New DNA techniques in

modern human identification

A guide to the latest in next-generation sequencing
to decipher highly discriminating forensic data

BROUGHT TO YOU BY INDEPENDENT SCIENCE PUBLISHER IN PARTNERSHIP WITH

Forensic genomics

Introduction
The forensic field, having relied on the
same DNA profiling technology for the
past 20 years, is long due for a technology Contents
transformation. Modern methods such as
next-generation sequencing (NGS), also •U
 se of modern forensic
known as massively parallel sequencing genomics to solve more crimes
(MPS), can overcome limitations posed by
traditional technologies, uncovering more •O
 btain data from severely
information from limited, damaged or mixed degraded nuclear DNA
DNA samples obtained from crime scenes. •F
 rom damaged DNA to
In this application eBook, we present human identification
an overview of methods used in modern
forensics offering better genetic resolution. •F
 rom mixed sample DNA to
We also discuss strategies to achieve accurate human identification
human identification from challenging DNA,
•G
 enetic genealogy as a
supported by examples from case studies.
forensic tool
Obtaining accurate investigative intelligence
using robust forensic methods ultimately • Featured products
helps generate accurate leads, capture the
• Additional resources
guilty, exonerate the innocent, and reopen
cold cases.

Use of modern forensic genomics to solve physical characteristics in a single test. Even
more crimes with mixed DNA samples from two or more
individuals, NGS technology provides higher
NGS, a modern molecular method, improves genetic resolution, helping resolve cases
efficiency and cost-effectiveness for forensic that were previously deemed inconclusive.
labs in both databasing and casework The Verogen MiSeq FGx Forensic Genomics
operations. It can probe simultaneously for Solution, which includes the MiSeq FGx
unique identity information and estimate Sequencing System, is the first fully validated

2
Forensic genomics

NGS workflow specifically designed for use  Advances in Mitochondrial DNA Analysis:
in forensic genomics applications. In this In this video, we introduce the ForenSeq
application note, learn more about what mtDNA portfolio and Dr. Bobby LaRue,
makes NGS so effective in criminal casework. Sam Houston State University (SHSU),
For support in choosing the most applicable presents on how the ForenSeq mtDNA
forensic route from a suspect to further Control Region Kit has been used for
investigation, an investigative decision tree internal validation at SHSU.
can help design an intentional workflow
equipped with modern molecular biology From mixed sample DNA to human
techniques. identification

STRs and SNPs: Analysis of short tandem

For further information on how NGS repeats (STRs), comparing allele repeats at
can help to solve more cases, see specific loci in different DNA samples, helps
the range of webinars available in discriminate one DNA profile from another.
By sequencing STRs, forensic scientists can
our additional resources section.
achieve an additional level of resolution to
determine differences between individuals,
including the ability to see intra-STR single
nucleotide polymorphisms (SNPs). A recent
Obtain data from severely degraded study presented an approach to establish STR
nuclear DNA and SNP analysis thresholds using the Verogen
ForenSeq DNA Signature Prep Kit with the
Recent improvements in extraction MiSeq FGx System2.
methods combined with NGS technology
have increased the likelihood of obtaining  Inside case files: With NGS, a forensics
informative nuclear DNA profiles from research team resolved a Dutch sexual
even low-quality or degraded samples. assault case upon observing minor
One workflow includes a novel dental alleles that implicated the suspect in
tissue recovery method, a highly sensitive a mixed sample. The suspect, who
real-time qPCR assay followed by NGS was previously released based on
analysis on the Verogen MiSeq FGx Forensic capillary electrophoresis data, was
Genomics System. A key advantage of this later found guilty based on the
workflow is its ability to simultaneously convincing NGS report.
generate phenotype and biogeographical
ancestry estimation from what was once Genetic genealogy as a forensic tool
unusable DNA samples. This eBook
provides more information on creating a A traditional familial genealogy search, used to
workflow to efficiently obtain results from find long-lost birth families or obtain details
challenging samples. of one’s family tree, differs from a forensic
investigative search, which leverages genetic
From damaged DNA to human identification information to establish kinship between two
individuals. Using DNA analysis in addition
Mitochondrial DNA (mtDNA): In cases where to genealogical databases, forensic genetic
nuclear DNA is damaged, for instance, from genealogy (FGG) is used to establish genetic
rootless hair samples, teeth, or disaster relationships that can produce investigative
victims, mtDNA can become a source of leads and identification.
forensic intelligence to advance investigations. The direct-to-consumer database from
A recent publication outlines important Verogen, GEDmatch, coupled with DNA
aspects of mtDNA for implementing analysis makes it possible to generate
NGS-based workflows using the Verogen the genetic intelligence that can lead to
ForenSeq mtDNA Control Region Kit1. identification in an unresolved case.

3
Forensic genomics

 Inside case files: Data from DNA collected scientists with reliable tools and better
at the crime scene in 1987, over 30 years workflows. In summary, modern genomics
ago, were uploaded to GEDmatch, helping techniques can help forensic labs to:
identify a murder suspect after decades
of anonymity. Resolved in 2019, this D
 evelop usable DNA profiles from limited
marked the first-ever guilty verdict for quantity, degraded or mixed samples
a case that relied on forensic genealogy
evidence. P
 rovide strong investigative leads to help
identify the guilty and exonerate the
Targeted NGS and other complementary wrongly accused
tools ultimately bring elevated capabilities
to resolve crime, while providing forensic  Obtain new insights to reopen cold cases

In this webinar, forensic laboratory directors discuss their decision to implement NGS-based methods and Verogen technology.

References

 olt. C, et al., Mitochondrial DNA data analysis strategies that

1. H  idier. M., et al., Establishing STR and identity SNP analysis
2. D
inform MPS-based forensic casework implementation. Forensic thresholds for reliable interpretation and practical implementation
Science International: Genetics Supplement Series Volume 7, Issue 1, of MPS gDNA casework. Forensic Science International: Genetics
December 2019, Pages 389-391. Supplement Series Volume 7, Issue 1, December 2019, Pages 363-364

In this study, using next-generation sequencing of human In this article, researchers present an empirical data-driven
mitochondrial DNA (mtDNA) and the ForenSeq mtDNA typing approach for establishing analysis thresholds that enable reliable
system, researchers focused on aspects of mtDNA important in interpretation of NGS-based data within the limitations of a
implementing NGS-based mtDNA typing into casework operations. system ForenSeq™ DNA Signature assay.

4
 Forensic genomics

Case Study

How Next Generation Sequencing Resolved a Difficult

Case, Leading to the First Criminal Conviction of its Kind
Peter de Knijff explains how his laboratory used NGS methods to resolve a landmark case in the Netherlands.

Introduction Peter de Knijff (PDK): It involves a sexual assault on a 28-year-

old girl that took place in 2015. She preserved her clothes after
Peter de Knijff is not unaccustomed to the role of pioneer in the the assault, and also took intimate samples from herself, and
field of forensic genomics. Twenty years ago, he and his forensic waited for three days until she was courageous enough to go to
laboratory staff at Leiden University Medical Center (LUMC) the police. The police sent all the samples to another forensic
in Holland were among the first to demonstrate the forensic laboratory in the Netherlands, and they started as usual with
utility of male specific Y-STR markers1—a method that is widely capillary electrophoresis (CE) analysis. A year later in 2016, the
used today. They went on to develop a sensitive method to CE results in one particular sample led to a database search
isolate DNA from fired ammunition casings2 (a sample type that in the Dutch convicted criminal database, which resulted in a
was widely considered too challenging for practical use), which hit. Based on the database hit, the police arrested a possible
they have now used to analyze over 15,000 items of firearms suspect.
evidence.

Therefore, it should come as no surprise that Dr. de Knijff was

one of the very first to see the potential of next generation In other cases, we were able to exclude suspects,
sequencing (NGS), also known as massively parallel sequencing
which were suggestively included based on CE...
(MPS), for forensic applications. His laboratory acquired an
Illumina MiSeq system soon after it was introduced in 2011, and That is as important as getting a conviction.
became the first forensic laboratory to receive an ISO-17025
accreditation to perform casework analysis using this platform
in 2015.
Soon after that, the method which led to the database hit was
Since then, they have used MPS on a wide range of challenging challenged by the defense lawyer because he thought that
cases, recovering critical data that could not be obtained using there were some irregularities with the interpretation of the CE
standard capillary electrophoresis (CE) methods, to support results. In Holland, suspects of a crime have the right to access
claims of innocence or guilt and generate “hits” (investigative
leads) using the Dutch criminal offender database.

In one recent case, they used MPS to decipher highly

discriminating data from a challenging mixed sample in a sexual
assault case, which ultimately led to the first and only criminal
conviction based on MPS data to be reported globally to date.

Verogen spoke with Dr. de Knijff recently regarding this

interesting case, and its potential ramifications for MPS
utilization moving forward.

Considering this particular case, I was convinced

that MPS would be able to outperform CE.

Q: Can you describe the case that led to this first criminal
conviction using MPS data? Peter de Knijff is head of the forensic laboratory for
DNA research (FLDO) at Leiden University Medical
Center (LUMC).

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

5
 Forensic genomics

Case Study

contra-expertise performed by an independent laboratory, for a single MPS reaction, and we got, as I expected, really good
which is also paid for by the Dutch government. So, the lawyer results. Meaning that, in all the mixed DNA samples, we got
contacted me for the possibility of performing the contra- much more clear results, predominantly implicating the suspect,
expertise. He explained that the samples previously analyzed because we could decipher whether the minor alleles belonged
by the laboratory were mixed DNA samples with a major to a stutter or not. We used likelihood ratio statistics to express
contributor consistent with the victim and a minor contribution the evidentiary value of each individual stain, and that was very,
less than 10%. very conclusive.

I then suggested to the lawyer a contra-expertise using Q: How did this affect the verdict?
massively parallel sequencing. Considering this particular case,
I was convinced that MPS would be able to outperform CE, PDK: The results were reported in December 2018, and the
with the potential to exonerate his client; however, of course, appeal court convened and extensively discussed the MPS
we could also include his client and support the prosecution’s results. I was not asked to come and give evidence because,
hypothesis further. Much to my surprise, the defense lawyer according to the judges, the prosecutor, and the defense lawyer,
declined the offer for MPS, and strictly wanted me to use CE as they had nothing further to ask in addition to what was written
the method of contra-expertise. in our report. On January 17th, the appeal court ruled the
suspect guilty for the crime. In the verdict, they clearly stated
According to Dutch law, we have to follow the request of the it was the MPS evidence combined with the likelihood ratio
lawyer, so we did a contra-expertise on all the samples using statistics which led them to the conviction of this particular
CE. Not very surprisingly, we found that indeed there were suspect, who was sentenced for three years.
mixed DNA samples with a very minor contributor, which could
possibly belong to someone with a DNA profile identical to
the suspect. However, we also noticed that there were many
alleles in the CE profile which were in the stutter position of a
major allele belonging to the victim. For those variants, we could I expect there will be more court cases using
not decide whether or not those alleles belonged to a stutter MPS data by my own lab or other labs that are
associated with the victim or to a minor contribution from the starting to use this technology. It’s just a matter
perpetrator. And that’s what we wrote down in our report, that
we had predominantly inconclusive results as to whether the
of time.
suspect did or did not contribute to the mixed DNA samples in
this particular case.

In April 2017, the judge, confronted with my report, found that

there was not enough convincing evidence and the suspect was Q: The ability of MPS to distinguish true minor contributor
alleles from stutter clearly played a large role here, but what
released.
about the ability to detect other alleles that were “masked”
Immediately thereafter, the prosecutor filed for an appeal, but it or undetectable with CE?
took more than a year to get permission from the appeal judges
PDK: In this particular case, we could only unmask two alleles
to use MPS. It was not that they didn’t trust the technology, but
with MPS where the victim and the suspect had the same
there was an enormous administrative backlog at the court.
fragment length but a different sequence composition. But at
Finally, in August 2018, our laboratory was given permission to
least that was two more which still helps. And we were able
use MPS in an effort to get the final answer in this case.
to identify more of the minor alleles which were unique to the
suspect. That led to the total package of much more alleles
present in the analysis that we could use for the likelihood
statistics. But the most power came from unmasking the
stutters in this case.
In the verdict, they clearly stated it was the MPS
evidence combined with the likelihood ratio Q: What accounted for being able to detect the additional
statistics which led them to the conviction of this minor peaks that were unique to the suspect?
particular suspect. PDK: It’s our experience that if you have a minor contribution in
a mixed sample, anywhere in the level of around 5-10%, which
is on the border of detection with CE, you almost always find all
the minor alleles with MPS.

Q: What did the MPS testing reveal? Q: And this is due to increased sensitivity?

PDK: We analyzed all the leftovers of the remaining samples PDK: Yes, this is due to the fact that most of the loci have a

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

6
 Forensic genomics

Case Study

more similar PCR fragment length (minimizing preferential because in the long term they do not seem viable or practical,
amplification), and you no longer depend on the sensitivity of or they simply are not picked up by the community. It’s always
a fluorescent label. You can simply count the molecules which exciting to see that something you’ve been working on—and in
have been sequenced. That combined effect in many cases leads this particular case, working on for close to 11 years—finally
to higher sensitivity. reaches the ultimate stage, and that is being accepted in court
as evidence either to exonerate or convict a suspect. You do not
Q: Do you believe you could have achieved these results with see that very frequently in a forensic genetics career.
any different method or platform?
I’m a very strong believer in MPS as a technology on its own, and
PDK: No. I know there are other platforms which allow this is a very simple application of MPS. I’m convinced that MPS
sequencing of micro-satellites, but, to my knowledge, those can add a lot of value in court. This is only the beginning, and
platforms use different PCR kits and software to call the with the beginning, you always run the risk that a court doesn’t
variants, and they do not allow you to download the raw data, accept it. If a court doesn’t accept it, then you have to work very
so you can’t analyze the full sequence reads which the machine hard to get it accepted, which might take years. Now that we
produces. For me, that is unacceptable, because I need to have have the acceptance, which is an important hurdle, it will make it
access to the raw data that allows me to understand what the more acceptable in other courts of law also, I think.
machine has been doing.
Q: Do you think this first court case is indicative of how
Q: Were you worried or concerned with how these MPS data other cases could be helped as well?
would be accepted in Dutch criminal courts?
PDK: Yes. My laboratory has already now used this technology
PDK: No, not for a second. I know a little bit how the Dutch legal in 35 different cases. Many of them were cold cases where we
system works. First, the fact that a method can only be used if generated profiles, which went into the database, but we simply
it is accredited by the Dutch Board of Accreditation is a major do not have a hit yet. In other cases, we were able to exclude
quality assurance. Second, they know my own laboratory is a suspects, which were suggestively included based on capillary
driving force behind forensic innovation, at least in the Dutch electrophoresis. Those cases were not sent to court simply
legal system. They know that if we introduce an accredited because we found that a potential suspect could be excluded.
method, it is reliable, and that I’m always available for further That is as important as getting a conviction; however, because
questions. But it is quite rare in Holland that a DNA expert is that never reaches the phase of a court appearance, it doesn’t
asked to answer questions in court for a criminal case. I only go count in terms of publicity, but it still counts for me.
to court here, at most, once or twice a year. In this particular
case, I expected that they may have some questions because it is I expect there will be more court cases using MPS data by my
a completely new technology, but they were just very impressed own lab or other labs that are starting to use this technology. It’s
by the good results, and the defense lawyer had no further just a matter of time.
questions. It turned out to be a very simple case. But it was
exciting that it came to this particular verdict for me, of course. Q: Do you think more forensic laboratories will start using
MPS for casework now?

PDK: I think this particular case might help to persuade

labs that there are situations where this is absolutely worth
It’s our experience that if you have a minor considering. That perhaps MPS is much better than just
exhausting your DNA extract with different CE attempts,
contribution in a mixed sample, anywhere in the
and that you should reconsider that strategy. If you see that
level of around 5-10%, which is on the border of you have a complex CE profile, it’s better to move to another
detection with CE, you almost always find all the technology to robustly get answers.
minor alleles with MPS.
And I think the fact that there are now SWGDAM Guidelines for
MPS3 initiated in the United States will certainly help the labs
in the United States to reconsider their initial reluctance... and
in Europe I see labs gradually are willing to introduce this. I’m
Q: Why was this so exciting for you? just leading the pack, and I hope that the pack will be big in a few
years, but I don’t have any idea how fast it will grow.
PDK: Well, I start a lot of forensic research projects and…
many research ideas never make it to this particular stage,

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

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 Forensic genomics

Case Study

References Learn More

1. Honda K, Roewer L, de Knijff P. Male DNA typing from To learn more about forensic genomics technology and applica-
25-year-old vaginal swabs using Y chromosomal STR tions, visit: [Link]
polymorphisms in a retrial request case. J Forensic Sci.
1999; 44:868-872.

2. Dieltjes P, Mieremet R, Zuniga S, Kraaijenbrink T, Pijpe

J, de Knijff P. A sensitive method to extract DNA from
biological traces present on ammunition for the purpose of
genetic profiling. Int J Legal Med. 2011; 125:597-602.

3. Addendum to the SWGDAM Interpretation Guidelines

for Autosomal STR Typing by Forensic DNA Testing
Laboratories to Address Next Generation Sequencing,
[Link]

Verogen • 1.833.837.6436 toll-free (North America) • +1.858.285.4101 tel • +44 (20) 399 28411 (United Kingdom) • info@[Link] • [Link]
©2019 Verogen, Inc. All rights reserved. Pub. No. VD2019024 Current as of November 2019

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

8
 Forensic genomics

Applica'on Note: Forensic Genomics

A Comprehensive Massively Parallel Sequencing

Workflow for Severely Degraded Nuclear DNA
Using the Dental Forensic Extrac/on Kit (DFKMR), InnoQuant® HY DNA Quantfica/on Kit and the MiSeq FGx™
Forensic Genomics System to efficiently obtain conclusive results from challenging samples.

Introduc)on and Methods • MiSeq FGx Forensic Genomics System –

o Simultaneous amplifica/on of up to 230 STR and SNP
Degraded DNA poses a cri/cal challenge for forensic DNA
markers, plus Amelogenin, with 200 amplicons
laboratories and is o6en encountered in crime scene samples,
between 63 - 180 bp in size [10]
cold case evidence, and uniden/fied remains associated with
o Highly discrimina/ng profiles can be consistently
missing persons cases or mass disaster tragedies.
obtained with ≤ 16 pg total DNA input [9]
Severely degraded samples, such as teeth and bones, are o Ability to simultaneously generate phenotype and
tradi/onally reserved for mitochondrial DNA processing due to biogeographical ancestry es/ma/on [9,10,11]
inadequate results achieved with standard nuclear DNA tes/ng
protocols. Recent improvements in upstream extrac/on
methods and quan/fica/on chemistries, coupled with massively
parallel sequencing (MPS) technology, collec/vely increase the
likelihood of obtaining informa/ve nuclear DNA profiles from
challenging samples.

The data described herein demonstrate the superior

performance of a comprehensive workflow on degraded DNA
samples. This workflow includes the following:

• Dental Forensic Kit (DFK) – a novel dental /ssue

recovery method developed at Universidad de los Andes,
San/ago, Chile (Figure 1) [1,2,3,4,5]
• InnoQuant HY DNA Quan/fica/on Kit (InnoGenomics) –
a highly sensi/ve real-/me qPCR assay for simultaneous
assessment of human and male DNA quan/ty, quality,
and integrity (Table 1) [6,7]
• MiSeq FGx Forensic Genomics System (Verogen) – a
DNA-to-answer MPS solu/on designed specifically for
forensic DNA applica/ons (Figure 2) [8,9,10,11,12]

The primary advantages for each method include:

• Dental Forensic Kit (DFK) –

o Faster, more efficient extrac/on with higher yield
o Decreased contamina/on risk by omikng the
tradi/onal grinding step Figure 1: DFK method overview (Universidad de los Andes,
o Preserves the tooth to allow for future re-extrac/on San<ago, Chile) – The DFK method improves DNA recovery
from dental remains with a unique and efficient dental /ssue
or dental examina/ons
extrac/on method. This versa/le method may also be
• InnoQuant HY DNA Quan/fica/on Kit – applied to bone samples. The process begins with radiograph
o Extremely sensi/ve and reproducible DNA imaging of the tooth, followed by washing and external
quan/ta/on down to < 0.001 ng/µL using high copy rehydra/on. A specialized dental instrument then perforates
number retrotransposable element (RE) targets the tooth or bone to allow for internal rehydra/on while
minimizing damage to the sample. Both root cement and
o Improved genotyoping success rate through accurate
dental pulp may be obtained for subsequent DNA extrac/on
degrada/on index, PCR inhibitor detec/on, and true using the Quick Extract™ FFPE DNA Extrac/on Kit
nega/ve screening (Lucigen®). The tooth is then sealed and retained, allowing for
future tes/ng if necessary. [1,2,3,4,5]

For Research, Forensic or Paternity Use Only. Not for use in diagnos/c procedures.

9
 Forensic genomics

Applica'on Note: Forensic Genomics

Degrada)on Study Experimental Design Results

To evaluate the combined performance of InnoQuant HY The progressively degraded blood samples resulted in short
quan/fica/on and MPS analysis with the MiSeq FGx System, an target concentra/ons ranging from 0.87 to 0.17 ng/µL and
ini/al degrada/on study was performed u/lizing DNA origina/ng long target concentra/ons from 0.87 to 0.0004 ng/µL. The
from an anonymous male blood bank donor. Degrada/on levels degrada/on indexes (DI) ranged from 1 (no degrada/on
from moderate to severe were apained via sonica/on at 50°C detected) to 460 (severely degraded). The samples were
ranging from zero to 16 hours. DNA extracts were quan/fied amplified with ForenSeq according to the InnoQuant HY long
with InnoQuant HY to obtain short and long target target quan//es, resul/ng in total DNA inputs ranging from
concentra/ons (ng/µL) and the corresponding degrada/on index 1.7 to 0.002 ng.
(DI = [Short/Long]) for each sample. Sequencing libraries were
prepared from the quan/fied DNA using the ForenSeq DNA Figure 3 shows the total number of STR alleles called and iSNP
Signature Prep Kit, u/lizing a maximum DNA input volume of 5 loci typed for samples with increasing levels of degrada/on.
µL, followed by sequencing on the MiSeq FGx Instrument and The total number of autosomal, Y- and X- STR alleles called for
data analysis using the ForenSeq Universal Analysis So6ware. each sample ranged from 85 (100%) with a DI of 1 at 1 ng
total DNA input to 23 alleles called (27%) with a 460 DI and 2
Following the ini/al degrada/on study with ar/ficially degraded pg of total DNA input. Total iSNP loci typed ranged from 94 to
DNA, several teeth and a jaw bone specimen with postmortem 70 (100% - 74%).
intervals (PMI) ranging from seven days to 45 years were
processed. In a collabora/ve experiment, Universidad de los
Andes, San/ago, Chile performed the DFK extrac/on method to
obtain root cement, dental pulp and jaw bone DNA. These
samples were then quan/fied with InnoQuant HY before
processing with the MiSeq FGx Forensic Genomics System.

Table 1: InnoQuant HY Kit Configura<on

Target Genomic Loca/on Amplicon Length Reporter Dye

Short Yb8 autosomal RE 80 HEX

Long SVA autosomal RE 207 Cy5

Male Y chromosome 79 FAM

IPC Synthe'c sequence 172 TAMRA

Table 1: InnoQuant HY Kit Configura<on - InnoQuant HY is a real-/me qPCR

system containing four DNA targets – two different sized RE autosomal targets,
male specific targets, and an internal posi/ve control (IPC) target. The use of high
copy number Alu and SVA REs (>1000 copies per genome) as the two autosomal
targets significantly enhances both sensi/vity and reproducibility of DNA
quan/ta/on and degrada/on detec/on in forensic samples. [6,7]

Figure 3: Total number of STR alleles and iSNP loci - Total number of
STR alleles detected (blue) and total number of iSNP loci called (green)
for ar/ficially degraded blood samples with DI ranging from 1 (not
degraded) to 460 (severely degraded).

In addi/on to STR and iSNP data, aiSNP and piSNP data provide
phenotype and biogeographical ancestry es/ma/on in the
ForenSeq Universal Analysis So6ware. Hair and eye color
Figure 2: The MiSeq FGx Forensic Genomics System - The MiSeq FGx System
phenotype es/ma/ons, which require 100% piSNP locus call
consists of the ForenSeq DNA Signature Prep Kit, the MiSeq FGx Instrument, and rates, were successfully generated for the first four samples
the ForenSeq Universal Analysis So6ware. The ForenSeq DNA Signature Prep Kit down to 90 pg total input and a DI of 16 [11]. Hair color
u/lizes PCR amplifica/on and magne/c bead-based chemistries to prepare targeted
DNA sequencing libraries for the MiSeq FGx instrument. MPS data are generated es/ma/ons for the four samples origina/ng from the same
for up to 230 gene/c markers plus Amelogenin per sample. Upon comple/on of anonymous blood donor indicated 74% red, 22% blond, 4%
sequencing, the ForenSeq Universal Analysis So6ware generates semi-automated brown and 0% black. Eye color es/ma/ons were 96 – 97% blue,
allele calls and displays genotype data for analyst review. In addi/on to short
tandem repeats (autosomal, Y chromosome and X chromosome STRs), single 2 – 3% intermediate and 1 – 2% brown. Biogeographical
nucleo/de polymorphism (SNP) markers are targeted, including iden/ty-informa/ve ancestry es/ma/on was consistently depicted as the European
SNPs (iSNPs), biogeographical ancestry-informa/ve SNPs (aiSNPs), and phenotypic-
informa/ve SNPs (piSNPs). [8,9,10,11,12]
popula/on group for all nine samples (Figure 4).

For Research, Forensic or Paternity Use Only. Not for use in diagnos/c procedures.

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 Forensic genomics

Applica'on Note: Forensic Genomics

The more challenging samples also produced highly

discrimina/ng STR and SNP results, despite more pronounced
degrada/on, extremely low levels of DNA input and indica/on of
inhibi/on as detected by the InnoQuant HY IPC.

One of the more challenging teeth samples with a DI of 76 and

total long target input of 0.001 ng resulted in 78 total STR
alleles and 85 iSNP loci typed (Figure 7). Addi/onally, a 45-year
PMI jaw bone extract was successfully typed with 48 STR alleles
and 59 iSNP loci called at a long target input of 0.01 ng and a DI
of 4.7 (Figure 7). This 45-year PMI bone sample had previously
yielded zero STR alleles with the Iden/filer Plus kit run on the
3100 instrument.

Figure 4: Phenotype and biogeographical ancestry es<ma<on - ForenSeq STR Alleles and iSNP Loci Called
Hair color, eye color and biogeographical ancestry es/ma/on in
the ForenSeq Universal Analysis so6ware for degraded blood with
90 pg input and DI of 16 (sample indicated with red dot).

Data from the dental remains study indicate that the root
cement or dental pulp recovered using the DFK method yielded
DNA of sufficient quality and quan/ty for the majority of the
teeth samples. Several extracts with PMIs ranging from seven
days to approximately six months were not significantly
degraded. As shown in Figures 5 and 6, teeth extracts with DI
ranging from 0.7 to 7.8 and long target total DNA inputs from
0.05 to 5.7 ng resulted in 46 to 49 ForenSeq autosomal STR Figure 7: Total number of ForenSeq STR alleles and iSNP loci
allele calls and 58 to 94 iSNP loci typed. By comparison, the detected in extremely low quan/ty/quality teeth and bone extracts.
same teeth extracts previously resulted in 25 to 31 autosomal
STR allele calls with Iden/filer® Plus amplifica/on using the
maximum 10 µL DNA input run on the Applied Biosystems®
3100 CE instrument.

Conclusions

The combined DFK extrac/on method, InnoQuant HY

quan/fica/on and MiSeq FGx Forensic Genomics System
provides a robust workflow capable of achieving successful
nuclear DNA results from challenging, low-level and degraded
samples. Sufficient DNA was recovered during DFK
extrac/on and InnoQuant HY provided accurate quan/fica/on
data to enable op/mal amplifica/on with the ForenSeq DNA
Signature Prep Kit. The applica/on of this workflow to real-
life, challenging dental remains generated a significant amount
Figure 5: Total number of ForenSeq (blue) and Iden/filer
Plus (grey) autosomal STR alleles detected in teeth extracts
of usable data for iden/fica/on.
with DI ranging from 0.7 to 7.8.

Figure 6: Total number of ForenSeq iSNP loci detected in teeth

extracts with DI ranging from 0.7 to 7.8.

For Research, Forensic or Paternity Use Only. Not for use in diagnos/c procedures.

11
 Forensic genomics

Applica'on Note: Forensic Genomics

Learn More References

To learn more about the DFK extrac/on method, visit:

1. Universidad de los Andes, San/ago, Chile DFK Patent:
hpps://[Link]/search/en/[Link]?
hpps://[Link]/search/en/[Link]?
docId=WO2014188345&redirectedID=true
docId=WO2014188345&redirectedID=true
To learn more about the InnoQuant HY Human and Male DNA 2. hpp://[Link]/[Link]?cat=282
Quan/fica/on & Degrada/on Assessment Kit, visit:
hpp://[Link]/ 3. P. Carrasco, C. Brizuela, I. Rodriguez, S. Muñoz, M. Godoy,
C. Inostroza, Histological transforma/ons of the dental
To learn more about the MiSeq FGx Forensic Genomics pulp as possible indicator of post mortem interval: a pilot
System, visit: hpp://[Link]/ study. hpps://[Link]/10.1016/[Link].2017.09.001
4. P. Carrasco, C. Inostroza, M. Godoy M, G. Gak, Innova/ve
method and kit DFK(MR) of sample prepara/on from dental
human remains for human gene/c iden/tyForensic Science
Interna/onal Gene/cs (fsigen 2018-7), Submiped January
12, 2018.
5. Applica/on of NGS in the analysis of human DNA from
dental remains with new Forensic Kit DFK(MR) Unpublished
results (manuscript in prepara/on)
6. hpp://[Link]/

7. A. Lo6us, G. Murphy, H. Brown, A. Montgomery, J. Tabak,

J., Baus, et al., Development and valida/on of
InnoQuant® HY, a system for quan/ta/on and quality
assessment of total human and male DNA using high copy
targets, hpps://[Link]/10.1016/[Link].2017.04.009
8. hpp://[Link]/
9. A. Jager, M. Alvarez, C. Davis, E. Guzman, Y. Han, L. Way,
et al., Developmental valida/on of the MiSeq FGx Forensic
Genomics System for targeted next genera/on sequencing
in forensic DNA casework and database laboratories
Forensic Sci. Int. Genet. 28 (2017) 52-70.
hpps://[Link]/10.1016/[Link].2017.01.011

10. ForenSeq DNA Signature Prep Reference Guide

hpp://[Link]/
11. ForenSeq Universal Analysis So6ware Guide v1.2
hpp://[Link]/

12. MiSeq FGx Instrument Reference Guide

hpp://[Link]/

Verogen • 1.833.837.6436 toll-free (North America) • +1.858.285.4101 tel • info@[Link] • [Link]

© 2018 Verogen, Inc. All rights reserved. Pub. No. 20180216.2003 Current as of February 2018.

For Research, Forensic or Paternity Use Only. Not for use in diagnos/c procedures.

12
 Forensic genomics

From DNA
to Identification
Multi-application sequencing solutions from Verogen

13
 Forensic genomics

Applications for Every Case

Generate data that lead to a human identification

When human identification matters, Verogen offers the technology that generates powerful investigative intelligence from a DNA sample.
Forensic genetic genealogy (FGG), mitochondrial DNA (mtDNA), short tandem repeats (STRs), and single nucleotide polymorphisms
(SNPs) can work together to solve cold cases, identify missing persons, or exonerate and convict—a portfolio of capabilities exclusive to
the Verogen MiSeq FGx® Forensic Genomics Solution.

To provide a unique, turnkey solution, Verogen is integrating the MiSeq FGx Sequencing System, the only next-generation sequencing
(NGS) instrument specifically designed and validated for forensic applications, with an expanding range of application-specific,
single-workflow library prep kits, matched software tools, and now, a genealogical database. Backed by validation and implementation
services, leveraging these tools is straightforward for any laboratory with access to a MiSeq FGx System. Maximize your ability to deliver
the data that lead to a human identification.

Forensic Genetic • The charging of Joseph DeAngelo with 13 cases of murder

and 13 cases of kidnapping. After decades of dead ends,
Genealogy GEDmatch assisted with the identification of DeAngelo as the
alleged Golden State Killer.
Expand your network of information
• The conviction of William Earl Talbott II of a 1987 double

FGG combines genetic and genealogy methods to identify people homicide was the first conviction to apply FGG. Data from

through relatives. The results provide investigative intelligence that DNA collected at the crime scene over 30 years ago were

exonerates the innocent, matches adoptees, and creates leads uploaded to GEDmatch and ultimately led investigators to

for cold cases. FGG is particularly useful when traditional methods Talbot.

are inconclusive, or all other options are exhausted.

• The exoneration of Christopher Tapp for the 1996 homicide

A recent Verogen acquisition, the genealogical database of Angie Dodge and the charging of Brian Leigh Dripps

GEDmatch aggregates DNA data files from known, voluntary con- marked the first genealogy-based exoneration. A DNA

tributors. Uploading a DNA data file yields a simple measurement sample from the crime scene was processed and compared

of relatedness to help estimate kinship. This type of result makes in GEDmatch, narrowing the focus to Dripps. A DNA sample

GEDmatch a valuable tool that can create leads or eliminate from Dripps then confirmed him as the source of the crime

suspects. scene material.

FGG and the GEDmatch database are gaining traction in re- Leveraging the MiSeq FGx System and working in concert with
al-world scenarios, providing meaningful investigative break- the forensic community, Verogen is developing FGG as an end-
throughs and resolving cases like the following. to-end, fully integrated solution. As the only portfolio to include
this capability, Verogen is uniquely able to bring the next era of
genealogy into your laboratory.

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

14
 Forensic genomics

Mitochondrial DNA STRs and SNPs

Extract more answers from challenging samples Translate samples into phenotypical traits

When nuclear DNA is compromised, mtDNA can be a plentiful STRs are the bedrock of current DNA profiles. Sequencing STRs
and robust source of additional intelligence. In some cases, se- grants an additional level of resolution for determining differences
quencing mtDNA might be the only way to obtain an association between individuals, including the ability to see intra-STR SNPs.
from degraded or limited samples. Rootless hairs, teeth, and other Instead of relying on fragment length alone, the MiSeq FGx
challenging sources are hallmarks of missing persons and disaster System generates the sequence of each fragment in a precise,
victim identification (DVI). By targeting mtDNA, these samples can base-by-base result, revealing variations within STR amplicons
still aid investigations. and providing identity-informative data beyond length variation.
These data are important for kinship analysis and mixture decon-
In Vietnam, for example, analysts used mtDNA to successfully
volution, particularly when DNA contributors share alleles of the
link two brothers. Working with bone fragments recovered from
same length.
a mass grave, they sequenced DNA extracted from both a bone
sample and the blood sample of a possible brother. The sequenc- In a recent Dutch case, Peter de Knijff and his staff at Leiden
es revealed a matrilineal connection, establishing kinship—and University Medical Center (LUMC) analyzed a mixed sample and
ultimately identity—for the previously anonymous remains.* generated discriminating data that proved decisive in a sexual
assault conviction. Because the capillary electrophoresis (CE)
mtDNA has the potential to become a standard forensic tool.
profile contained alleles in stutter positions of alleles belonging
Quality and scaling issues have confined mtDNA to a limited role
in the hands of a few specialists. The ForenSeq™ mtDNA portfolio to the survivor, the LUMC team could not determine whether the

opens up mtDNA to a broader range of applications and laborato- suspect contributed DNA to the mixture. Subsequent NGS results
ries. A simple, efficient workflow underpins all Verogen chemistry, were much clearer: the team saw minor alleles that implicated the
minimizing training and implementation hurdles and facilitating suspect. The verdict stated that the NGS evidence, combined
the transition from traditional methods—especially for laborato- with likelihood ratio statistics, led to the conviction.†
ries already using ForenSeq kits. The ForenSeq mtDNA portfolio
features highly curated, extended primer sets that generate the When DNA is too degraded for STRs to yield conclusive results,

lowest mean amplicon size of any commercial workflow to max- identity-specific SNPs can provide additional means of identifi-

imize outcomes on degraded and unknown samples. Integrated cation. Biogeographical informative SNPs (aiSNPs) and pheno-
Verogen software reduces complex bioinformatics to data that are type-informative SNPs (piSNPs), which estimate traits such as hair
easy to review and interpret. and eye color, help sketch a physical description and generate
leads. For example, if phenotypic piSNPs indicate blue eyes, an
The Verogen mtDNA workflow centers on the MiSeq FGx System.
investigation can reduce the scope of the suspect pool.
Laboratories already processing ForenSeq libraries can leverage
the same instrument, protocols, and infrastructure to quickly and Verogen offers the first and only kit that interrogates all of these
easily insource mtDNA capability. For those new to the MiSeq FGx markers—including a range of SNP types and autosomal, X-, and
System, ForenSeq mtDNA offers a straightforward access point to
Y-STRs—in one test to recover more useful genomic data from
mtDNA and NGS capabilities.
low-quality DNA and mixtures. This approach preserves precious
sample while maximizing the impact the data has on forensic
investigations.

* For details, see A Comprehensive Massively Parallel Sequencing Workflow for Severely Degraded Nuclear DNA (Pub. No. 20180216.2003).
† For details, see How Next Generation Sequencing Resolved a Difficult Case, Leading to the First Criminal Conviction of Its Kind (Pub. No. VD2019024).

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

15
 Forensic genomics

MiSeq FGx System

Start the complete solution

The MiSeq FGx System is the first fully validated, NGS system
designed for forensic laboratories. Dedicated library prep kits, reagent
kits, and software generate the data that can lead to a human
identification.

Combining proven data quality and ease of use, the MiSeq FGx System
is the key to a unique, single-platform solution built on gold-standard
sequencing technology. Prepare libraries, sequence libraries, and
analyze data in a single workflow that’s built to scale. With the system
in place, a laboratory is ready to realize the full power of NGS for a
variety of applications, including FGG, mtDNA, STRs, and SNPs.

This multi-application solution starts with the MiSeq FGx System:

• Set up a sequencing run through an intuitive touch-screen interface.

• Minimize hands-on time with load and go reagents packaged in a pre-filled cartridge.

• Interrogate hundreds of STRs and SNPs in one run, with higher resolution results.

• Target DNA for forensic applications in Forensic Genomics mode or use Research Use Only (RUO) mode for greater flexibility.

Discover more at [Link]

Ready to learn more about NGS

solutions and human identification?

Contact us at info@[Link]

Verogen • 1.833.837.6436 toll-free (North America) • +1.858.451.4187 tel • +44 (20) 399 28411 (United Kingdom) • info@[Link] [Link]
For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.
© 2020 Verogen Inc. All rights reserved.
Pub No. VD2020009 Current as of January 2020

16
 Forensic genomics

Application Note: Forensic Genetic Genealogy

Forensic Genetic Genealogy with GEDmatch

Forensic genetic genealogy is a new method for creating investigative leads that realizes the
collective power of community, DNA science, and sequencing technology.

Highlights
• Powerful genetic intelligence
Data from DNA samples and genealogical databases help
identify people through relatives
• Closure for cold and contemporary cases
New investigative leads for missing persons identifications,
innocence projects, and criminal investigations
• Turnkey genealogy solution
End-to-end solution packaged in a familiar workflow and
optimized for genealogy applications

Introduction
Genealogists, historians, and adoptees have traditionally used genealogy
to find birth families and build family trees. The rise of direct-to-consumer
(DTC) genetic tests, such as 23andMe, creates the potential for law Figure 1: In criminal investigations, FGG compares crime-scene DNA to
enforcement to partner with these communities and forensic laboratories volunteer DNA in GEDmatch. Shared segments inform the creation of a
multi-generation family tree that can lead investigators to a suspect. The
to solve cold cases. Forensic genetic genealogy (FGG), also called
process is similar for unidentified remains.
investigative genetic genealogy (IGG), combines genealogy with DNA
analysis to produce investigative leads in cases of unidentified remains or
unsolved crimes. Kinship Insights

Verogen is developing an end-to-end, next-generation sequencing (NGS) Identity by descent (IBD) describes a DNA segment that two people
solution to empower FGG by aiding identification in ways traditional have inherited from a common ancestor—a concept that provides
methods cannot. This application note describes how FGG uses DNA the framework for FGG work. Two samples sharing overlapping IBD
data and the Verogen GEDmatch database to generate the genetic segments across chromosomes allows genealogists to approximate the
intelligence that can lead to an identification. It also presents real-world degree of kinship, with recombination events helping distinguish whether
examples of FGG results linking perpetrators to crimes and exonerating DNA is inherited or shared by chance (Figure 2). The length of an IBD
the wrongly convicted with equal levels of assuredness. segment is measured in centimorgans (cM), which measure genetic
distance. The larger the cM value, the more DNA is shared between two
Genetic Genealogy as a Forensic Tool people and the more likely they are to be related (Table 1).

FGG employs genealogical databases, such as GEDmatch, and family

Table 1: Centimorgan Values as a Measure of Kinship
trees to develop intelligence in cases of missing persons or unsolved
IBD Segment Length Matching Chromosomes
violent crimes. As an impartial, science-driven tool, FGG is particularly Relationship (cM) (%)
useful when traditional methods are inconclusive or all other options
Identical twins 3800 100
are exhausted.
Parents 3400 50
The FGG process starts after results from a forensic database, such as
Full siblings 2500 37.5
CODIS, are inconclusive. The DNA profile from an unidentified sample
is uploaded to GEDmatch for comparison against profiles compiled Grandparents 1700 25
from known, voluntary contributors. GEDmatch results indicate potential Aunts and uncles 1700 25
relatives based on the amount of shared genetic material. Using these Great-grandparents 850 12.5
relatives, a genealogist constructs a family tree that extends over multiple
First cousins 850 12.5
generations to narrow results for the unidentified sample (Figure 1).¹
Second cousins 212.5 3.125

First cousins twice 212.5 3.125

removed

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

17
 Forensic genomics

Application Note: Forensic Genetic Genealogy

Targeted Assays for Challenging Samples

Although the GEDmatch database is a powerful tool, the large-scale
SNP array methods that generate the data it contains are incompatible
with the challenging samples typical in forensic casework. Verogen
leverages GEDmatch data to identify an optimum set of compatible
markers to combine in a dense NGS SNP assay designed for low-
quantity, low-quality samples. The amplicons are small—ideal for
degraded DNA—and selected to be the most informative. Sequencing
these amplicons on the MiSeq FGx® Sequencing System targets the
data relevant to FGG. In turn, GEDmatch uses only the critical markers
to find relatives. The Verogen NGS workflow ultimately requires less
sample than traditional methods and limits data to the specific set
needed for FGG.

Justice and Public Safety

FGG plays a growing role in correcting miscarriages of justice and
identifying violent offenders who evaded detection. After decades
of dead ends, GEDmatch assisted with the identification of Joseph
James DeAngelo as the alleged Golden State Killer. His arrest in
April 2018 cast a spotlight on FGG, highlighting its potential as a
mainstream forensic tool.

In June 2019, the conviction of William Earl Talbott II for the 1987
Figure 2: IBD segment lengths approximate the amount of shared DNA
between two samples. GEDmatch converts the SNP calls (A, G, C, or T) murder of a young Canadian couple, Tanya Van Cuylenborg and Jay
from a DTC genetic test into IBD segments.² Cook, marked the first-ever guilty verdict for a case that relied on
FGG evidence. Data from DNA collected at the crime scene over 30
years ago were uploaded to GEDmatch, helping identify Talbott after
Expanded Database Capability
decades of anonymity.4
Where permitted, forensic databases already enable short-range
In another cold case, FGG helped to not only identify the offender,
familial searches to find close relatives. However, they use only a small
but release the man wrongly convicted of the crime. Unsatisfied with
number of markers and restrict the pool of DNA profiles to people
the conviction of Christopher Tapp of the 1996 rape and murder
who have come into contact with law enforcement. When a forensic
of her daughter, Angie Dodge, Carol Dodge pushed for a proper
database fails to produce a hit, a genealogical database can broaden
resolution of the case. Fraught with inconsistencies, the original case
the search by increasing the DNA profile pool and expanding the
included an alleged coerced confession that Tapp later recanted and
search criteria.
a lack of direct evidence. Critically, none of the crime-scene DNA
GEDmatch is the only genealogical database that aggregates DNA matched Tapp.
profiles from all DTC genetic testing companies—an approach that
In May 2019, an FGG breakthrough led to the arrest of Brian Leigh
allows it to compare data from unknown samples against a diverse
Dripps and the definitive clearing of Tapp. A DNA sample from the
and extensive set of volunteer data. A long-range familial search finds
crime scene was processed and compared with profiles in GEDmatch,
distant relatives or sets of relatives within this extended data set. The
identifying Dripps as a possible suspect. Investigators then obtained
results yield a simple measure of kinship based on IBD segments that
a discarded cigarette butt for comparison to hair and semen samples
can, for example, identify second cousins of a sample contributor.³
from the 1996 crime scene, which matched. When questioned, Dripps
Figure 3 illustrates an example GEDmatch query result with graphical admitted to the crime.5
representation of two shared segments on human chromosome 6. For
each of the two segments, the table shows start and end locations
on the chromosome, total length in centimorgans, and the number of
Conclusion
shared SNPs. SNP allele calls, which are not necessary for FGG, are Without an identification, cases can languish for decades,
excluded from GEDmatch results. suspending survivors, families, and the wrongly convicted in a state
of uncertainty while offenders go free. The ability of Verogen NGS
technology to advance challenging samples, limit data exposure, and
find relatives through shared DNA unlocks an opportunity to push
genealogy further. FGG has already demonstrated the capability to
generate investigative intelligence that can identify the anonymous
Figure 3: An association between two people on a segment of and reanimate cold cases.
chromosome 6. The horizontal dark green bars indicate two shared
segments, while the table displays information about each segment.

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

18
 Forensic genomics

Application Note: Forensic Genetic Genealogy

Working in concert with the forensic community, Verogen is leveraging

the MiSeq FGx System and GEDmatch to develop FGG as an end-to-
end, fully integrated solution. Based on gold-standard NGS technology,
the solution integrates instrument, chemistry, analysis software, and
database—backed by evaluation and implementation services—to equip
forensic laboratories with more ways to generate investigative intelligence
from DNA. As the only portfolio to include this capability, Verogen is
uniquely able to bring the next era of genealogy into your laboratory.

Learn More
To learn more about the GEDmatch database, visit:
[Link]/a-message-to-verogen-customers-about-the-
gedmatch-partnership

To learn more about the MiSeq FGx System, visit:

[Link]/miseq-fgx/

References
1. Callaghan, Thomas F. 2019. “Responsible genetic genealogy.”
Science 366(6462):115. doi:10.1126/science.aaz6578.
2. Greytak, Ellen M., CeCe Moore, Steven L. Armentrout. Genetic
genealogy for cold case and active investigations. Forensic Science
International. 2019;299:103–113. [Link]
forsciint.2019.03.039.
3. Erlich, Yaniv, Tal Shor, Istik Pe’er, Shai Carmi. 2018. “Identity
inference of genomic data using long-range familial searches.”
Science 362(6415):690–694. doi:10.1126/science.aau4832.
4. Murphy, Heather. 2019. “Genealogy Sites Have Helped Identify
Suspects. Now They’ve Helped Convict One.” New York Times,
July 1. [Link]
[Link].
5. Bishop, Shane. 2019. “Police arrest Idaho man in 23-year-old cold-
case murder of Angie Dodge.” NBC News, May 16. [Link]
[Link]/dateline/police-arrest-idaho-man-23-year-old-cold-
case-murder-n1006726.

Verogen • 1.833.837.6436 toll-free (North America) • +1.858.285.4101 tel • +44 (20) 399 28411 (United Kingdom) • info@[Link] • [Link]
© 2020 Verogen, Inc. All rights reserved.
Pub No. VD2020005 Current as of January 2020

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

19
 Forensic genomics

Info Sheet: The Expanded Genetic Toolkit

Modern Forensic DNA Testing Capabilities: Your Genetic Toolkit

How to use state-of-the-art forensic genomics techniques to solve more crimes.

Introduction
DNA testing capabilities have expanded to offer greater
insight from biological evidence. This guide is intended to
provide a basic overview of the modern technology land-
scape, and help clarify the scenarios and sample types that
may benefit from new and emerging methods.

What new tools are available and how are they different
from previous technology?
The forensic field has used the same DNA profiling
technology for the past 20 years. Modern molecular
biology advancements such as massively parallel
sequencing (MPS, also known as next generation
sequencing) are able to overcome limitations of the
previous technology to reveal more information
from evidence samples. For example, MPS can probe
simultaneously for unique identity information and
estimate physical characteristics in a single test. By
typing all relevant forensic markers in a single reaction,
MPS improves efficiency and cost effectiveness in both
databasing and casework operations. As a result, the
data are more informative with higher genetic resolution,
allowing samples deemed inconclusive to be actionable
when re-tested with MPS. This includes mixed DNA
samples from two or more individuals.

Another modern molecular method is microarray

genotyping, a tool for large scale analysis of many
thousands of genetic markers. This method has been used
to generate data for forensic genealogy searches in several Figure 1: The New Investigative Workflow- The decision tree when using the ex-
panded genetic toolkit enabled by modern molecular biology techniques. (*certain
high-profile cases when all other options were exhausted. investigation methods may not be available in all jurisdictions; forensic genealogy
However, this method has technical limitations, including a requires a sufficient quantity of intact DNA)
requirement for a sufficient quantity of intact DNA without
complex mixtures. When pursued, this method can produce MPS can:
many leads that require extensive resources to investigate,
using both genetic and non-genetic information. • Develop usable and probative DNA profiles from low
quantity, degraded, and mixed crime scene samples
How can MPS be used? • Provide law enforcement officials with strong investi-
MPS helps transcend inherent limitations of older DNA gative leads, help identify the guilty, and exonerate the
technologies to solve more cases. Previous DNA technol- wrongly accused
ogies can fail to provide usable information from samples
• Provide new insight and information to reopen and
that are low in quantity, quality, or contain complex mixed
pursue cold cases
genetic profiles from multiple DNA contributors. As a
result, crimes may go unsolved and victims may be denied
justice.

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

20
 Forensic genomics

Info Sheet: The Expanded Genetic Toolkit

What makes MPS more effective in criminal casework? In certain unique cases where other efforts have failed,
MPS simultaneously targets a larger number of specific investigators may wish to pursue additional avenues. These
forensic markers within human DNA to provide genetic include familial searching, i.e. “genetic proximity testing”,
information. MPS data allow more confident inclusions and and forensic genealogy. Forensic genealogy uses microarray
exclusions, and can benefit many types of forensic DNA genotyping rather than MPS, and requires a relatively
casework. The current DNA markers are included in MPS high quantity of intact DNA for analysis. These practices,
tests so that profiles remain compatible with current law which are not permitted in some jurisdictions due to their
enforcement databases, such as CODIS. MPS also provides controversial nature, are typically only used in special
a critical advantage for sexual assault cases by simultane- circumstances. However, when successful they have led to
ously typing male-specific (Y chromosome) DNA within a perpetrator identifications.
sample—even in the presence of large amounts of survivor
or decedent DNA. Mixed DNA profiles from two or more
persons are commonly encountered in sexual assaults How do I gain access to the complete genetic toolkit and
and other crime scene samples. Because of the refined assist my cases?
information from MPS, these complex samples are better Verogen is aware of at least 150 forensic laboratories
interpreted, such that more profiles may be searched for globally that have implemented MPS or are in the process
the unknown DNA contributor on a database or compared of implementing it, with more expected in the coming
directly to a suspected perpetrator. months. As more crime labs bring MPS online, you may be
able to obtain expanded services locally. In the meantime,
several leading forensic institutions and fee-for-service
Are additional options now possible for no-suspect cases laboratories are offering testing for jurisdictions who need
without a DNA database “hit”? help. By reviewing open cases and prioritizing them with
Yes. MPS data expands the genetic toolbox for investiga- the “best fit” genetic method, you can move new and old
tions when a suspect has not been developed by conven- cases forward today.
tional means or no database “hit” is obtained. MPS can
produce actionable investigative leads by providing hair
color, eye color, and biogeographical ancestry estimations. How can I get more information?
This can support or refute eyewitness accounts, helping Contact Dr. Cydne Holt, Chief Scientific Officer at Verogen
investigations stay on the best path. at cholt@[Link] about how modern tools may assist
you.

Figure 2: The Verogen Forensic Genomics Solution, which includes the

Illumina MiSeq FGx sequencing instrument, is the first fully validated MPS
workflow specifically designed for use in forensic genomics applications.

About Verogen
Verogen serves those who pursue the truth using genetic tools. Powered by Illumina’s gold standard sequencing
and array-based technology, and working in partnership with forensic laboratories, Verogen is advancing science
to unlock the true potential of forensic genomics. For more information, visit [Link].

Verogen • 1.833.837.6436 toll-free (North America) • +1.858.285.4101 tel • info@[Link] • [Link]

© 2018 Verogen, Inc. All rights reserved. Pub No. VD2018018 Rev B Current as of November 2018

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

21
Forensic genomics

Featured Products
The MiSeq FGx Forensic Genomics System

MiSeq FGx Universal Analysis

Sequencing System Software and Server
by Verogen by Verogen
The MiSeq FGx System is based on the most widely The ForenSeq Universal Analysis Software (UAS)
adopted sequencing chemistry in the scientific contains comprehensive data management and
community to produce the highest yield of error- analytical capabilities, including sample and index
free reads at the lowest base-by-base price. The management, application-specific workflows,
intuitive setup and flexible sequencing workflows data visualization at sample and locus levels,
make it easy to use NGS for your specific application quality flags to simplify data analysis, and easily
with minimal requirement for additional exportable reports.
infrastructure or equipment.

ForenSeq DNA Signature Prep Kit

by Verogen
The ForenSeq DNA Signature Prep Kit includes
all the required reagents to prepare sequencing
libraries from forensic DNA samples. By
simultaneously targeting over 200 markers
with small amplicons in a single reaction,
analysts can obtain discriminating profiles
from degraded and low- level DNA samples.

Additional Resources
Webinars:

Advances in Mitochondrial DNA Analysis

How Can NGS Help Law Enforcement Solve More Cases?

Why NGS, Why Now? A Q&A with forensic laboratory directors

22