it's actually able to help us reach a broader audience.
Oh, that's great. Sometimes in the adaptation to the pandemic, we've definitely
found ways to get in touch with each other. So that's really great.
Jamie, let's turn our attention to you quickly. The tech studio had fewer
spectral artifacts.
Could I contribute that to the inclusion of marker-to-marker correction?
Yes, so that is part of the reason. The second level of pull-up reduction
offered with the SEEC Studio for Applied Biosystems Kit is that marker-to-mark
er correction factor that you enable by selecting the AB kit that you're running
when you're setting up the SEEC Studio.
But in addition, because the optics are different on the SEEC Studio and I
talked about instrument-specific pull-up on the 3500, which has to do with the
way the fluorescently labeled molecules move across the detection cell, you don
't see that due to the optical design on the SEEC Studio.
So you don't see that type of instrument-specific pull-up, which also takes
away a portion of a pull-up on the SEEC Studio that you see on the 3500, just
by the nature of the optical design.
Gotcha. Okay. So what is the benefit of using sample-specific spectral versus
using the generic spectral?
I'll take it that one for me too. So using the sample-specific spectral, so
that is how the pull-up reduction algorithms are working.
The benefit is that you're doing more of an apples-to-apples comparison when
you're using a matrix to deconvolute the dye data from your sample.
So when you use a manual calibration or the traditional spectral calibration
that we set up on the 3500, remember those dyes are pure dyes.
They're not attached to any fragments. So it's more of an orange-to-apples
correction.
And when you use sample-specific data, you're actually getting a matrix based
on dyes that are connected to fragments of DNA, just like your sample. So that
's where that more apples-to-apples deconvolution comes in.
Gotcha. Okay. Robert, let's get you back in the fold. Do you have any data
testing touch DNA samples on the rapid hit?
So we haven't really done a full study on the rapid and touch DNA. I mean, from
what I showed you, yes, we planted as low as one microliter of saliva on to a
swab and we were able to get a result.
We have done stuff paired with basically cell phones, but it's just been like
maybe just one cell phone, one firearm.
We've done water bottles, cigarette butts, and we've gotten good results.
Basically, we've gotten results that we're able to definitely eliminate someone
and even go so far to make the inclusion with it.
We do have plans to do larger studies to accomplish a lot more scenarios.
So that's also something that, you know, from the community, if you ever have
any ideas, if there's any need that you think that you would like to use the
rapid ID for, and you can send those directly to me.
When we design a study, we can try our best to include your suggestions so that
when we do produce something, it'll be something that actually the community
wants to see, and not just something that we sat down hand thought would be
best.
We're always looking for suggestions on what we can do to make our studies
better.
That sounds great.
Are Jamie over to you? How does the C-Studio do with difficult natures?
Nixtures.
Did you get the same results compared to 3,500?
I will answer from the C-Studio developmental validation that we did.
We did look at some nixtures.
We focused on two person nixtures up to one to seven and seven to one mixture
ratios, and the performance was similar between the 3,500 and the C-Studio.
I'm not sure if Robert had any more experience in the work he did, but...
We only did for right now just a two person mixture down to the one to 16, and
we got comparable results from the 3,500 and the C-Studio.
They performed the same.
We could do that later on doing more complicated mixtures, three and four
person mixtures, but we just had a two person and the lowest proportion we did
was a one to 16.
Okay, great.
Now you guys can both answer this question based on the instruments that you
talked about.
So Robert, we'll start with you.
What is the performance of Rapid Hit in analyzing samples exposed to extreme
environments?
So we've done limited testing on that.
I don't know if we've done anything with extreme environments to be honest with
you.
That is once again something that we are going to be doing more testing on,
because we do have those questions.
I know we've done some teeth, but they were from bodies that were left in the
ocean, and they didn't perform well, whereas teeth that were dried, and even
for 30 years, were stored in the dried environment.
We actually got results on, but just respect to extreme heat or mold or
bacteria, or we have not really done a lot of those studies yet.
It's still studies and progress, what we do here.
So like I said, once again, anything, there's any environment, or any
conditions that people specifically want to know about, please let us know so
that when we do plan these studies, we can incorporate them into our testing.
And Jamie, what about you?
What was the performance of CQEO like in analyzing samples exposed to extreme
environments?
So we actually just released an application note as it relates to bone testing,
and in that application note, we talk about bones that have been exposed to
some harsh environments, some formic acid.
There are some DVI cases in that case study, and that's focused on the rapid
hit ID with our Intel cartridges, and performance for some of those lower
quality bone samples that were exposed to harsh conditions.
Wasn't as great as the better quality samples, where we may only have seen a
partial profile, or just a few peaks to no peaks.
We also put out a poster that ran some of the same bone samples on the SEK
Studio using a PrEPFILER BPA extraction, and the bone performed, those low
quality bone samples exposed to harsh conditions for that subset of data.
I think there were 18 different bones. Did fare better on the SEK Studio in the
traditional CE workflow than in the rapid workflow?
I'm not sure if we have the contact information of the person who asked that
question, but I'd be happy to send both the application note and the poster to
them so that they could have a closer look at the data.
Great. Thank you, Jamie.
Yeah, we audience, don't worry. We do have your info from when you registered,
so I think we could definitely get that info to you, which is very kind.
All right. Next one, Jamie.
You mentioned the J6T die set. Our audience member, David, saw that it has the
TED die instead of the Ned die from the earlier J6 die set.
What was it? Was this just an upgrade, or does each die set apply to different
STR kits?
Yeah, so it is a die change. The TED die is slightly quieter die. It'll still
fluoresce in the yellow die channel.
We have certain kits that are designed using the J6T dies in particular. That
is our Verifiler Plus Kit.
One of our kits that is used solely with our Chinese customers, Global Filer,
Global Filer IQC, those are both J6, our original J6, and MGM the TED is a J6T
die set also.
That's used mostly by our European customers.
Robert, we have a few questions about RapidHit coming up for you. So tell us,
does the RapidHit ID automatically check the hit in the DNA database? If you have the application, so with the RapidID and the RapidLink software, if
it's in the RapidLink software, it automatically important to file something
into the software, and then you have the different applications.
The matching software, you actually have to ask it, tell it to make a match.
For the familial, you can do a familial search where it will search everything
that's in the RapidLink for familial match.
For the kinship, you have to put the stated relationship of what you are
checking to make sure that that relationship is what it's supported to be.
The only thing it automatically searches is the staff elimination database. So
once you set up your staff elimination database and you have those people in
there, anytime you run a sample, it will automatically tell you if that person
profile, if the profile develop, ends up matching someone in the staff
elimination database.
Okay, while we're getting close to time here, so Jamie, I'm going to ask you
the last question. Do you advise having seek studio for lab with many samples?
So the seek studio is a four-cap instrument, so a lab would definitely want to
consider their throughput when deciding what CE option would be best for them,
whether they're a high throughput lab and maybe a 3500 Excel with 24 caps might
be the right answer, or if they could use the seek studio with the four caps
can meet their throughput needs.
Of course, compared to the rapid hit ID, you're talking about four capillaries
versus a single capillary, but then again, you may have some more upfront steps
to do before running those four samples on the CE.
So it all depends on the overall workflow, and then looking at the labs
throughput and needs, I think, when you're making that decision.
All right, audience, that about wraps up all the time we have today. I'd like
to thank Thermo Fisher Scientific for sponsoring this webinar, our speakers,
Robert and Jamie, and of course you, the audience, for your attendance and
participation.
In 48 hours, this webinar will be available on demand if you would like to
watch it again, or share it with friends and colleagues.
Additionally, you will receive an email with information on how to obtain CE
credit documentation for your participation today.
The fourth webinar in this six part Future Trends in Forensic DNA Technology
series will be held on August 20th at 8am Pacific 11am Eastern.
You can register for tips, tricks and best practices for gaining efficiency in
your forensic DNA laboratory on the forensic website, www.forensicmag.com,
where you can also view other webinars in the series on demand.
Thank you and have a wonderful day.