Improving Sample Prep Efficiency and Data Quality of Challenging DNA Samples
The purification of DNA from forensic samples is usually a time-consuming process, especially for challenging forensic samples. Dr. Sheree Hughes—along with her graduate students Natalia Czado, Kayli Carrillo and Jennifer Snedeker—will present results from a recent study that compares manual DNA purification methods with recently developed automated processes for compromised samples. The studies demonstrated that the newly developed automated method (Applied Biosystems HID NIMBUS Presto), resulted not only in faster turnaround time from purification to results, but also high-quality DNA recovery and streamlined interpretation from multiple challenging forensic sample types. In this webinar, you will be presented with: •Examples of challenging forensic samples such as highly degraded bone, hair, nail and tooth samples from human remains and “touch” evidence and fired cartridge cases purified using the automated system •Data demonstrating high-quality DNA results •The benefits of using an efficient automated DNA purification workflow for challenging samples
Hello and welcome to Improving Sample Prep Efficiency and Data Quality of Chall
enging DNA Samples,
Brought to you by Forensic and Sponsored by Thermo Fisher Scientific.
My name is Michelle Taylor, Editor-in-Chief of Forensic and I would be your
moderator throughout.
For today's webinar, you will 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 lineup scheduled to present to you today, but
before we begin,
I'd like to take just a moment to cover a few logistics. At the end of the
presentation,
we will hold a question and answer session. To ask a question, click on the "
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with your friends
and colleagues. Today, you will hear from Dr. Shari Hughes, Associate Professor
of Forensic
Science at Sam Houston State University in Texas, as well as a few of her
graduate students.
Dr. Hughes completed a PhD in Health Sciences/Frenzic Genetics at Bond
University on the Gold Coast in
Australia, investigating forensic DNA typing methods for highly degraded
samples, such as those
recovered from mass disasters, missing persons, and historical cases, in
conjunction with DNA
repair techniques and phenotypic SNP analysis. Natalia Chado is currently a
doctoral student at
Sam Houston State University. She earned her Master's in Forensic Science in
2015 and completed her
bachelor's degrees in Biological Sciences and Criminology at North Carolina
State University in 2013.
She was a lecturer in Forensic Science at Fayetteville State University from
2015 to 2020.
Natalia's research interests include DNA profiling of challenging and degraded
samples,
and she has authored/coauthored several papers in this area. She is hoping to
branch out into
genetic genealogy and has recently completed Boston University's Genealogical
Principles course.
You'll also hear from doctoral student Kaylee Carrillo. Kaylee's research
interests include
optimizing DNA collection, extraction, and enhancement methods for low-tem
plates samples.
Prior to attending Sam Houston, Kaylee received a Bachelor of Science in
Biology from the University
of North Texas. The last doctoral student you will hear from today is Jennifer
Snedeker.
She is interested in advancing forensic DNA and human identification methods by
optimizing
DNA extraction procedures from skeletal remains and examining novel
investigative methods for
these sample types. Her research is focused on highly degraded and challenging
skeletal samples
that closely mimic those seen in forensic case work. Prior to beginning her PhD
research,
Jennifer received a Bachelor of Science in Human Biology from Michigan State
University.
Thank you for joining us for today's webinar. I keep your eyes open for an
invitation to our
next one. Now, without further ado, I'll hand it over to Dr Hughes to get us
started.
Thank you very much for the opportunity to speak today.
So first of all, our quick disclaimer is to say that the data and the opinions
that we are going
to present today are that of our own and not of of them are fish or scientific.
Also, I'd like to
just claim that a lot of the instrumentation and kits are provided by them are
fish today.
However, there was no financial enumeration at all. And last but not least was
also because of
some of the sensitive nature and imagery in this presentation. We also request
that there is no
screenshots or pictures taken of any of those images throughout this
presentation. Thank you.
So of course, this work would not be possible without my fantastic team. And I
am three of my
PhD students that I have presenting today on each of their representative areas
, Natalia,
Kaylee and Jennifer. And you'll hear from those throughout this presentation.
So overall, the objective of the work that we are presenting today was really
looking at how we
can examine the utility of the HID Nimbus Presto system for its effective and
efficient DNA
purification from really difficult and challenging sample types that you may
find in a lot of
casework. So these were include five and unfired cartridge casings and DNA
recovered from those
touched items. Also, other touched items that may be commonly retrieved from
casework and crime
scenes such as water bottles and computers and phones, etc. And then also a bit
of a plethora
of challenging samples from decomposing cadavers. So we're going to look at
recovery from
hair, nails and teeth, as well as some bones from skeletons that have been
environmentally
challenged in different ways. So a little bit about the Nimbus Presto system.
So starting with
the Kingfish Presto purification system, and this is working on an automated
magnetic
powder-cooled base purification system. As you can see, working behind you
there,
it's really efficient and effective. And it's quite a swanky little thing that
works well.
It actually has a really neat magnetic rod system that has these these tip com
bs
that work for the purification. And this nifty little turntable system that
allows for sample
handling and being able to load your samples and move them around the deck.
And of course that system is then coupled with a liquid handling system. And
the way we've done
it in our lab is with the Hamilton micro lab Nimbus HD. And it can process, as
you expect,
up to 96 samples and does that in a very, very effective way in combination
with the Kingfish
Presto. And it's extremely adaptable. It's an adaptable system to into whatever
platforms that you would
like in the formats that work for you. And we've found it to be working very
seamless and efficient
in our lab once it was up and running. And the scripts that we have on this
system that we're
going to talk about today are able to use both the PrEPFiler and the PrEPFiler
BTA chemistry for
our sample types. And again, these samples are lysed manually before we put
them on the machine.
So we have the lysus step performed first, and then we put it onto the system
for all of the
purification after that. So we really does allow for this high throughput
capability,
as you would want in a current in a crime working lab, or even in a very high
throughput research
lab. And it's a very flexible format. You can do anywhere from four to eight
samples all the way
up to 96 on a deck without very little time difference. So it does reduce the
analyst's
hands-on time quite significantly. And if you're looking at batches of, say, 12
samples is what
you may be looking at, similar to automated, say, on the Automate Express. It's
essentially 40 minutes
versus almost two hours if you're doing it even manually by hand, which we will
be drawing a
couple of comparisons and conclusions on throughout this presentation. Another
really handy tool that
we find is extremely useful in the lab, particularly even just starting with a
lot of students getting
familiar with the instrumentation, the software, how to have it working
properly. The GUI system
really does make this a piece of cake. It's really quick and easy to follow. It
's quite intuitive.
It enables you to custom the adaptability of what you want to use for your user
preferences
in terms of plastics and workware. It eliminates the risk of a lot of setup
error,
as well as obviously handling contamination. Being able to set up with this GUI
system allows you
to make sure that you've got all the chemistry, all of the chemistry, all the
consumables and all the plastics all in the right place doing the right thing.
It has a sample and
reagent tracking capabilities. If that's something that's really important for
your lab
and working in with your limb system, etc. Also, a lot of guidance for setup.
In essence,
it prevents anything from going wrong. We found that extremely useful and very
advantageous when
it comes to the good handling side of things. As you can see, you can just
choose,
allows you to walk you through the deck setup. It's very, very easy. It's got
this swanky little
color system and it allows you to set up the deck efficiently, effectively and
correctly every time.
The two chemistries that we are talking about and that we're looking today and
we're looking at is
our prep-filer BTA for all of our bone samples and some of our other casing
samples as well,
and also prep-filer for our stock standard, your blood and swabs, etc. Both of
these
chemistries for these various types of samples. The scripts are able to very
effectively work
your solutions from start to finish on the Kingfisher Presto and limb systems.
For DNA quantification, after our extracts, with our illusions, run through our
quantifiler trio quantification kit. Most of you will be familiar with all of
the targets that are
used. We have our two human targets, our small and large, which of course
allows for the degradation
index to be able to give you some sort of indication of your DNA quality. So,
DI's 1 to 10 is indicative
of some slight to moderate degradation, whereas our DI value is greater than 10
would definitely tells you that there's a significant degradation there.
Sometimes you would get this undetermined, you're unable to determine your DI.
Well, of course,
extreme degradation and or potentially some significant inhibition there as
well.
But in terms of other targets, you also have your male target, of course, and
your IPC,
which allows you to obviously detect PCR inhibition when your delta IPC from
your controls, of course,
is greater than one cycle. In terms of PCR implications, so our STI kit we're
using for this work
is the Verifyla Plus PCR implication system. And this is great system for
really low template
and challenging samples, which we are going to cover today, is the ability to
increase your input
volume, so up to 17 and a half microliters. So this really does allow some
increased sensitivity
and being able to put as much of that really low template extract into PCR into
the PCR reaction.
We have our 23 autosomal markers, as you would expect in these in these new
kits.
We have a Y in Dell and of course a melagenin. And also we have our now two
internal quality
control markers. We have one short at around 70 base pairs and then the longer
at 451.
And of course, these are used to help basically confirm the success of your PCR
and also
confirm some inhibition and degradation within your sample. So as a quick
reminder of how these
internal quality control markers work, this is essentially what they look like.
We've got a nice
beautiful sample here. Our control markers are nice and balanced. All of our
peaks look good.
We've got a really nice nice sample. When we have a sample with a heart with
some significant
amount of PCR inhibitors involved, you can see that we have now our large
quality control marker
has now dropped out. So this would be either very small or dropped out and
confirming that
we've got some significant inhibition here as opposed to degradation where our
markers are
nice and reasonably balanced. And however, we do see that significant ski slope
and decrease in
our peak heights, particularly and drop out of our larger markers, as you'd
expect. So of course,
these markers are extremely useful for being able to get a snapshot and
confirmation of the
quality of your sample and your PCR reaction, how it's worked. And of course,
then your PCR
profile in front of you. In terms of our analysis, here are the students in
their room
heart at it. So of course, with the Verifala Plus, just very quickly, we
targeted a 500 picogram
DNA amount for our PCR. And only throughout all of these studies, only samples
that yielded some
detectable DNA, so one or greater than one picogram per microliter were
continued on for STR typing.
All of these samples were run and were separated and detected using with the
fragment analysis on
3500. And our data analysis was performed with gene macro IDX version 1.6, with
our analytical
threshold being 100 RFUs and our ST being 700 RFUs. So overall, the workflow
for this study that
we're presenting today is looking at a combination of difficult samples. So
here are our brass casings
that were collected with using various methods, which Natalia is going to talk
about. We then have
some swab touch items in some challenging samples from decomposed cadavers that
Kaylee is going to
discuss. And then lastly, with our skeletal remains, we have some buried
surface burials,
some different types of bones, et cetera, from different out from different
environmental
insults that Jennifer is going to talk about. All of these samples were then
extra DNA extraction and purification was performed on the nimbis presto.
HIV nimbis presto and then run through DNA quantification with quantifier trio
as we as I explained,
and also STR typing with performed with varicyla plus. We also ran some
comparisons by performing
all of these samples were ran concurrently with using Prpfyla or Prpfyla BTA as
determined by
sample type and these were performed manually as well. So we've got comparison
of on the automated platform versus manually performed in the lab and we're
going to present
those results too. So I'm now going to hand over to Natalia to run you through
all of the
results from the brass casings. Thanks Natalia over to you. Thank you Dr.
Hughes.
As many of you know firearms and ammunition are some of the most common types
of evidence submitted
to crime laboratories. DNA can be deposited on the surfaces of guns and
cartridges during the
handling and firing of a weapon. Using this DNA to generate profiles can help
investigators
identify suspects and persons of interest in violent crimes. However, obtaining
DNA profiles
from these samples has historically been difficult. The difficulties stem from
several factors.
First, these types of samples normally have low amounts of DNA deposited on the
surfaces.
They are most often thought of as touch or low template samples. In addition to
this,
any DNA present could exist as a mixture further complicating DNA profile
interpretation.
Lastly, the metal composition of the weapons and ammo plays a role in DNA
recovery and
genotyping. Metal ions combined at several different sites on the DNA template.
These
strong bonds form metal ion complexes that can make DNA recovery from the metal
surface more
difficult. So not only do analysts have to consider how best to recover low
amounts of template,
they must also consider whether their method will adequately release the DNA
from the surface.
Also, these complexes can break one or both strands of the DNA template
depending on the binding site.
Although many types of metals interact with DNA, obtaining DNA profiles from
ammunition with
copper or brass surfaces is most challenging. This is due to copper's ability
to bind up
more sites on the DNA and produce reactive oxygen species that lead to
oxidative damage of the
DNA template. Damage and degradation of the DNA is detrimental to our size-
based detection methods,
and the presence of metal ions in PCR can inhibit the polymerase. So to
summarize this,
we have an issue collecting enough DNA. The DNA we collect is often fragmented
and bound to ions,
and the ions are inhibiting the amplification of the low amounts of good
quality DNA template,
not really a recipe for success. For these reasons, there has been a lot of
research over
the years in how best to collect low template DNA, especially from the surface
of ammunition.
One of the most popular DNA collection methods is double swabbing, where the
first swab is moistened
with sterile water, and a subsequent dry swab is used to collect any remaining
genetic material.
Although this method is inexpensive and works for a variety of surfaces,
there has been mixed success reported for collecting DNA from ammunition.
To improve DNA recovery, soaking has been tested. There have been several
different
protocols proposed. The one developed by San Diego PD's Forensic Biology Lab is
probably the most
well-known. However, increased success was only minimal using this collection
method.
Both swabbing and soaking prior to DNA lysis and purification do have the
inherent risk of losing
precious amounts of DNA. Therefore, direct PCR has been examined. By bypassing
extraction
and adding the swab directly into a PCR reaction, about 60 to 70 percent more
DNA could be amplified.
Although direct PCR works great for other low template samples, success has
been inconsistent for
DNA recovered from metallic surfaces and ammunition. And this is probably due
to the continued presence
of metal ions in PCR without the purification step. Because of the impact that
metal ions are thought
to have on downstream genotyping, the use of metal ion chelators has been
examined. One study
examined moistening a swab with .5 molar EDTA rather than sterile water. The
researchers reported
that less oxidation took place and enough good quality DNA template was
recovered to successfully
sequence mitochondrial DNA. More recently, a rinse swab protocol using a
solution comprising bovine
serum albumin and glycine, glycine, histidine, tripeptides. Both great metal
ion chelators
has been proposed. The solution is referred to as BT mix and has been
moderately more successful
than previous collection methods, but is time consuming and does not
consistently generate
complete STR profiles. Currently, no method has consistently recovered enough
good quality
DNA to generate full profiles. Collecting the small amounts of DNA deposited is
the most important
consideration for these types of samples, therefore more research in
determining a more
effective collection method is warranted. In a recent study completed at Sam
Houston State University,
we determined that automated extraction methods based on magnetic beads could
adequately remove
inhibitory metal ions. Using prepphiler BTA chemistry, we can still obtain full
DNA profiles
in the presence of .1 molar copper and .03 molar zinc, which are concentrations
much higher than
what would be expected from the surface of ammunition. Because of this, we
wanted to examine whether
including prepphiler chemistry during the DNA collection stage and pairing it
with extraction
on the HID nimbus presto system could increase both the amount of DNA we
collect and the number of
samples we can process in a short period of time. For this portion of study, we
sterilized 36 cartridges.
Through some miracle, we were able to get enough rounds of the same type of
ammunition in the middle
of an ammunition shortage. We selected a brand of brass cartridges since these
are the most problematic
for DNA profiling. We made buckle cell suspensions from a single donor and
spotted the equivalent
of 10 nanograms of DNA onto a marked location on the cartridge. All of our
samples were fired by
a male law enforcement officer using a Glock 19. We took 12 of the 36 casings
and used them to test
the prepphiler lysis buffers as moistening agents. So six were moistened with
prepphiler BTA lysis
buffer and continued on to the BTA extraction protocol on the HID nimbus presto
system.
The other six underwent the same process using the standard prepphiler lysis
buffer.
The other 24 casings went on to comparison testing. 12 were moistened with
water and then
divided between prepphiler and prepphiler BTA extraction on the nimbus. The
other 12 underwent
the BT mix rinse swab protocol followed by each type of prepphiler extraction.
Because the BT mix protocol requires the use of two swabs, they had to be
placed into separate
extraction tubes. And after extraction on the nimbus, LU-wits from the same cas
ings were pooled
and concentrated to a final volume of 50 microliters. All samples were then
quantified with
quantified lertrillo with the addition of BSA. I want to mention that a similar
workflow was
repeated for the samples that underwent manual extraction.
We compared the DNA quant data for all of the automated samples examining DNA
yield,
inhibition, and degradation. We did not observe any inhibition,
but all samples exhibited some level of degradation. DNA was only recovered for
22 of the 36 samples,
meaning that 14 of the casings resulted in zero or undetermined amounts of DNA,
which is not unexpected for fired brass casings. Extremely low amounts of DNA
were recovered,
with 134 picograms being the highest total amount recovered. The combination of
BT mix
rinse swab collection paired with prepphiler extraction was the most successful
When we compared manual extraction to automated, there was no trend observed
for DNA recovery.
For most samples, automated extraction was able to recover more amplifiable DNA
However, any differences were not statistically significant.
We then examined genotyping success. Again, only samples that recovered at
least one
picogram per microliter of DNA were amplified. For casings, this means we only
had three samples
for both manual and automated extraction move forward to DNA amplification.
None of the samples generated a complete profile with allele recovery ranging
between
two and 17% for our automated samples. Although samples for manual extraction
were amplified,
all three exhibited a mixture of an unknown contributor. We have not yet
completed mixture
analysis, so these results are not listed here. I think it's important to note
that none of the
automated samples exhibited mixtures. This example highlights that processing
samples in an automated
workflow decreases the risk of contamination. So we recommend using an
automated platform for
DNA extraction from low template samples. That wraps up the results for the cas
ings portion of
this study, so now I'll be handing it over to Kaylee to discuss the next
section.
Thank you, Natalia. As mentioned, the next sample type we will be discussing is
touched items.
Touch DNA is often referred to DNA that is transferred to an object or another
person during
physical contact, which is often encountered during property-related crimes.
Touch items present
their own unique set of challenges due to the limited amounts of DNA present.
With low amounts
of DNA, there can be stochastic effects such as allelic peak height imbalance
or allelic or locus
drop-in or drop-out. That can further complicate interpretation. As you can see
in the photo,
touch DNA is typically composed of cell-free DNA, endogenous or exogenous nucle
ated cells,
fragment-associated residual DNA, which is the result of apoptosis or cell
death,
and a nucleic corneocytes. The outermost layer of your skin consists of keratin
ocytes,
also known as corneocytes, that have undergone keratinization, so it is thought
that corneocytes
do not have a lot of nuclear DNA, and therefore do not constitute a significant
source for touch DNA.
However, DNA deposited on touched items is highly variable and not easy to
predict due to the
wide variety of factors that can affect touch DNA yield, such as the skin
shedding status of an
individual. An individual could have sweaty or dry hands, or the frequency of
hand washing can
all lead into this idea of a good versus bad shutter. Another factor to take
into account
is the type of contact. Does this person use a little, or a lot of pressure
when touching the item?
Also, the substrate surface can affect DNA yields, as DNA yield tends to be
greater with porous or
rough surfaces. All these combined factors impact DNA transfer, persistence,
prevalence,
and recovery, or TPPR. In order to help recover as much as possible from these
touch samples,
there has been an improvement on the increased sensitivity of PCR kits, such as
global filer
IQC and verifiler-plus. In this project, two swabs were moistened with DI water
, and were used to
sample cell phones, keyboards, and bottles from six individuals. One swab was
processed using the
HID Nimbus Presto and the other swab underwent manual extraction. Both methods
followed the PrEP
filer kit protocol. Now for our results, we can see that most DNA was recovered
from our keyboards
and the lowest from our cell phones. But all of our samples had at least .0067
nanograms per
microliter amounts of DNA recovered. And degradation was not an issue for our
samples,
as we did not see a scree slope effect in our STR profiles, and our degradation
index only
ranged from 1.36 to 3.46. Now if we take a closer look, we can see 13 out of 18
samples
indicated mixtures. As you can see in this electrophoreogram of the cell phone,
of the clear appearance of
mixtures. And mixtures in our samples is not unexpected because just think of
how many times you
either shake hands with a co-worker or touch a doorknob, and just how much you
touch your cell phone in
general, and all that transfer of DNA that is possible. Even though there were
mixtures, we did
see 100% a little recovery from the major contributor or the owner of the item.
So right now we are
currently running through a mixture interpretation software for the minor
contributor.
Now when we compare the HID Nimbus Presto and manual extraction, we can see
that the touched
samples extracted with the HID Nimbus Presto generated extracts with
significantly higher DNA yields.
Next we will be discussing challenging samples from decomposed cadavers.
For this portion of the project, two samples were collected from each cadaver,
two hair, nails, and teeth, from six cadavers that were placed on the surface
for up to three months.
But these challenging samples come with its own unique set of challenges. While
hair is a commonly
encountered evidence type, it may be limited in its DNA quantity and quality,
especially if the
root is not present. The keratin that is present in both hair and nails is
known for degrading and
inhibiting DNA as well. And for teeth while it is more resistant to degradation
, it is still
subject to environmental contamination that can negatively impact DNA yield.
Additionally,
the calcium and enamel present in teeth can also act as PCR inhibitors.
To prepare these samples, hair was cut three to five millimeters and if the
root was present,
was included. Hair was washed with a turgazim detergent in order to wash away
any exogenous DNA.
Then with water and following with ethanol. Hair was extracted using the PrEP
filer protocol.
Our nail samples were not simply just nail clippings, but the entire nail bed
itself,
from either the thumbs or the big toe. We received both a left and right nail
for each cadaver.
And as you can see in the photo, we cut each nail in half and from that half of
five millimeter cut
was made so that we could proceed with the HID Nimbus Presto workflow or manual
extraction.
Similarly to hair, nails were extracted using the PrEP filer protocol.
Finally, for our teeth samples, premolars and molars were washed with 10%
bleach for three seconds,
then water and ethanol. Teeth were wrapped in a kim wipe and pulverized with a
mallet before
being powdered with a freezer mill. Teeth samples were then extracted using the
PrEP filer BTA protocol
for both manual and automated extraction.
Now if we look at our results for the challenging samples, our highest amount
of DNA was recovered
from our nails. This can be explained because like I previously mentioned, we
were using our
nail beds and not necessarily just nail clippings. Nail beds have adhering soft
tissue, which can
explain the higher amounts of DNA available. But in our samples, there is no
inhibition detected,
but DNA degradation was observed in our nail and teeth samples.
And while we know that these are challenging samples, we were still able to
achieve over 80%
allele recovery from our hair, nails, and teeth. And here is a picture of an
electrophiragram of
one of our nail samples with 100% allele recovery. And we can see that there
are good peak heights
and a balanced profile throughout this sample. When we look at the hair samples
extracted with
the HID Nimbus Presto versus manual extraction, the HID Nimbus Presto generated
statistically,
significantly higher DNA yields in comparison to manual extraction.
Additionally, more hair samples
met our one-peak gram per microliter threshold to proceed with STR analysis.
Similarly, for
nails, the HID Nimbus Presto also yielded significantly more DNA yields than
manual extraction. And also
yielded more samples that met our threshold. For our teeth samples, there was
no statistically
significant difference between DNA yields between the HID Nimbus Presto and
manual extraction.
All of our manual extracts and 10 out of 12 samples using the HID Nimbus Presto
met this
threshold. But in summary, you can see that the HID Nimbus Presto either
performed similarly
or significantly better than manual extraction.
Now, on to Jen, who will discuss the much anticipated bone results.
Thank you, Kaylee. As she mentioned, the last sample type we'll be discussing
throughout this
webinar are our skeletal samples. These samples are very important because they
are often the only
remaining pieces of evidence in several situations, including cold cases,
missing persons, or mass
disaster victim identification. These samples are unique in that the reason
they are often the last
source of evidence is the same reason they can be so challenging to process.
The inherent nature
of these samples allows for that DNA to be protected within the TUF hydroxyapat
ite matrix of
the bone. But this requires a very complicated sample preparation process to
actually access
that DNA. Not only that, but these samples are also often highly inhibited from
either
endogenous sources like calcium or collagen or exogenous contaminants like hum
ic acid found in
the soil. Additionally, these samples are usually highly degraded and naturally
contain low levels
of DNA. Therefore, we wanted to investigate the success of the HID Nimbus Prest
o system
for DNA extraction from these challenging sample types. We collected our
samples from
Staphs, the Southeast Texas Applied Forensic Science Facility, which is a world
-bodied donor
program at San Houston State University. Individuals or family members can
choose to donate their
bodies or their loved ones to this facility for research or education purposes.
I'm lucky enough
to work out at Staphs and utilize it for my research. We're very thankful for
our donors,
and this is what I would like to remind you all to please refrain from taking
any photos
of the slides that contain cadaver images. With that being said, this project
we collected from
three different insult categories, surface decompose remains, burned remains,
and buried remains.
Six cadavers were collected for each insult group and five skeletal elements or
bones were
collected from each cadaver. Like I mentioned previously, the sample
preparation for skeletal
samples can be very tedious and time-consuming, but it is doable. First, a
window cut needs to be
taken from our larger skeletal elements like this femur you see here. This is
done using a
Dremel Power tool. Smaller samples skip this window cut step and instead the
entire sample
is processed as a whole. Here you see this window cut taken away, and once we
have that window cut
or the entire sample that will be processed, we sand down the outside surface
in order to
remove any exogenous DNA to limit contamination moving forward. This is again
done using a Dremel
Power tool, but with a sanding bit attachment. At this point, we're ready to
chip our bone into
smaller pieces to make our powdering process easier. In these chips go through
a series of wash steps
to again limit contamination. At this point, we're ready to begin our powdering
. At SAM, we use a
freezer mill, and this works by pouring our chips into the vial you see on the
left. In that vial,
there's a magnetic rod. So you place this vial in the freezer mill, which is
located in the center
image, in this freezer mill spill with liquid nitrogen. When we turn it on,
that magnetic rod
is going to move back and forth very, very quickly, pulverizing the chips into
a fine powder like you
see here. At this point, we're ready to begin our extraction process. You can
see there's a lot of
hands-on time that occurs up front during the sample preparation process, and
that's why we
were interested in looking at the HID Nimbus Presto system for DNA extraction
from these samples to
limit hands-on time at a later step. The first insult type we'll be discussing
for this project
are our surface decomposed remains. At staffs, upon intake, the cadavers are
placed on the surface
under cages for extended periods of time. The samples collected for this
project were on the
surface from anywhere between half a year to about two years. While in the
field, these cadavers are
exposed to a variety of environmental insults, including sun bleaching or UV
damage from that hot
Texas sun. Additionally, these samples can interact with the soil leading to
potential inhibition,
as well as interactions with different microbial communities or even scavengers
if they're able to
make it under the cage. They're also exposed to moisture from rain or humidity,
and they have
that extreme Texas heat impacting them. So anything that the natural
environment goes through, these
samples go through, which is going to lead to a very challenging sample type.
For this project,
we collected three long bones, the humerus, femur, and tibia, in two foot bones
or tarsals,
the medial cuneiform, and the first metatarsal. For each cadaver, we collected
either the left
or the right side, but not both. Our burn samples are our next type of insult,
and these were processed
by individually burning each element separately, as you see in that top image
here. So the skeletal
elements were placed on a makeshift grill of wood, charcoal, and chicken wire
to hold the bone,
and the bones were burned until they reached a black or brown color. At this
point, we see that
there's DNA damage and degradation induced on these samples. For our burn
samples, we again
collected our three long bones, the humerus, femur, and tibia, and then two
other foot bones,
the cuboid, and then the first metatarsal. The last insult category is our
buried samples.
At staffs, cadavers are buried at various depths and for extended periods of
time
based on the research study. For this particular study, the cadavers were
buried for a length of
between just a few months to almost four years. We often find that these are
our most challenging
sample types as they are highly degraded and inhibited, likely due to the acid
ity of our soil
from our pine trees, as we are part of the piney woods and fences. Additionally
, these samples
often have adiposeure formation, which is commonly seen in buried samples where
there is a lot of
moisture. This is when the decomposing soft tissue forms a waxy fatty substance
that can further
complicate the DNA extraction process from these sample types. For our buried
samples,
we collected the humerus, femur, tibia, mediocre cuneiform, and first metatars
al.
So overall, for this project, a total of 30 samples were collected for each
insult type
and extracted in duplicate on the HID Nimbus Presto system using the PrEPFiler
BTA chemistry.
Here we are looking at the average DNA yield per insult, and you can see that
the burn samples
had the largest variation with some having relatively high yields, but on the
other hand,
our buried samples had a limited spread with a much lower recovery. From the
180 extracts,
133 of these proceeded forward for STR typing, meaning they met that one pic
ogram per microliter
input value. 57 of these were surface samples, 44 burned, and 32 buried.
When we compare our allele recovery versus DNA input, as expected, the more DNA
we're able to target,
the higher allele recovery we see. For our surface and burn samples here, we
see that if we could
target at least 0.2 nanograms of sample, we have an 80% allele recovery or
greater.
The only exception is that burn sample that you see has 0.5 input value, but
about a
allele recovery of 63%. And I want you to keep the sample in mind when I move
to the next slide.
When we look at our buried samples, we see that this trend isn't very
consistent,
and we have a much lower allele recovery regardless of our DNA input.
And much of this can be explained if we examine the degradation index of these
samples.
So the majority of our surface bones you see here have a DI between 1 and 5,
which means they're
slightly degraded, and a few samples pass that threshold of 10 DI, meaning that
they are severely
degraded. And that same trend is consistent with our burn samples with the
majority between 1 and 5,
and we see one sample has a DI around 15. So this is actually that sample I
mentioned on the previous
slide that had an input of 0.5 nanograms, but a lower allele recovery, and that
's due to this
high degradation index in the dropout of those larger low side. So this
highlights the some of
the challenges we see when processing these sample types. We have to look at
the sample as a whole
picture. So we have to look at the amount of template DNA if these samples are
degraded,
and then furthermore, we have to look at our inhibition of these samples. And
all of this
will predict our success in allele recovery. When we look at our burn or our
buried samples,
we see that we have much higher degradation indices indicating those lower
allele recoveries.
And this is not unexpected at this, as this is a common issue, we face with
these challenging
sample types at Staphs. So from here, we're going to compare our HID Nimbus
Presto extractions
to our manual extractions using the PrEPbiler BTA chemistry. And in this figure
here, we're
looking at our quant data. And we see that overall, there was no significant
difference in DNA yield
for our surface or burn samples. But when we compare our buried samples, we
found that this
sample process using the HID Nimbus Presto system had a statistically
significant increase in DNA
yield. However, regardless of the method used, these values were still very,
very low.
for these manual
When we compare which samples meet that one picogram per monthulator threshold
for PCR amplification,
we see that the Nimbus had more surface samples and buried samples proceed
forward.
This demonstrates that even if the DNA yield was not significantly better with
the Nimbus,
it resulted in a higher chance of success in continuing through the HID
pipeline.
Unfortunately, at this time, we are still working to complete our STR typing
extractions. So we were unable to compare our real recovery data.
However, we were able to process a subset of these samples using other bone
extraction methods
that we commonly use in our lab. This includes the automated express, which
utilizes the PrEPbiler
BTA chemistry, and our lab's total demon method, which is an adaptation of the
Laurier method.
And when we compare these four methods, we found that we had no statistically
significant
difference in DNA yield, regardless of which extraction method was used.
This means that the HID Nimbus Presto system performed on par with the other
DNA extraction
methods we implement in our lab. But outside of DNA recovery, there are some
other variables
that should be considered when choosing which DNA extraction method to use for
our skeletal samples.
For instance, using the HID Nimbus Presto system allows for 96 samples to be
processed at once,
compared to the only 13 that can be processed on the automated express or
comfortably processed
manually. Furthermore, utilizing the PrEPbiler BTA chemistry, whether that be
on the Nimbus Presto,
the automated express, or manually, only requires 50 milligrams of bone powder
versus the 250
milligrams used for our total demon method. And this is saving our precious
sample type.
And lastly, utilizing the partial demoneralization chemistry
of the PrEPbiler BTA kit saves time reducing the extraction process to hours
rather than days.
And if the HID Nimbus Presto is used, the hands-on time can be further reduced
as Dr Hughes is about
to summarize. Thank you, Jen. So now I'd like to now draw your attention to
really the workflow
comparison altogether. So if we start with the PrEPbiler BTA system, whether we
perform that
manually or on the Automate Express or the HID Nimbus Presto, if we look at a
batch of say 12 samples
to compare, it's a great place to start. So with a set of 12 samples manually,
there is really no
difference in the beginning steps. So from sample PrEPb going from bone bone
chips down to the
bone powder, it's exactly the same. Going through to the digestion or cell l
ysis step, it's the same.
Now moving that onto extraction and purification, performing that manually with
12 samples is that
step is a rough just shy of two hours. So when you look at PrEPbiler BTA
solution, chemistry being
performed manually, it's a total time of around about 16 hours and just over
two and a half hours
of hands-on time. When you compare that to the Automate Express and the HID Nim
bus Presto work
solutions with those 12 samples, there's a vast difference. Even though in
totality is about 14
hours, it's really the hands-on time down to less than half an hour or so,
really does allow the
analyst to free up their time to do other things. And even though the 12
samples on the Automate
Express and the HID Nimbus Presto is comparable in terms of time and hands-on
time, that's with 12
samples. The real neat thing with the HID Nimbus Presto, of course, being able
to increase the
throughput up to those 96 samples, the increase in time on the instrument is
actually quite negligible.
Another 10, 15 minutes and you've gone from 12 to 96 samples. So that is the
real advantage here
using this combined Kingfish Presto and the HID Nimbus system as a whole. The
same holds true with
PrEPbiler chemistry, slightly different. We have our, of course, moving through
from Cell Lysis is
the same again, down through the automated platform extraction and purification
. Those differences,
you can see, arranged from everything from an hour and a half performing it
manually, down to
about half an hour or 40 minutes on the automated systems. And again, exactly
the same advantages.
Over two and a half, like two hours or so for the hands-on time, with the PrEPb
iler kits,
performing it manually versus your half an hour on the Automate Express and
your HID Nimbus Presto.
But of course, remembering that you can increase that throughput very, very
easily with negligible
time. So it's a really fantastic solution for all of you DNA extraction and pur
ification samples
in the lab. So in summary, the HID Nimbus Presto system really did work really
well in our laboratory
with these types of really challenging samples. They performed, as we would
expect, with such
difficult samples. They were able to purify DNA from a variety of all of the
different difficult
samples that we encounter in our lab all the time. When we compared DNA yield,
the Nimbus Presto really
did perform, as we would expect, to see. And very similarly, if not better,
with manual extraction
methods that we use in our lab for those different types of samples. And
overall, the big advantage
is the HID Nimbus Presto system really does save a lot of analysts' time by
limiting those hands-on
requirements and allowing for that 96th sample throughput all in one time very,
very effectively
and efficiently. So that is it from us. I'd like to, of course, acknowledge a
few very important
people. This work wouldn't have been possible without the immense support from
the FOMO Fisher
team that work with us, namely, Yvonne, Angie, Han, Laura, of course, Andrew
and Patrick on this
project. Of course, I have to acknowledge Southeast Texas Applied Forensic
Science Facility or Staphs.
For providing us with some samples from their donors, I need to thank their
donors and their
loved ones, because, of course, without them, this type of research is just not
possible. And
work is very, very difficult to do without these types of real samples. And of
course,
the Department of Forensic Science and the Institute of Forensic Research
Training and Innovation for
their support at San Houston State University. So if you have any questions,
please feel free
to reach out to any of my three PhD students here in their respective areas, or
myself,
if you have any other generic questions or general questions in this type of
research
that we perform here at San Houston State with all of our very, very difficult
and challenging
samples. Otherwise, if you have any instrumentation questions, please feel free
to reach out to Han.
His email is there or your local representative, I'm sure, but we'll be able to
help you with anything.
Thank you so much. I'd like to hand back to Michelle now, and I think she's
going to
moderate the Q&A session. Thank you. Thank you, Cherie and panelists for those
interesting
and informative presentations. We do have time for Q&A, so if you have a
question for the panelists,
please submit it through the Q&A panel on your screen. I also want to draw your
attention to
the Resources tab on the right side of the screen. There are a few resources
there connected
to today's webinar that you'll probably find really interesting. While I wait
for everyone to
submit their questions, I have a couple polling questions for you.
Our first polling question is, does your lab use automation?
Answers are A through F, and please check all that apply.
All right, let's see. Most of you do not use automation, but let little less
than half
are. Let's go to the next one. Which areas are your greatest need for
automation? And again,
please feel free to check all that apply. A through E. Well, everyone says all
of the above,
100% on that. Last question before we get to the Q&A. Would you like to receive
more information
about the Nimbus Presto system? All right, thank you audience so much for
participating in our
polling questions. Now we get on to the extra fun part, the Q&A. Kaylee, our
first question is
for you. Are you ready for us? Yes, ma'am. All right, so are there major
differences between
bone and teeth? I would say that the main difference between like bone and
teeth process,
mainly like the size of the sample. So with bone, you know, you have a larger
sample usually, but with
teeth, we use, as we saw in the presentation, usually a mallet to pulverize the
bone. So that
would save a lot of time. You just kind of smash the tooth instead of using
maybe like a drum roll
that we do with bones. But you still do the same protocols of washing it and
powdering it.
So I would say just that sample prep is the main difference. Gotcha. Okay. Nat
alia, you're up next.
All right. How long can the swabs from cartridges be stored and still have
success
in isolating DNA? And how do you store those cartridges?
Okay. So those are all fantastic questions. And there are a bunch of different
variables we have
to consider when answering that question. So as many of you know, the longer
that DNA is exposed
to the surface of copper, the more detrimental those effects are going to be on
the DNA. And so
I would definitely recommend that if your lab does use standard swabbing as the
collection method
of choice, that you don't just leave the casing stored, but you do go ahead and
swab and then
store the swabs. Now we haven't measured or tested storage time here at FHSU.
And a lot of that
really does depend on the collection method. So with standard swabbing, storage
is definitely an
option. However, if your lab is going to adopt the BT mix rinse swab protocol,
you can't really
store the swabs for that. And the reason is because you are using that rinse
solution in the same
tube as your swab. So it all gets collected together. And so if that is your
method of collection,
then I would actually recommend that you go straight through extraction and
then store the
extract. And that way you're preventing any more damage and degradation from
those copper ions.
And you're at a place where you're not storing potential ions in that rinse
solution as well.
Hopefully that answered the question. Absolutely. Thank you so much.
Alright Jennifer, let's get you into the mix. We have a question for you.
Sounds good. How long does sample preparation for bones take?
So it's really dependent on the method and sample type being used for each lab.
So for instance,
for our lab we're processing that entire window cut because we want to have a
large pool of powder
to sample from. But other labs that maybe are just trying to target a enough
powder to actually,
you know, work that sample, might choose to only process a few chips at a time.
So that could
change the sampling process time. But of course every sample is going to
require washing because
we want to make sure we're removing any contaminants. And once we wash we're of
course going to have
to dry. We opt to dry overnight just because the timing of it, it gives you a
little more
variability. But you can also choose to use an oven which will speed things up.
But of course
there still is that wait time for that. So pretty much from beginning to end
for each sample you're
going to take roughly a day or a day and a night. So it is a time consuming
process.
And it's one that is very tedious. But if you do it right it helps things out
quite a bit.
That's it. Okay, thank you Jennifer. Dr. Hughes, this is a question for you or
pretty much anyone
else. But maybe we'll start with you since you haven't heard from you in a bit.
How difficult or time consuming was it to set up the instrument, add reagents,
tips and plays,
and what about running the software, etc. That whole kind of process.
Yeah, well setting it up obviously and everyone getting familiar with the way
it all works in
the scripts running smoothly. Obviously is what took the longest time but we
had immense support
from the thermophisher team to be able to make sure we had everything up and
running. But of course
the GUI system, the GUI makes it so easy to put everything on correctly and
step it through.
So it does it did take us quite a while to get up and running and everyone
familiar with the
process. But the actual time of putting the plastics on and getting everything
run that was
really dependent on of the samples number that you were batching the batch
number you were going
through and also the type of format in the plastics you wear. But as I said, I
mean whether you're
only starting off with your minimum sort of eight samples or your entire plate,
you know,
was a lot more of after the 96 samples. There was such minimal increase in time
of running the
instrument. So the setup at the front-hand is a little bit longer and a little
bit more
longer to put in there. However, once we had the platform up and running
smoothly in the script
saw running and everything working well, it was actually extremely streamlined.
But any of my
students can pipe in on the actual logistics more if they have anything to say.
Yes, so the actual--
Oh, go ahead.
No, no, go ahead.
The actual processing and setting everything up on the instrument, once you
know what you're
doing is very, very quick. You can actually leave things on the deck to kind of
make things a
little bit quicker for the next setup. If you know multiple people are going to
be doing runs that day
and then you can load in ample tips ahead of time. So you don't have to keep
reloading tips. It has
locations for three full plates of the big tips and then the smaller tips as
well. So there really
is that ability to kind of keep things set up and moving forward, speeding up
the process.
Perfect. Thank you. Jennifer, we have another bone question for you.
What was the amount of bone that you used in the method for the Nimbus Presto?
So the Nimbus Presto is using that Preppiler BTA chemistry for bone and so it
follows the same
input value of 50 milligrams for each sample type.
Bacha, okay. Another question here. Someone's asked saying we process fingern
ail clippings in
our lab by swabbing each clipping for any DNA. Have any of you ever tried
processing entire
fingernail clipping much like the nail bed and received successful results?
So I'll include this one. I have not done either swabbing other fingernails
clicking or
the entire fingernail clipping personally. We just follow, I follow the Prepp
iler protocol for
nail clipping and so I know we do collect clippings for our staff database. So
we have them on file.
I think we were able to receive some pretty successful results but considering
we were using
the nail bed, they were highly more successful than just simply the nail
clipping. I don't know if
anyone else would agree or disagree with that. Yeah, we have actually used fing
ernail clippings
in the past but not on the numbers presser. We have with the BTA chemistry on
the Automate
Express and manually and success was hitting this and of course a lot of n
ixtures but with that
was directly soaking the fingernail clippings, not swabbing.
Perfect, thank you. Another bone question, Jennifer.
How many days you keep buried bones in EDTA solutions for demineralization? Is
there any special
preening protocol for buried bones before demineralization? So for this project
since we were using
the Preppiler BTA chemistry, it's a partial demon process so there really is no
pre-soaking
of the buried bones using this method. However, other methods reviews have kind
of been
different total demon methods so an adaptation of the lorry method where we
stoked our sample and our lysis buffer which is EDTA and then pro-K pretty much
And we've only done that for 24-hour periods, so an overnight period. Same
thing with an adaptation
of the Daphne method, again just 24 hours. I know there are other methods that
do a little bit
longer but personally I have not played around with this at all. Okay thank you
Jennifer.
I'm going to tell you we have another question for you. Okay.
The size, copper, and brass, is there a great success with other types of cas
ings such as
nickel, steel, aluminum, anything else?
So here at Sam Houston we haven't examined other types of ammunition. However
what we've seen
in the literature is that there is greater success with non-brass and non-co
pper ammunition
and this really has to do with that metal ion composition. So without copper's
deleterious effects
labs have been generating more STRs in their profiles after swabbing from
aluminum or steel
casings. That's definitely something that we can explore further and do some
comparative research.
However for this project we really wanted to stick to the most challenging type
of ammunition.
Of course why not make it the most challenging. Okay thank you so much for that
Here's a process question. What's the BSA concentration used in the quant
ification step?
So for that we did use BSA at 4.5 milligrams per milliliter and that's not the
final
concentration that is the input concentration. We used one microliter of that
in that standard
trio reaction. Bye thank you. We'll tell you another question for you.
I'm on a roll. Let's go. You are. Since the DT mix rinse/swab protocol
recovered the most DNA,
would it be possible to remove some of the hands-on time by just using the
solution as a swab
moistening agent? Okay so yes so the BT mix rinse/swab protocol is extremely
hands-on.
Does take a little bit more time than the standard swabbing and so that's a
great question.
We actually did test this out here. We
looked at it and we quantified how much we eliminate, how much DNA we recovered
from those swabs
after just doing that and we found that no we couldn't really eliminate that
hands-on time.
And I think actually the original article that the research was published in
also examined the same
thing. They used four different collection methods and one of those happened to
be just using that
BT mix solution as a moistening agent and although it did perform better than
moistening with water
and double swabbing that way it did not outperform that rinse/swab protocol.
And so when we're talking
about really low template samples like DNA from these casings I would recommend
just sticking
to that standard BT mix rinse/swab protocol in full to get the most DNA out of
your sample.
Perfect thank you so much. We had time for one more question Jennifer this one
's yours.
I know we were talking about the berry bones earlier. How deep were the could
divers bones buried?
So we actually did not have record of this at that time but based off of
photographs of the
recovery and at the burial it's roughly between a foot and a half to three feet
and this kind of
goes to show that we're still seeing these challenges with buried bones even at
these more shallow depths
which I thought was kind of interesting. Absolutely that is. All right well
audience that about
wraps up the time we have for Q&A today but I'd like to take our fantastic
presenters as well as
thermophusher scientific for sponsoring today's webinar. In 24 hours or less
this webinar will be
available on demand if you'd like to watch it again or share it with your
friends and colleagues.
Additionally you will receive an email with a URL to obtain CE credit
documentation for your
participation today. We look forward to seeing you at the next webinar. Thanks
so much.