BOSTON UNIVERSITY

SCHOOL OF MEDICINE

Thesis

AMPLIFICATION REPRODUCIBILITY AND THE EFFECTS ON DNA MIXTURE

INTERPRETATION ON PROFILES GENERATED VIA TRADITIONAL AND MINI-STR

AMPLIFICATION

by

ELISSE RUIZ CORONADO

B.A., Boston University, 2003

M.S., University of Massachusetts Boston, 2007

Submitted in partial fulfillment of the

requirements for the degree of

Master of Science

2011

Approved by

First Reader ____________________________________________________

Catherine Grgicak, M.S.F.S., Ph.D.

Instructor, Program in Biomedical Forensic Sciences

Second Reader __________________________________________________

Robin Cotton, Ph.D.

Associate Professor, Program in Biomedical Forensic Sciences

iii

AMPLIFICATION REPRODUCIBILITY AND THE EFFECTS ON DNA MIXTURE

INTERPRETATION ON PROFILES GENERATED VIA TRADITIONAL AND MINI-STR

AMPLIFICATION

ELISSE RUIZ CORONADO

Boston University School of Medicine, 2011

Major Professor: Catherine Grgicak, M.S.F.S., Ph.D., Instructor, Program in Biomedical

Forensic Sciences

ABSTRACT

Mixture interpretation of complex DNA evidence samples remains a challenge.

Determination of the number of contributors and the relative input of said contributors

remains one of the most important and difficult tasks to accomplish. This is exacerbated

when the DNA is inhibited and/or degraded. Currently the ability to characterize the

number and relative ratios of contributors depends on a number of assumptions which

include but are not limited to the following;

1) Peak heights and peak height ratios do not significantly differ between loci,

amplifications or kits.

2) The peak heights and peak height ratios do not significantly differ between

amplifications regardless of input amount. More specifically, calculating the

peak height ratio between two alleles originating from a single source medium-

high end target is applicable to samples with low template levels.

iv

3) Lastly, generalized peak height ratios, derived from validation studies using

single source samples are similar to those for mixture profiles, whereby it is

assumed that the DNA amplified independently during amplification when more

than one contributor is present.

This study was designed to assess the validity of the aforementioned

assumptions by testing the reproducibility of the peak heights and peak height ratios of

single source and mixture samples amplified at various targets. More specifically, these

assumptions were evaluated by amplifying single source samples using various target

amounts (4ng-0.0625ng), in quadruplicate with the AmpFℓSTR® Identifiler® and

MiniFiler® Amplification Kits. Analysis of these results focused on examining the

reproducibility of the peak heights and peak height ratios between amplifications, loci

and target amounts. The peak height ratios between amplifications and target amounts

were examined by utilizing the F-test, which tested whether the error in peak height

ratios remained constant despite target amounts.

The mixtures were created using a series of 2-person DNA mixtures. The DNA’s

were mixed at known ratios and amplified with the aforementioned STR chemistries

with varying amounts of DNA. The profiles generated were analyzed with GeneMapper

ID® v. 3.2 and the peak heights and peak height ratios were compared to the single

source samples.

Although Minifiler® had a lower limit of detection it also had a lower sensitivity

at a given target, suggesting that Identifiler® is the recommended kit for obtaining a full

v

DNA profile from non-compromised samples. Both AmpFℓSTR Identifiler® and Minifiler®

Amplification Kits demonstrated increased peak height variance with a decreasing target

amount; however Minifiler® showed more variance across all targets when compared to

Identifiler®. Peak height ratios significantly differed at low targets for both kits. Peak

heights and their ratios did not vary between mixture and single source conditions,

suggesting peak height ratios obtained through single source validation studies are

appropriate to use for mixture deconvolution.

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Table of Contents Title Page i

Reader Approval Page ii

Abstract iii

Table of Contents vi

List of Tables vii

List of Figures ix

List of Abbreviations xi

Introduction 1

Materials and Methods 15

Results and Discussion 22

References 54

Vita 58

vii

List of Tables Table 1 20

Minimum Distinguishable Signal ( blY + 3 bls ) for each locus and Minimum

Quantifiable Signal ( blY + 10 bls ) calculated via analysis of the baseline derived from blanks. Table 2 22 STR profiles for both samples used in this study, determined by amplification via AmpFℓSTR® Identifiler® and AmpFℓSTR® Minifiler® Kits. Table 3 25 Minimum and Maximum Peak Height and Range of Average Peak Heights for each target for all loci amplified with both AmpFℓSTR® Identifiler® and Minifiler® for single source data. Table 4 27 Number of alleles below the minimum distinguishable signal (16 RFU) at 0.0625 and 0.125 ng targets for AmpFℓSTR® Identifiler® and Minifiler® Kits. Table 5 28 Analytical and Calibration Sensitivities for AmpFℓSTR® Identifiler® and Minifiler® at 0.5 ng. Table 6 36 Average Peak Height Ratio Range for each target amplified with the AmpFℓSTR® Identifiler® and Minifiler® Kits. Table 7 41 F-test for the comparison of the variances of peak height ratio for loci in the

AmpFℓSTR® Minifiler® kit.

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Table 8 44 F-test for the comparison of the variances of peak height ratio for loci in the AmpFℓSTR® Identifiler® kit. Table 9 46 Number of loci at each target where the Ho was rejected for AmpFℓSTR® Identifiler® and Minifiler® using a 0.05 confidence interval.

Table 10 51 Mixture ratios and resulting peak height ratios at varying target amounts for loci D16S539 and D21S11.

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List of Figures Figure 1A 23 A sample electropherogram of the blue-dye channel of AmpFℓSTR® Identifiler® and Minifiler® Amplification Kits for one sample at a 1 ng target with a 5 second injection. Figure 1B 24 3*RSD (Relative Standard Deviation) of all peak heights at 9 loci (Amelogenin, CSF1PO, D13S317, D16S539, D18S51, D21S11, D2S1338, D7S820, and FGA) included in AmpFℓSTR® Identifiler® and Minifiler® Amplifications Kits. The results are from four repeat amplifications of two single source samples with targets ranging from 0.0625 to 4 ng. Figure 2 32 Locus specific average peak heights and 3 standard deviations for four repeat amplifications of 2 single source samples with targets ranging from 0.0625 to 4 ng for AmpFℓSTR® Minifiler® and Identifiler®. A) Amelogenin B) CSF1PO C) D13S317 D) D16S539 E) D18S51 F) D21S11 G) D2S1338 H) D7S820 I) FGA J) Identifiler®-specific loci.

Figure 3 34 An example of average peak heights and one standard deviation for four repeat amplifications of 2 single source samples with targets ranging from 0.0625 to 1 ng for AmpFℓSTR® Minifiler® and Identifiler®.

Figure 4 38

Locus specific average peak height ratios and 3 standard deviations for heterozygote loci A) Amelogenin B) CSF1PO C) D13S317 D) D16S539 E) D18S51 F)D21S11 G) D2S1338 H)D7S820 I)FGA J) Identifiler®-specific loci with error bars showing 3 standard deviations from the mean. The results depicted are from four amplifications of 2 single source samples of DNA with targets ranging from 0.0625 to 4 ng for AmpFℓSTR® Minifiler® and Identifiler®.

Figure 5 48 Peak height ratios at heterozygote loci D16S539 and D21S11 at varying nominal targets.

x

Figure 6 49 An example of peak height comparisons of each allele at heterozygote locus D21S11 from one contributor at varying nominal targets.

Figure 7 50 An example of peak height ratios for the major and minor contributor in 1:2, 1:4, and 1:9 mixture ratios at varying targets.

xi

List of Abbreviations

µL - Microliter

bp - Base Pair

° C - Degrees Celsius

Cal/Calc - Calculated

CE - Capillary Electrophoresis

CODIS - Combined DNA Index System

DNA - Deoxyribonucleic acid

dNTP - Deoxynucleotide triphosphate

EDTA - Ethylenediaminetetraacetic acid

HCl - Hydrochloric Acid

Hi-Di - Highly Deionized

Ho - Null Hypothesis

KCl - Potassium Chloride

M - Molar

MgCl2 - Magnesium Chloride

MDS - Minimum Distinguishable Signal

ml - Milliliter

MQS - Minimum Quantifiable Signal

ng - Nanogram

PCR - Polymerase Chain Reaction

xii

RFLP - Restriction Fragment Length Polymorphism

RFU - Relative Fluorescent Unit

RSD - Relative Standard Deviation

SD - Standard Deviation

SDS - Sodium Dodecyl Sulfate

SSC - Saline Sodium Citrate

STR - Short Tandem Repeat

Taq - Thermus aquaticus

TE - Tris-EDTA

VNTR - Variable Number Tandem Repeat

1

INTRODUCTION

Human identification via the analysis of length polymorphisms at short tandem

repeat (STR) loci utilizing the polymerase chain reaction (PCR) has proven ideal for

forensic DNA analysis. In 1985 Dr. Alec Jeffreys first described variable number tandem

repeats (VNTRs) and their characterization via restriction fragment length

polymorphisms (RFLPs) (1). Jeffreys, et al., described how these length polymorphisms

are a result of the number of tandem repeats present in a minisatellite locus (1).

Pairwise comparisons of DNA fingerprints obtained from a number of unrelated

individuals showed that minisatellite patterns were highly specific to an individual and

very few fragments were shared between randomly selected individuals (1). More

importantly, the DNA fingerprints’ obtained from this study showed that RFLP analysis

using core minisatellite sequences as probes are reproducible and suitable for individual

identification.

Also described in 1985, the polymerase chain reaction (PCR) significantly

revolutionized forensic DNA analysis, and has become the most widely used, rapid and

sensitive method to obtain DNA information (2). In its infancy, single-copy genomic

sequences were amplified by a factor of more than 10 million, and were highly specific,

and DNA segments up to 2000 base pairs were easily amplified. In addition, the method

was capable of amplifying and detecting a target DNA molecule present only once in a

sample of 105 cells (3).

2

Although RFLP-based DNA analysis, such as that used by Jeffreys et al., is highly

discriminating, it has its limitations. RFLP forensic methods require large amounts of

undegraded DNA and several days to weeks to complete the hybridization with radio-

labeled probes (4). In contrast, by utilizing PCR-based methods, a specific DNA

sequence can be exponentially amplified by a factor of 2 with each cycle, where as many

as 40 cycles may be used (5). The use of this kind of technology allows for smaller

amounts of DNA to be detected and can be performed in significantly less time.

Currently, the amplification of short tandem repeats (STRs) are preferred over

RFLP-based methods. Forensically relevant STRs, also known as microsatellites, are DNA

regions with repeat units that are 2-6 base pairs in length versus the 10-100 bases in

length of VNTRs (6). STRs are tandemly repeated from approximately half a dozen times

to several dozen times (6). There are many STR markers but only a core set has been

chosen for forensic DNA and human identity testing. These core loci/STRs allow for the

comparison of genetic information, are easily amplified via PCR when used in

combination, and are highly variable among individuals.

Multiplex-PCR

Multiplex PCR is the simultaneous amplification of two or more regions of DNA.

It is an important tool in forensic DNA analysis because it allows for multiple STRs to be

amplified at the same time thus creating a genetic profile using one reaction tube (7).

Commercially available PCR reaction kits include pre-mixed primers, DNA polymerase,

and a PCR reaction mix which contains the rest of the necessary PCR components (i.e.

3

dNTPs, MgCl2, etc.,). STR amplification kits such as AmpFℓSTR® Cofiler® and Profiler

Plus® which include these three components allow laboratories to measure out each

component individually and depending on the number of samples, combine them to

create a master mix. The master mix can then be aliquoted to the appropriate tubes

followed by the addition of the sample DNA, and subsequently placed in a thermocylcer

for amplification (7). A typical thermocycling protocol includes denaturing the target

DNA to separate the two strands (i.e. ~95°C), annealing at lower temperatures to allow

the primer to bind to the target (i.e. ~60°C), and finally extension which is the point at

which the DNA is synthesized (i.e. ~70°C) (3).

A primer is a short synthetic oligonucleotide which is used in many molecular

techniques such as PCR and DNA sequencing. Properly designed primers have a

sequence that is the reverse complement of a specific region of template to which it will

anneal (8). The annealing characteristics of the primers directly affect the molecular

mass of DNA that is amplified because the target region of DNA template is defined by

the position of the primers. Efficient PCR reactions require primers be specific to the

target region, have similar annealing temperatures, not significantly interact with each

other or themselves to form “primer dimers”, and be structurally compatible (8).

Many parameters need to be considered when designing primers such as: primer

length, primer melting temperature, GC content, self-complementarity,

complementarity to other primers (primer dimer), distance between two primers on

target sequence, the oligonucleotide sequence, the difference in melting temperatures

4

between the forward and reverse primer pair, and finally that there are no long runs

with the same base. Primers are generally 18-25 nucleotides long and target the

flanking sequences around the area of interest. A number of considerations are taken

into account when designing primers to target a specific sequence. First, the GC content

of the primer is considered because its melting temperature can be affected; the lower

the GC content the lower the annealing temperature during amplification. Also,

ensuring lack of self-complimentarity is essential since primers may form hairpin loops

or dimers resulting in less primer available during the reaction resulting in less product.

Additionally, for the purposes of human identification, it is crucial to ensure that the

region to which the primers bind be single copy with little to no mutation because if the

sequence changes from one DNA template to the next then the primers will not bind

appropriately in all samples.

Multiplex PCR uses primer pairs with a dye label on one primer in the same

amplification reaction to allow for simultaneous detection of loci that are of similar size.

The process of amplifying multiple loci simultaneously is accomplished by adding one

primer pair per locus amplified to the amplification reaction mixture. In order for a

multiplex reaction to work well the optimization of reaction conditions and primer

sequences is necessary to avoid one locus with its respective primer pair from

preferentially amplifying over another. However, this requires extensive optimization of

annealing conditions and primer design for maximum amplification efficiency of the

different primer–template systems. Multiplexing requires the same care in primer

5

design as regular PCR but with more primers to consider. Ideally the amount of

amplified product from each locus needs to be similar to the amount of product from

the other loci. In addition to the primers, other parameters which also need

optimization include: cycling conditions, buffer, MgCl2, and polymerase concentration

(7).

Annealing temperature is one of the most important cycling parameters and

needs considerable attention during the optimization process. Each commercially

available STR-kit has its own PCR cycling protocol because different primer sequences

have different hybridization properties and therefore anneal to the DNA template

strands at different rates. Also, the extension times are usually increased to give the

polymerase time to copy all of the DNA targets (7). Due to the relationship between GC

content and primer annealing temperature, precise and accurate heating and cooling is

essential for efficient amplification in order to produce consistent results within and

between amplifications and laboratories (9).

The remaining factors to consider for obtaining optimal results for a multiplex

PCR amplification are the buffer, DNA polymerase, and dNTPs. The buffer may contain

Tris-HCl, magnesium chloride (MgCl2), potassium chloride (KCl), and bovine serum

albumin (BSA) (9). The Tris-HCl has a specific pH which can affect the melting

temperature of specific DNA fragments. The MgCl2 is crucial to the multiplex PCR

reaction because it is needed by the polymerase to sustain enzyme activity. The

concentration of KCl affects the hybridization stringency (the degree of mismatch of

6

bases); the higher the salt concentration the lower the stringency which increases

potential mismatches (10). The BSA is a necessary component because it is a “sticky”

protein and impurities in the extracted DNA solution adhere to it. The

deoxyribonucleotide triphosphates (dNTPs) are needed as they are the building blocks

of the newly synthesized DNA.

DNA polymerase is responsible for adding the building blocks (the dNTPs) in the

proper order based on the template DNA sequence. AmpliTaq Gold® DNA polymerase is

commonly utilized for PCR reactions due to its thermal stability. Because non-modified

DNA polymerases may exhibit activity below their optimal working temperature,

primers can anneal non-specifically to the template at room temperature while the PCR

reaction is being set-up, resulting in non-specific amplified product (11). To avoid non-

specific products from forming, AmpliTaq Gold® is used because it has been chemically

modified to render it inactive until heated. An extended pre-incubation period of 95° C

for approximately 10 minutes is typically used to activate the AmpliTaq Gold®. When

the temperature is increased, the pH of the buffer decreases and the chemical moieties

of the AmpliTaq Gold® used to render it inactive are modified (11).

Multiplex PCR has become important in forensic DNA analysis because it offers

numerous advantages over the amplification of one locus at a time. One advantage is

the amount of labor involved and the time it takes to obtain results is decreased making

it an economical choice. Another advantage is the total amount of input DNA required

to obtain equivalently discriminatory results is also decreased. As previously discussed,

7

the development of an efficient multiplex PCR reaction requires extensive

experimentation in the area of primer design and optimization of reaction component

concentrations.

AmpFℓSTR® Identifiler®

The AmpFℓSTR® Identifiler® PCR Amplification Kit is capable of amplifying fifteen

STR loci and the sex-determining marker Amelogenin in a single amplification (11). The

Identifiler® kit amplifies the fifteen tetranucleotide STR loci; CSF1PO, D2S1338,

D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11,

FGA, TH01, TPOX, and vWA, as well as the sex-determining marker Amelogenin (11).

The fifteen locus combination included in Identifiler® is consistent with many worldwide

databases and includes all thirteen core loci utilized in the Combined DNA Index System

(CODIS) database, which contains the U.S.’s convicted offender database (13).

In addition to primer component design considerations mentioned previously,

non-nucleotide linkers are added to one of the primers in the following Identifiler® loci:

CSF1PO, D2S1338, D16S539 and TPOX. These non-nucleotide linkers provide

appropriate spacing between adjacent loci within a given color channel thereby

increasing the ability to separate fragments between loci (7). The combination of the

five-dye system and non-nucleotide linkers allows the same primer sequences to be

used that were previously developed for other amplification kits (12). Identifiler® is

improved compared to previous amplification kits because there are more alleles

represented in the allelic ladder, which decreases the number of “off-ladder” alleles

8

therefore reducing the amount of re-runs that would need to be performed on samples

(7). This multiplex kit uses a five flourophore chemistry that allows for the amplification

of more loci that are of similar size. The five dye chemistry allows these similar sized

loci to be differentiated. The five dyes used are 6-FAM™, VIC™, NED™, PET™, and LIZ™,

whereby LIZ™ is used to label the internal size standard. Each primer set is fluorescently

labeled with a flourophore at the 5’ end of one of the primers. Since each dye emits its

maximum fluorescence at a different wavelength, DNA amplicons of the same size can

be distinguished from emission differences between the fluorescent dyes (12).

AmpFℓSTR® Minifiler®

Although Identifiler® is a robust STR PCR multiplex system, forensic biological

samples are commonly degraded and/or inhibited, or simply contain little DNA. This is a

constant challenge that arises during forensic DNA typing, and often times larger loci do

not amplify resulting in partial genetic profiles. In response to this challenge, research

into the amplification of Mini-STRs has recently garnered a significant amount of

attention. As a result the AmpFℓSTR® Minifiler® PCR Amplification Kit was introduced to

the forensic arena in 2007. The kit has the capability to amplify the eight largest loci

contained within the AmpFℓSTR® Identifiler® kit (D13S317, D7S820, D2S1338, D21S11,

D16S539, D18S51, CSF1PO, and FGA) and the sex-determining marker, Amelogenin, in a

single PCR reaction (14).

The probability of obtaining genetic information from compromised samples

using Minifiler® is purported to be enhanced due to design changes. By decreasing the

9

PCR amplicon size and modifying reaction components, the amount of recommended

input DNA decreased from 0.5-1.25 ng to 0.5-0.75 ng for AmpFℓSTR® Identifiler® and

Minifiler® respectively. Additionally the amplicon size range changed from 101-356 to

70-283 nucleotides (12, 14). One of the reasons for Mini-STRs increased success in

amplifying degraded samples is the small size of the STR amplicons which have been

reduced by moving the PCR primers closer to the STR repeat region thereby closely

flanking the repeat regions of interest (15).

Studies with degraded and inhibited samples have shown that Minifiler® is

capable of producing a profile of 8 loci when traditionally used amplification kits have

resulted in partial profiles for 8 or fewer loci. More specifically, profiles have been

obtained using Minifiler® from degraded buccal swabs and samples inhibited by

nicotine, while the SGM Plus® kit was only able to produce a partial profile where only 2

loci amplified (16). Additionally, compromised samples which included bone, hair,

teeth, degraded blood, and saliva produced no profile or a partial profile when amplified

with Identifiler®. These same samples resulted in full profiles when amplified with

Minifiler® (17). Minifiler® was also able to verify the presence of a false homozygote

and artifact peaks that were observed when AmpFℓSTR® Identifiler® was utilized (17).

Based on the design and comparison studies, the use of truncated PCR amplicons or

“miniSTR” technology may prove useful for compromised forensic casework samples.

10

Capillary Electrophoresis

Following amplification of DNA samples, the resulting fluorescently labeled STR

regions need to be separated, sized, and genotyped. DNA samples are typically

separated using capillary electrophoresis. To accomplish this, the first step is to

combine a size standard and Hi-Di (highly-deionized) formamide with a portion of the

amplified sample. The deionized formamide is a denaturing solution that disrupts the

hydrogen bonds between the complementary strands of the PCR products (8). The

formamide is an essential component in keeping the samples in a denatured state and

dilutes any salts that are present which in turn aids the injection process, leading to

good resolution of closely spaced alleles (18). It is critical for the DNA to remain

denatured because only one strand of the DNA is fluorescently tagged and if the strands

are still in a double-stranded state it will alter the molecular weight of the strand,

modifying migration during electrophoresis and leading to a multitude of issues (8).

During electrophoresis, the salt content is also an important factor to consider. As the

salt levels in the sample increases, fewer DNA molecules are injected because they are

competing with the salt ions during electrokinetic injection (8). Therefore, it is

important to add the appropriate amount of formamide because it helps increase the

amount of DNA that is injected into the capillary by reducing any competing salts (8).

Since the multiple fluorescent dyes can be spectrally resolved, the various dye

colors are separated and the peaks representing the DNA fragments of interest are

identified and associated with the appropriate color. For both AmpFℓSTR® Identifiler®

11

and Minifiler® STR amplification fragment analysis, the internal size standard is LIZ™.

The internal size standard is used to appropriately size the DNA fragment using a Local

Southern and 3rd order least squares algorithm for Identifiler® and Minifiler®

respectively (12, 14, 19). To determine the number of STR repeats (i.e. allele call), the

size of the unknown allele is compared to the sizes of the known ladder alleles. Known

and unknown sizes must fall within a window of +/- 0.5 bp of each other in order for a

peak to be genotyped as a known allele (8). In summary, the basic steps involved in

estimating the sizes of the fragments and, hence the allele calls are as follows: after the

instrument collects the data points (scans) which includes the information about

fluorescent intensity at the various wavelengths, color separation is performed and

peaks are detected based on shape and user set threshold (20). For each sample the

resultant peaks are sized by either Local Southern or 3rd order least squares algorithms

and the sizes are compared to an allelic ladder as previously described (19, 20).

Mixture Analysis

STR Analysis in Forensic Casework

Many biological samples deposited and collected from crime scenes contain

mixtures from two or more individuals. The elucidation of individual donors in mixed

biological samples has traditionally been a challenge for forensic DNA analysts, and

remains one today. Determining the total number of contributors, the relative ratio of

DNA from each contributor, and whether a known individual is included or excluded as a

source continues to be difficult.

12

Although training for DNA analysts are offered and a number of publications

with information regarding mixture interpretation (21-24), several of these analyses are

constructed on a foundation of assumptions which assume amplification reproducibility.

Many of these studies use biological samples that are in pristine condition, however,

crime scene samples are often inhibited, degraded, and/or of low copy number. Since

many laboratories have only validated the manufacturer’s recommended protocol,

there continues to be a lack of additional validated procedures, which may result in

improved DNA profiles and more rigorous interpretation guidelines. Therefore,

incorporating a defined set of complex DNA mixture guidelines based on validation and

research with commercially available PCR human identity chemistries is critical.

Purpose of Study

In this study, amplification reproducibility was evaluated for single source

samples using the AmpFℓSTR® Identifiler® and Minifiler® amplification kits. Mixtures

were produced using various ratios of component DNAs. These findings were then

related to results observed in the single-source samples. More specifically, this project

focused on verifying and validating various assumptions used by analysts when

interpreting low level samples as well as mixture samples. The assumptions tested are

as follows:

1) Peak heights and peak height ratios do not significantly differ between loci,

amplifications or between kits (AmpFℓSTR® Identifiler® versus AmpFℓSTR®

Minifiler®).

13

2) The peak heights and peak height ratios do not significantly differ between

amplifications using a variety of target DNA amounts. That is, when deducing a

DNA contributor, is it reasonable to assume a generalized peak height ratio is

appropriate to use across all targets, some targets or does each individual target

result in a specific, but quantifiable peak height ratio?

3) Lastly, is a generalized peak height ratio, derived from validation studies

using single source samples at a particular target, is an appropriate value when

evaluating a DNA mixture profiles. That is, does the DNA of 2 or more individuals

amplify independently?

In this study the aforementioned assumptions were tested by amplifying single

source samples (male and female) using various target amounts (0.0625 - 4 ng), in

quadruplicate using the AmpFℓSTR® Identifiler® and Minifiler® Amplification Kits

(Applied Biosystems). Analysis of results focused on examining the reproducibility of

the peak heights and peak height ratios between amplifications, loci and input levels.

Statistical analysis using the F-test was utilized to determine whether the error in peak

height ratios remained the same despite target thereby assessing the validity of

assumption 2.

The DNA mixtures consisted of a 2-person mixture of human DNA from the same 2

sources. The samples were mixed at known ratios and amplified with AmpFℓSTR®

Identifiler® and Minifiler® with varying amounts of input DNA. The amplified products

were prepared for injection onto a 3130 Genetic Analyzer (Applied Biosystems) using a

14

five second injection. The profiles generated were then analyzed using GeneMapper®

ID version 3.2 (Applied Biosystems) and the peak heights and peak height ratios were

compared to the single source samples to assess the viability of assumption 3.

15

MATERIALS & METHODS

Extraction

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated.

An organic extraction was performed on whole-blood samples from two

individuals, one male and one female. A volume of 1 ml of each whole blood specimen

was aliquoted to 2 ml tubes and the volume adjusted to 1.5 ml using saline sodium

citrate (SSC). Each tube was mixed, then centrifuged for one minute and 1 ml of

supernatant was removed from each sample. An additional 1 ml of SSC was added to

each tube and gently shaken to re-suspend the cell pellets that formed at the bottom of

the tube after being centrifuged. The samples were centrifuged for one minute and 1.4

ml of supernatant was removed and discarded. A volume of 375 µL of 0.2 M sodium

acetate, 25 µL of 10% sodium dodecyl sulfate (SDS) and 3.2 µL of 31.5 mg/ml proteinase

K solution were added to each sample to re-suspend the pellets. The tubes were

vortexed and centrifuged to bring all of the liquid to the bottom of the tube. The

samples were then incubated overnight at 56° C.

The next day a phenol-chloroform purification was performed on the samples. A

volume of 500 µL of phenol-chloroform was added to each tube and mixed via hand

shaking. The samples were centrifuged for 2 minutes and the organic phase was

discarded. An additional 500 µL of chloroform was then added to the aqueous phase

and centrifuged for 2 minutes. The aqueous phase was removed and placed in a new

microcentrifuge tube. Next, 50 µL of 2 M sodium acetate and 0.8 µL of 20 mg/ml

16

glycogen were then added to the aqueous phase and the solution was mixed gently. A

volume of 500 µL of isoproponal was added and the tubes were gently hand shaken

until the mixing patterns disappeared. The samples were incubated overnight at -20° C.

After incubation, the samples were microcentrifuged for 30 minutes. The supernatant

was discarded and 1 ml of 80% ethanol was added and the samples were

microcentrifuged for 5 minutes. The supernatant was removed and the pellet air dried

and dissolved in 50 µL of tris-EDTA (TE) buffer at 56° C until dissolved.

Quantitation

On the Applied Biosystems 7500 Sequence Detection System (Foster City, CA), a

plate document was created to represent the arrangement of the samples and

standards on the reaction plate using the manufacture instructions (25). DNA standards

were prepared by obtaining and properly labeling 8 1.5 mL tubes and diluting the stock

standard from 200 ng/µL to 50 ng/µL by adding 10 µL of stock to 30 µL of standard.

Seven three-fold serial dilutions followed resulting in 8 standards ranging in

concentration from 50 ng/µL to 0.023 ng/µL.

The samples were quantified using the Applied Biosystems, Quantifiler Duo®

DNA Quantification Kit (Foster City, CA). Master Mix was prepared for the standards,

samples, reagent blanks, and a negative, according to the manufacturer instructions,

and the standards were run in duplicate. Master Mix (23 µL) was aliquoted into the

appropriate wells of a 96-well PCR microtitre plate. A volume of 2 µL of standard or

sample was pipetted into the appropriate wells based on the created plate document.

17

The plate was then sealed and vortexed to ensure no bubbles were present. The plate

was then placed in a 7500 Sequence Detection System and run according to the

manufacturer instructions (25).

Amplification

Using the data provided from the quantitation, a dilution series was created for

each of the samples where the target DNA amounts were 0.0625, 0.125, 0.25, 0.5, 1.0,

1.5, 2, and 4 ng. Each DNA target was amplified in quadruplicate by pre-mixing the

appropriate amount of master mix and DNA together for four reactions. The master mix

and sample was pre-mixed to reduce the pipetting error, hence reducing imprecision in

the amplification. Amplifications were performed using AmpFℓSTR® Identifiler® and

Minifiler® Amplification Kits (Applied Biosystems). Mixtures of 1:1, 1:2, 1:4, 1:9, and

1:19 (male: female) were prepared using the single source DNA extractions. Target

amounts of 0.0625, 0.125, 0.25, 0.5, 1.0, 2, and 4 ng of each mixture were amplified

once using the AmpFℓSTR® Identifiler® Kit and Minifiler® Kit.

AmpFℓSTR® Identifiler®

Each amplification was performed by adding 10.5 µL AmpFℓSTR® PCR Reaction

Mix, 0.5µL AmpliTaq Gold® DNA Polymerase, and 5.5 µL AmpFℓSTR® Identifiler® primer

set and a total of 10 µL of sample. All reactions were performed on a GeneAmp PCR

System 9700 (Applied Biosystems) using the 9600 emulation mode. The thermal profile

consisted of an 11 minute incubation at 95° C, followed by 28 cycles of three-step PCR at

18

94° C, 59° C, and 72° C each for 1 minute, and concluded with a final hold at 60° C for 60

minutes (12).

AmpFℓSTR® Minifiler®

Each amplification was performed by adding 10 µL AmpFℓSTR® Minifiler® Master

Mix and 5 µL AmpFℓSTR® Minifiler® primer set multiplied by the number of samples.

The thermocylcer was programmed with the following conditions: an initial incubation

of 95° C for 11 min followed by 30 cycles of 94° C for 20 seconds, 59° C for 2 minutes,

72° C for 1 minute, and a final extension of 60° C for 45 minutes (14).

Electrophoresis, Detection, and Analysis

All PCR products were separated using the Applied Biosystems 3130 Genetic

Analyzer using POP-4™ polymer (Foster City, CA). A plate map and results group were

created using the Applied Biosystems 3130 collection software along with the

appropriate instrument protocol for use with the AmpFℓSTR® Identifiler® and

AmpFℓSTR® Minifiler® amplification kits respectively (12, 14).

A master mix of Hi-Di Formamide (8.3 µL/sample) and GeneScan™ 600 LIZ™ Size

Standard (0.7 µL/sample) was aliquoted into a 96 well plate and 1 µL of amplified

product was added to the appropriate wells according to the plate document. A septa

was placed on the plate, the plate was then vortexed and pulse-spun to remove all

bubbles. The samples were denatured at 95° C for 3 minutes and cooled at -20° C for 3

minutes. The plate was then placed in the 3130 Genetic Analyzer and injected for 2, 5,

19

and 10 seconds at 3 kV. Only data from the five second injection were analyzed in this

work.

Minimum Distinguishable Signal

To determine a minimum signal/relative fluorescent unit (RFU) at which to

analyze the samples on GeneMapper® ID v 3.2, approximately 32 blanks (Hi-Di

formamide and LIZ™ 600) were injected on the 3130 Genetic Analyzer at 2, 5, and 10

seconds. This data was analyzed using the Genemapper® ID v. 3.2 using an RFU

threshold of 5. Peaks that were within +/- 2 bases from the LIZ™ 600 size standard were

removed. The largest baseline peak at a given locus was used to calculate the average

baseline signal per locus. Additionally, the standard deviation, the average plus three

times the standard deviation, and the average plus ten times the standard deviation

were calculated (26). The RFU threshold for single source samples amplified with both

AmpFℓSTR® Identifiler® and Minifiler® was set at 16 RFU, based on the highest mean

plus 3 standard deviations. A threshold of 30 RFU for mixture samples was based on the

highest mean plus ten times the standard deviation of the blank signals. Table 1 shows

the averages of the three injection times plus 3 and 10 standard deviations calculated

across all loci. Shown in Table 1, FGA has a minimum distinguishable signal (MSD) of

15.8 and TPOX has a minimum quantifiable signal (MQS) of 28.19, which were rounded

up to 16 and 30 RFU respectively for analysis.

20

Table 1. Minimum Distinguishable Signal ( blY + 3 bls ) for each locus and Minimum

Quantifiable Signal ( blY + 10 bls ) calculated via analysis of the baseline derived from blanks.

Locus Average + (3*SD) Average + (10*SD)

CSF1PO 7.20 11.40

D21S11 6.21 8.55

D7S820 6.91 10.34

D8S1179 7.87 13.35

D13S317 8.01 13.62

D16S539 7.56 12.13

D2S1338 8.64 15.45

D3S1358 10.06 19.11

TH01 8.71 15.23

AMEL 14.08 26.74

D5S818 14.70 23.83

FGA 15.80 28.15

D18S51 13.49 21.17

D19S433 13.87 22.68

TPOX 13.93 28.19

vWA 13.31 20.84

Data Analysis

The data collected during capillary electrophoresis was imported into Applied

Biosystems GeneMapper® ID software v 3.2 (Foster City, CA). The Local Southern sizing

method was used for samples amplified with AmpFℓSTR® Identifiler® and 3rd order least

squares sizing was used for samples amplified with AmpFℓSTR® Minifiler®. Artifact

peaks such as bleed through, stutter, spikes, etc., were removed before exporting the

data for statistical analysis. Once all labeled artifact peaks were analyzed and removed,

the sample name, locus, allele label, and peak height were exported from GeneMapper®

21

ID into Microsoft® Excel 2003. The Data Analysis Tool Pak add-in was used for statistical

analysis.

Average peak height and peak height ratios at each locus were calculated for

each individual amplification (8 total – 2 samples amplified in quadruplicate) and

charted. The average peak height across the 8 amplifications at each target amount was

calculated as well as three times the standard deviation. Additionally, the variation in

peak height ratios between target amounts was examined by utilizing the F-test

performed in Excel using the Data Analysis Tool Pak add-in (Equation 1) (28).

(Equation 1)

The F-test was used to compare precision between peak height ratios for two

sets of data. In this case, it was utilized to test whether the null hypothesis (Ho) that

there is no significant difference between the variance of peak height ratios among

target amounts. If the Ho is rejected it suggests there may be observable stochastic

effects and/or amplification variation between targets. If significant differences of

variances of peak height ratios between targets exist, the F-test will allow for the

elucidation of the presence of stochastic effects and at which target significant

differences are to be expected. Comparisons were made between the errors of the

target showing the least variance versus the remaining sample sets.

2

2

2

10 : H 2

2

2

1:

calcF

22

RESULTS & DISCUSSION

Five second injection times were analyzed for samples amplified by AmpFℓSTR®

Identifiler® and AmpFℓSTR® Minifiler® Amplification Kits. Table 2 shows the genotypes

assigned at each locus for both samples used in this study, as well as the dye color

associated with each locus.

Table 2. STR profiles for both samples used in this study, determined by amplification via AmpFℓSTR® Identifiler® and AmpFℓSTR® Minifiler® Kits.

Locus Sample 1

Sample 2 Dye Color

Identifiler® Minifiler®

AMEL X, Y X Red Green

CSF1PO 12, 14 10, 12 Blue Red

D13S317 12 10, 13 Green Blue

D16S539 8, 11 9, 13 Green Yellow

D18S51 15 14, 20 Yellow Yellow

D19S433 14, 15 13, 14 Yellow

D21S11 28, 29 30, 31 Blue Green

D2S1338 16, 18 16, 19 Green Green

D3S1358 14, 15 16 Green

D5S818 11 13 Red

D7S820 10, 11 10 Blue Blue

D8S1179 13, 14 12 Blue

FGA 21, 24 21, 23 Red Red

TH01 9, 9.3 9, 9.3 Green

TPOX 8 8, 11 Yellow

vWA 17 15 Yellow

Peak Heights

Figure 1A shows a sample electropherogram of the blue-dye channel for both

AmpFℓSTR® Indentifiler® and Minifiler® amplification kits. Figure 1B shows three times

the relative standard deviation (Equation 2) across all loci included in the AmpFℓSTR®

23

Identifiler® and Minifiler® Amplification Kits at varying target amounts. Minimum and

maximum peak heights and the range of average peak heights for each target for loci

included in both amplification kits are presented in Table 3.

Identifiler®

Minifiler®

Figure 1A. A sample electropherogram of the blue-dye channel of AmpFℓSTR® Identifiler® and Minifiler® amplification kits for one sample at a 1 ng target with a 5 second injection.

24

0

50

100

150

200

250

300

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

3*R

SD %

Identifiler Minifiler

Figure 1B. 3*RSD (Relative Standard Deviation) of all peak heights at 9 loci (Amelogenin, CSF1PO, D13S317, D16S539, D18S51, D21S11, D2S1338, D7S820, and FGA) included in AmpFℓSTR® Identifiler® and Minifiler® Amplifications Kits. The results are from four repeat amplifications of two single source samples with targets ranging from 0.0625 to 4 ng.

25

Table 3. Minimum and Maximum Peak Height and Range of Average Peak Heights for each target for all loci amplified with both AmpFℓSTR® Identifiler® and Minifiler® for single source data. Target

(ng)

Identifiler®

Minifiler®

Min-Max Peak Height (RFU)

Range of Average Peak Heights (RFU)

Min-Max Peak Height (RFU)

Range of Average Peak Heights (RFU)

0.0625 16-245 27-49 16-431 56-139

0.125 19-238 47-83 16-504 109-184

0.25 42-473 98-189 33-751 209-387

0.5 137-902 208-350 154-1626 443-808

1 300-1277 375-695 251-3085 850-1482

2 561-2638 752-1383 109-7033 1831-2814

4 1073-4409 1307-2796 N/A N/A

N/A=Not Applicable (Minifiler® results at 4 ng were not included in this study)

The relative standard deviation (RSD) is widely used to express the precision and

repeatability of an assay and is calculated as follows:

RSD = (standard deviation/average) × 100% (Equation 2)

In this case three times the relative standard deviation is charted. Table 3 shows that at

a given target Minifiler® has higher average peak heights suggesting it is more sensitive

at a given target amount, especially when less than 0.25 ng of DNA is amplified.

However, it should be noted that Table 3 also shows the overall range (min-max) and

the range of average peak heights, which is larger for Minifiler® as corroborated by the

larger RSD of peak heights for Minifiler® shown in Figure 1B.

Although Minifiler® seems to have increased variability over Identifiler®, both

kits show increased variability at lower DNA input levels. Qualitatively, Figure 1B shows

26

that Identifiler® and Minifiler® RSD’s appear to be insignificantly different between kits

when less than 0.25 ng of DNA is amplified. Additionally, using a five second injection

time and a threshold of 16 RFUs (for single source data), full and partial profiles were

obtained at 0.0625 ng and 0.125 ng for both kits. Table 4 shows the number of alleles

that were below the MDS at each locus at the 0.0625 ng and 0.125 ng target for each

kit. Drop out of alleles was not observed at targets greater than 0.125 ng for either kit.

Qualitatively, Figure 1B shows Identifiler® variation seems to increase at 0.25 ng and

increases from 113% to 135% to 195% at 0.5, 0.25 ng, and 0.0625 ng respectively. This

is in contrast to Minifiler® which shows a significant increase in RSD starting at 0.125 ng.

Although Minifiler® appears more stable regarding peak height over a larger target

range; it consistently has larger variation than Identifiler® at every target. This effect of

variation is seen when comparing drop out rates between Identifiler® and Minifiler®,

whereby at 0.0625 and 0.125 ng Minifiler®’s drop out rate is not significantly better than

that of Identifiler®. This suggests that although Minifiler® on average produces higher

signal, its amplification variability may not necessarily lead to samples with more

powerful discrimination capability.

27

Table 4. Number of alleles below the minimum distinguishable signal (16 RFU) at 0.0625 and 0.125 ng targets for AmpFℓSTR® Identifiler® and Minifiler® Kits.

Locus 0.0625 ng 0.125 ng

Identifiler® Minifiler® Identifiler® Minifiler®

AMEL 1 1

CSF1PO 2 1

D13S317 1

D16S539 2 2

D18S51 1

D19S433 3 N/A N/A

D21S11 3 1

D2S1338 2 3 1

D3S1358 3 N/A N/A

D5S818 N/A N/A

D7S820 2 1

D8S1179 1 N/A 1

FGA 1 2

TH01 1 N/A N/A

TPOX N/A N/A

vWA N/A N/A

N/A=Not Applicable (Minifiler® does not contain these loci) Blank cells indicate that alleles at that locus were above the MSD

To further study this, Figures 2A to 2I show the peak heights for each locus for

four repeat amplifications of a single source sample with targets ranging from 0.0625 to

4 ng for Identifiler® and from 0.0625 to 2 ng for Minifiler® with error bars of +/- 3 SD.

As indicated in Table 3, Minifiler® appears to be more sensitive at a given input amount;

however, for most loci Minifiler® has increased variance even at the larger target

amounts. As an example, Figure 2B shows the average peak height obtained over 8

amplifications for CSF1PO, the 1 ng Identifiler® average peak height is 663 RFU while for

Minifiler® it is 1482 RFU suggesting a more sensitive test, but if one is to define

28

sensitivity by the ability to discriminate the change in RFU (signal) with the change in

target while taking into account standard deviation at a given concentration, the

sensitivity of Minifiler® is not greater than the sensitivity of Identifiler®. To elucidate

further, Figure 3A shows the calibration sensitivity (defined as the slope of a signal

versus concentration (Co) plot) of locus CSF1PO is 1473 RFU/ng and 659 RFU/ng for

Minifiler® and Identifiler® respectively. However, the analytical sensitivity is defined as:

Analytical Sensitivity = Slope/Standard Deviation Co (Equation 3)

As an example, the analytical sensitivity at 0.5 ng for Minifiler® and Identifiler® for

CSF1PO is 7.18 and 13.18 respectively. Similarly, Figure 3B shows the calibration

sensitivity and variances of Minifiler® and Identifiler® for locus D16S539. In summary,

the analytical and calibration sensitivity at 0.5 ng for each locus and kit are listed in

Table 5. At all but two loci, FGA and D21S11, Identifiler® has a higher analytical

sensitivity, suggesting that in fact even though its calibration sensitivity is lower it is the

more sensitive of the two kits at 0.5 ng.

Table 5. Analytical and Calibration Sensitivities for AmpFℓSTR® Identifiler® and Minifiler® at 0.5 ng.

Locus Analytical Sensitivity Calibration Sensitivity

Identifiler® Minifiler®

Identifiler® Minifiler®

CSF1PO 14.58 7.62 659 1473 D21S11 8.05 11.21 558 1256 D7S820 12.18 6.46 571 1062 D13S317 7.66 5.56 517 844 D16S539 10.44 8.30 682 1092 D2S1338 13.38 5.25 525 1256 AMEL 10.16 7.04 464 1268 FGA 11.66 14.33 373 1260 D18S51 8.03 6.37 502 1183

29

Amplification of a 4 ng target amount was performed for the Minifiler® kit;

however, that data was not used to due to detector saturation or excessive pull-up.

Pull-up is the observation of a single peak in more than one color because of the

spectral overlap between the fluorescent dyes used to tag the DNA strand that can not

be spectrally resolved; hence, it is then visualized as extra peak(s) in the data. In

addition to having pull-up peaks, Moretti, et al., suggests avoiding large DNA template

amounts since it can lead to non-allelic amplification, such as stutter and minus-A

products and the percent stutter may appear to be increased if an allele exceeds the

instrument’s detection limit (28).

Although the 4 ng data is not included here, it demonstrates Minifiler®’s ability

to successfully and efficiently amplify DNA. Mulero et al., performed a validation of the

Minifiler® amplification kit. During the validation, a sensitivity study using serial

dilutions of two DNA samples was performed. It was found that the optimal quantity of

template DNA ranged from 0.5 to 0.75 ng (29), which is in concordance with the

suggested input range from the Minifiler® Users Guide (ABI). Full profiles were obtained

with as little as 0.125 ng. The injection time for this particular study was 10 seconds and

allele peaks were interpreted when greater than or equal to 50 relative fluorescence

units (RFUs) (29). This is in concordance with the findings of this study, where full

profiles were obtained with a 5 second injection time and out of the 4 amplifications

only 3 alleles were below detectable levels at 0.125 ng (Table 4). Additionally, the

majority of the allele peaks in this study were greater than 50 RFUs. Andrande et al.,

30

amplified challenging samples with Minifiler® that had previously resulted in partial

profiles or no profiles with the use of Identifiler® (17). Minifiler® produced complete

profiles and verified false homozygotes and artifact peaks produced with Identifiler®

(17). Similar results were produced with the Minifiler® kit which resulted in full profiles

for DNA input amounts as low as 40 pg. This was in contrast to SGM Plus® which was

unable to produce complete profiles at such a low target (16). However, it should be

noted that not only does the size of the amplicon change, but also the thermocylcing

parameters (i.e. 28 versus 30 cycles, 2 minutes versus 1 minute annealing time). The

better performance is due to both factors and cannot be attributed solely to the

amplicon size.

The Identifiler® kits recommended target amount is 0.5 to 1.25 ng (12), which

does not coincide with the results in Figures 1B and 2. Figure 1B shows that the

variation of 2 and 4 ng samples are not significantly different from 1 and 0.5 ng, but the

peak heights are; therefore, the recommended target amount for this laboratory is 2 ng

using a 5 second injection time. Collins et al., also tested the Identifiler® amplification

kit, where amplification performance was assessed using a range of DNA input amounts

from 0.03125 to 1.25 ng (7). Triplicate amplifications were performed and full profiles

were generated for all but the two lowest template levels using a 50 RFU peak threshold

(7), again corroborating the findings of this study which suggests full profiles can be

obtained with the recommended input range of 0.5 to 1.25 ng. It also showed the

Identifiler® kits ability to obtain full profiles at 0.125 ng. However, it is important to

31

note that Collins et al., did not specify the injection time for the samples, only that they

were injected twice (7). Generally, these findings are similar to the results of this work,

which suggest Identifiler® is a capable and powerful human identification chemistry

which can successfully be utilized for forensic purposes. However, due to differences in

instrument sensitivities and quantification practices the optimal DNA input was

determined to be 2 ng which is larger than the 0.5-1.25 ng suggested by others.

Others have attempted to determine an optimal input of DNA for other

commercially available chemistries. Using a variety of amplification kits including

AmpFℓSTR® Profiler Plus®, AmpFℓSTR® Cofiler®, and Geneprint™ PowerPlex®, Moretti et

al., obtained the best results using a DNA template amount between 0.5 and 2 ng and

was able to obtain partial profiles with 0.078 ng (28). Another validation study using the

PowerPlex® 16 system and data from 19 different laboratories found that full profiles

were consistently generated when using 0.25 to 2 ng of input DNA template (30).

Twenty-four laboratories genotyped single source samples using PowerPlex® 16. All

labs produced reliable genotypes using 0.5 and 1 ng of input DNA. Only two of the

laboratories had difficulty detecting all alleles when using 0.25 ng of DNA; though this

was probably because they had a threshold of 150 RFU (30). The use of multiplex kits

across different laboratories shows the reproducibility and reliability of STR systems.

Based on the peak heights and RSD’s, Identifiler® and Minifiler® target

amplifications should be 2 ng for a 5 second injection time. The RSD for Minifiler®

shows little variability from 0.25 ng to 2 ng as can be seen in Figure 1B. The drop-out

32

rate and analytical sensitivity suggest that Identifiler® is the more sensitive of the two

kits and is a more robust kit because it is capable of obtaining a full 16 loci profile for

samples as low as 0.125 ng. Therefore, Minifiler® should only be considered for samples

that are severely compromised, since a kit-to-kit comparison suggests Identifiler®

amplification chemistries do not lead to a higher drop-out rate for pristine samples and

is more sensitive than Minifiler®.

A) B)

Amelogenin

-1000

0

1000

2000

3000

4000

5000

6000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler

CSF1P0

-1000

0

1000

2000

3000

4000

5000

6000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler C) D)

D13S317

-1000

0

1000

2000

3000

4000

5000

6000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (R

FU)

Identifiler Minifiler

D16S539

-500

0

500

1000

1500

2000

2500

3000

3500

4000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak H

eig

ht

(rfu

)

Identifiler Minifiler

33

E) F)

D18S51

-1000

0

1000

2000

3000

4000

5000

6000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler

D21S11

-1000

-500

0

500

1000

1500

2000

2500

3000

3500

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak H

eig

ht

(rfu

)

Identifiler Minifiler G) H)

D2S1338

-1000

0

1000

2000

3000

4000

5000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler

D7S820

-5000

50010001500200025003000350040004500

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler I)

FGA

-1000

0

1000

2000

3000

4000

5000

6000

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t (r

fu)

Identifiler Minifiler

34

J)

Average Peak Height for Identifiler-Specific Loci

-1000

0

1000

2000

3000

4000

5000

6000

7000

0.0625 0.125 0.25 0.5 1 2 4

Target DNA (ng)

Pe

ak H

eig

ht

(rfu

) D3S1358

TH01

D19S433

TPOX

D8S1179

vWA

D5S818

Figure 2. Locus specific average peak heights and 3 standard deviations for four repeat amplifications of 2 single source samples with targets ranging from 0.0625 to 4 ng for AmpFℓSTR® Minifiler® and Identifiler®. A) Amelogenin B) CSF1PO C) D13S317 D) D16S539 E) D18S51 F) D21S11 G) D2S1338 H) D7S820 I) FGA J) Identifiler®-specific loci.

A)

CSF1PO

y = 1473x + 27.086

y = 658.84x + 3.9479

-500

0

500

1000

1500

2000

2500

0 0.25 0.5 0.75 1

Target (ng)

Pe

ak H

eig

ht

(RFU

)

Identifiler

Minifiler

Linear (Minifiler)

Linear (Identifiler)

35

B)

D16S539

y = 1092.4x - 9.7794

y = 681.73x - 2.5938

0

200

400

600

800

1000

1200

1400

1600

0 0.25 0.5 0.75 1

Target (ng)

Pe

ak H

eig

ht

(RFU

)

Identifiler

Minifiler

Linear (Minifiler)

Linear (Identifiler)

Figure 3. An example of average peak heights and one standard deviation for four repeat amplifications of 2 single source samples with targets ranging from 0.0625 to 1 ng for AmpFℓSTR® Minifiler® and Identifiler®. Peak Height Ratios

Perfect PCR amplification would result in a peak height ratio of 1 between sister

alleles at a locus. However, imbalances occur when there is little DNA, degraded DNA,

and/or inhibited DNA, in the extreme results in loss of one or both alleles. Allelic

imbalance can become a problem when trying to interpret a DNA mixture. This

imbalance can make it difficult to determine the number of contributors and difficult to

identify a specific contributor (31). Characterizing allelic imbalance for an amplification

kit using a single source sample is important in determining an amplification kit’s

reliability, and in turn determining guidelines on how to use peak height ratios for

mixture interpretation.

36

Peak height ratios were calculated by dividing the peak height of the smaller

allele (RFU) by the peak height of the larger allele (RFU) at a locus. Peak height ratios

were calculated for each heterozygous locus and amplification for both samples

resulting in an n = 4 or n = 8 (assuming no dropout) depending on the locus. Those

ratios were then averaged for each input amount and 3 standard deviations from the

mean were calculated. Table 6 shows the range of ratios at a given target for both

Identifiler® and Minifiler® amplifications. As expected, peak height ratio decreases with

a decreasing target. Unexpectedly, Identifiler® had better peak height ratios at the

lower target amounts and maintained it throughout the larger target amounts as well.

Table 6. Average Peak Height Ratio Range for each target amplified with the AmpFℓSTR® Identifiler® and Minifiler® Kits.

Target (ng)

Identifiler® Average Peak Height Ratio Range

Minifiler® Average Peak Height Ratio Range

0.0625 0.52-0.88 0.38-0.64 0.125 0.54-0.91 0.43-0.78 0.25 0.60-0.86 0.63-0.87 0.5 0.76-0.95 0.63-0.86 1 0.79-0.94 0.72-0.85 2 0.86-0.95 0.84-0.90 4 0.85-0.96 N/A

N/A=Not Applicable (Minifiler® results at 4 ng were not included in this study)

Figure 4A to 4I shows the peak height ratios for each locus for both amplification kits.

Qualitatively, when examining the error bars it is observed that some of the loci show

extreme variation at the lower target amounts for both kits, for example CSF1PO and

D16S539 have large error bars at the 0.0625 ng and 0.125 ng targets for both kits. Also

there appears to be only minor differences in peak height ratios between targets

37

greater than or equal to 0.5 for both kits. These results coincide with the suggested

input amounts for Minifiler® (0.5 – 0.75 ng) and Identifiler® (0.5-1.25 ng). The peak

height ratios were also balanced between loci within the 0.5-2 ng target amounts for

both kits. Below 0.5 ng, the Identifiler®-specific loci begin to show greater peak height

ratio variation between loci, suggesting imbalance at lower target amounts. The

Identifiler® user guide reports a range of 0.43 to 1.00 for their minimum and maximum,

and an average peak height ratio range of 0.82 to 0.90 (12). However the peak height

ratios were only determined for heterozygous samples with a peak height greater than

200 RFU (12). The minimum and maximum average peak height ratio range for

Identifiler® in this study for 0.5 to 2 ng was 0.76 and 0.95 respectively, seen in Table 6.

Using a threshold as a high as 200 RFU can contribute to the well balanced peak heights

that ABI reported, by reducing the possibility of stochastic effects seen at lower

thresholds.

Collins et al., also observed similar peak height ratio results with 0.0625 to 1.25

ng of input DNA and found little variation between template amounts (7). Using the

PowerPlex® 16 System, Krenke et al., examined peak height balance by twenty-four

laboratories and found relatively consistent peak balance at 1 ng, where the mean peak

height ratio was 0.9. This is similar to the results obtained for a 2 ng target for

Identifiler® shown in Table 6. The mean peak balance was 0.87 at 0.5 ng and 0.84 at

0.25 ng (30). Peak height ratios of 0.91-0.92 for database samples and 0.84 to 0.90 for

casework samples using a 2.5 ng target have also been reported (31).

38

A) B)

Amelogenin

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t R

ati

o

Identifiler Minifiler

CSF1PO

-1

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak H

eig

ht

Ra

tio

Identifiler Minifiler

C) D)

D13S317

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t R

ati

o

Identifiler Minifiler

D16S539

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pea

k H

eigh

t R

atio

Identifiler Minifiler E) F)

D18S51

-1

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pea

k H

eigh

t R

atio

Identifiler Minifiler

D21S11

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pea

k H

eigh

t R

atio

Identifiler Minfiler

39

G) H)

D2S1338

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t R

ati

o

Identifiler Minifiler

D7S820

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pea

k H

eigh

t R

atio

Identifiler Minifiler I)

FGA

-1

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

t R

ati

o

Identifiler Minifiler

40

J)

Average Peak Height Ratios for Identifiler-Specific Loci

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.0625 0.125 0.25 0.5 1 2 4

Target DNA (ng)

Pe

ak H

eig

ht

Rat

ios

D19S433

D8S1179

THO1

TPOX

D3S1358

Figure 4. Locus specific average peak height ratios and 3 standard deviations for heterozygote loci A) Amelogenin B) CSF1PO C) D13S317 D) D16S539 E) D18S51 F)D21S11 G) D2S1338 H)D7S820 I)FGA J) Identifiler®-specific loci with error bars showing 3 standard deviations from the mean. The results depicted are from four amplifications of two single source samples of DNA with targets ranging from 0.0625 to 4 ng.

To quantitatively assess differences in peak height ratio variances, the F-test was

used to compare precision between two sets of data. In this case, it was utilized to test

the Ho that there is no significant difference between peak height ratio variances of

varying target amounts. If the Ho is rejected it suggests there is an observable stochastic

effect. The F-test allows for the elucidation of which target the stochastic effects were

expected to become significant. A symbol () indicates the null hypothesis is accepted

and () indicates the null hypothesis is rejected. Comparisons were made between the

41

errors of the target amount showing the least variance versus the remaining sample

sets. Confidence intervals of 0.05 and 0.003 were compared for each target.

The F-test was performed on each locus where one or both samples were

heterozygote at that specific locus; this was performed for each amplification kit. Table

7 shows Minifiler® amplification kit results. The F-test table for D13S317 shows that

using a 0.05 confidence interval the Ho is rejected for a target amount less than 0.25 ng,

but the Ho is not rejected for any of the target amounts when using a 0.003 confidence

interval. Peak height ratios significantly differ at low targets. For the Minifiler® kit using

the 0.05 confidence interval the Ho is accepted at a target amount equal to or greater

0.25 ng for 6 of the 9 loci. However, if using the 0.003 confidence interval the null

would be accepted at a target amount equal to or greater than 0.125, for 8 of the 9 loci.

Table 7. F-test for the comparison of the variances of peak height ratio for loci in the

AmpFℓSTR® Minifiler® kit.

Amelogenin CSF1PO

Target

vs 2

Fcal Fcritical Ho Target

vs 0.5

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

1 1.4 9.3 67.2 2 8.9 3.8 10.5

0.5 2.9 9.3 67.2 1 1.2 3.8 10.5

0.25 3.5 9.3 67.2 0.25 2.1 3.8 10.5

0.125 2.3 9.3 67.2 0.125 8.8 3.8 10.5

0.0625 5.1 9.6 70.6 0.0625 7.6 4.0 10.5

42

D13S317 D16S539

Target

vs 2

Fcal Fcritical Ho Target

vs 2

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

1 7.9 9.3 67.2 1 1.3 3.8 10.5

0.5 5.2 9.3 67.2 0.5 4.9 3.8 10.5

0.25 8.3 9.3 67.2 0.25 3.0 3.8 10.5

0.125 9.4 9.3 67.2 0.125 6.2 3.8 10.9

0.0625 10.4 10.1 70.6 0.0625 3.9 4.0 11.3

D18S51 D21S11

Target

vs 0.5

Fcal Fcritical Ho Target

vs 2

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 2.9 9.3 67.2 1 2.6 3.8 10.5

1 1.4 9.3 67.2 0.5 6.2 3.8 10.5

0.25 3.8 9.3 67.2 0.25 21.3 3.8 10.5

0.125 5.9 9.3 67.2 0.125 21.2 3.8 10.5

0.0625 26.8 9.3 67.2 0.0625 20.1 3.9 10.9

D2S1338 D7S820

Target

vs 2

Fcal Fcritical Ho Target

vs 2

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

1 4.3 3.8 10.5 1 3.7 9.3 67.2

0.5 4.1 3.8 10.5 0.5 3.3 9.3 67.2

0.25 9.5 3.8 10.5 0.25 3.4 9.3 67.2

0.125 5.6 3.9 10.9 0.125 3.5 9.3 67.2

0.0625 12.1 4.1 12.0 0.0625 8.8 9.6 70.6

43

FGA

Table 8 shows the Identifiler® amplification kit F-test results for 14 loci; the loci

D5S818 and vWA were homozygote for both samples. Peak height ratios significantly

differ at low targets. The lowest target amount that had the least amount of variance

for any of the loci was 0.5 ng. Using the 0.05 confidence interval the null hypothesis is

rejected at a target amount equal to or less than 0.5 ng for 9 of the 14 loci. This is a

significant increase from 2 or 3 of 14 loci at 1 or 2 ng respectively and suggests that

stochastic effects begin to become observable for Identifiler® at targets as high as 0.5 ng

and become worse as input levels decrease when compared within a kit. In contrast,

Minifiler®’s stochastic effects start to become observable at 0.125 ng at a 0.05

confidence interval. However, this may be a direct result of the overall large variance

seen in Minifiler®’s peak height ratios even at large targets. The comparison of the F-

test values is simply used as a measure to distinguish at which point stochastic effects

become observable within a kit. It is expected that lower target amounts would

experience greater variation due to stochastic effects that occur in lower input amounts

Target

vs 1

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 9.9 3.8 10.5

0.5 2.1 3.8 10.5

0.25 3.8 3.8 10.6

0.125 7.1 3.9 10.9

0.0625 12.3 4.1 11.975

44

of DNA. Lower targets experience more drop out events, especially with an allele at a

heterozygous locus where one allele may be preferentially amplified over the other.

Table 8. F-test for the comparison of the variances of peak height ratio for loci in the AmpFℓSTR® Identifiler® kit.

Amelogenin CSF1PO

Target

vs 1

Fcal Fcritical Ho Target

vs 1

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

4 2.8 9.3 67.2 4 5.1 3.8 10.5

2 12.3 9.3 67.2 2 5.4 3.8 10.5

0.5 3.6 9.3 67.2 0.5 25.0 3.8 10.5

0.25 16.7 9.3 67.2 0.25 13.2 3.8 10.5

0.125 30.0 3.8 67.2 0.125 65.1 3.9 10.9

0.0625 16.1 9.6 14.0 0.0625 52.0 3.8 10.5

D13S317 D16S539

Target

vs 2

Fcal Fcritical Ho Target

vs 4

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

4 8.1 9.3 67.2 2 1.7 3.8 10.5

1 5.6 9.3 67.2 1 1.2 3.8 10.5

0.5 30.1 9.3 67.2 0.5 1.3 3.8 10.5

0.25 43.8 9.3 67.2 0.25 1.1 3.8 10.5

0.125 147 9.3 67.2 0.125 6.9 3.8 10.5

0.0625 21.9 9.3 67.2 0.0625 5.9 3.8 10.5

45

D18S51 D21S11

Target

vs 4

Fcal Fcritical Ho Target

vs 2

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 1.4 3.8 10.5 4 4.1 3.8 10.5

1 2.7 3.8 10.5 1 5.3 3.8 10.5

0.5 4.6 3.8 10.5 0.5 20.0 3.8 10.5

0.25 1.1 3.8 10.5 0.25 24.5 3.8 10.5

0.125 20.9 3.8 10.5 0.125 45.8 3.9 10.9

0.0625 10.3 9.3 67.2 0.0625 18.3 4.1 12.0

D2S1338 D7S820

Target

vs 4

Fcal Fcritical Ho Target

vs 0.5

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 1.4 3.8 10.5 4 10.5 9.3 67.2

1 2.7 3.8 10.5 2 5.3 9.3 67.2

0.5 4.6 3.8 10.5 1 7.9 9.3 67.2

0.25 1.1 3.8 10.5 0.25 43.6 9.3 67.2

0.125 20.9 3.8 10.5 0.125 2.6 9.3 67.2

0.0625 13.9 4.0 11.3 0.0625 7.9 9.3 70.6

FGA D3S1358

Target

vs 4

Fcal Fcritical Ho Target

vs 4

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 1.7 3.8 10.5 2 2.0 9.3 67.2

1 4.4 3.8 10.5 1 8.8 9.3 67.2

0.5 6.9 3.8 10.5 0.5 4.4 9.3 67.2

0.25 11.7 3.8 10.5 0.25 11.0 9.3 67.2

0.125 9.2 3.8 10.5 0.125 3.6 9.3 67.2

0.0625 17.2 3.866 10.9 0.0625 N/A N/A N/A N/A N/A

N/A= Not Applicable due to high # of samples with allele drop out

46

D8S1179 D19S433

Target

vs 4

Fcal Fcritical Ho Target

vs 2

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 3.53 9.28 67.2 4 1.03 3.79 10.5

1 19.9

3

9.28 67.2 1 1.29 3.79 10.5

0.5 12.2

6

9.28 67.2 0.5 3.70 3.79 10.5

0.25 11.9

8

9.28 67.2 0.25 5.12 3.79 10.5

0.125 49.6

6

9.55 70.6 0.125 6.67 3.79 10.5

0.0625 88.0

5

9.55 70.6 0.0625 5.24 4.37 13.0

TPOX THO1

Target

vs 4

Fcal Fcritical Ho Target

vs 4

Fcal Fcritical Ho

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

α=

0.05

α=

0.003

2 9.2 9.3 67.2 2 2.8 3.8 10.5

1 7.5 9.3 67.2 1 1.9 3.8 10.5

0.5 15.2 9.3 67.2 0.5 6.7 3.8 10.5

0.25 4.8 9.3 67.2 0.25 8.7 3.8 10.5

0.125 1.2 9.3 67.2 0.125 23.7 3.8 10.5

0.0625 5.6 9.3 67.2 0.0625 20.1 3.9 10.9

Table 9. Number of loci at each target where the Ho was rejected for AmpFℓSTR® Identifiler® and Minifiler® using a 0.05 confidence interval.

Target

(ng)

Identifiler®

# of loci rejected/total # loci

Minifiler®

# of loci rejected/total # loci

0.0625 11/14 6/9 0.125 11/14 6/9 0.25 10/14 2/9 0.5 9/14 3/9 1 3/14 1/9 2 2/14 1/9

47

Based on Tables 7 and 8 it is expected that a significant change in peak height ratio

balance would be seen at a target of approximately 0.125 ng for Minifiler® and 0.5 ng

for Identifiler® using a confidence interval of 0.05. Table 9 gives a summary of the

number of loci where the Ho was rejected at the 0.05 confidence interval at each target

for both kits.

Mixture Analysis

The assumption that DNA amplifies independently when more than one

contributor is present was evaluated. A mixture analysis was performed by comparing

the resultant peak height ratio derived from the single source samples versus the 1:1

mixture amplifications. Figure 5 shows the peak height ratios at two loci at a nominal

target of DNA for the Identifiler® and Minifiler® kits. Loci D16S539 and D21S11 were

used for comparison since there were no overlapping alleles between the two samples

at these locations. The error bars were included for the single source data and

represent the 3 standard deviations of the 8 samples. It should be noted that the

nominal target in this case is the presupposed amount of DNA for that target. In this

case the male in the 1:1 mixture at a nominal target of 1 ng indicates that the peak

height ratio used was that from a target of 2 ng, indicating that the male and female in a

1:1 ratio at a 2 ng target equally contributed to the mixture.

Peak height ratios between the single source and mixture data did not

significantly differ at any condition suggesting amplification of DNA is independent, at

48

least for the purposes of the peak height ratios assessed. Due to drop out of alleles at

lower target amounts, Identifiler® mixtures could not be interpreted at nominal target

amount less than 0.25 ng. Although the single source data did not show drop out at

0.25 ng, the mixture data does because it was assessed at an RFU of 30 instead of the 16

RFU used for the single source data. The drop out for each nominal target can be seen

in Table 10 for loci D16S539 and D21S11.

D16S539 Identifiler

0

0.5

1

1.5

0.25 0.5 1 2

Nominal Target (ng)

Pe

ak

He

igh

t R

ati

o

Female Single Source

Female in 1:1

Male Single Source

Male in 1:1

D16S539 Minifiler

-1

-0.5

0

0.5

1

1.5

2

0.063 0.125 0.25 0.5 1

Nominal Target (ng)

Pe

ak

He

igh

t R

ati

o

Female Single Source

Female in 1:1

Male Single Source

Male in 1:1

D21S11 Identifiler

0

0.5

1

1.5

0.25 0.5 1 2

Nominal Target (ng)

Pe

ak

He

igh

t R

ati

o

Female Single Source

Female in 1:1

Male Single Source

Male in 1:1

D21S11 Minifiler

-1

-0.5

0

0.5

1

1.5

2

0.0625 0.125 0.25 0.5 1

Nominal Target (ng)

Pe

ak

He

igh

t R

ati

o

Female Single Source

Female in 1:1

Male Single Source

Male in 1:1

Figure 5. Peak height ratios at heterozygote loci D16S539 and D21S11 at varying nominal targets.

49

Figure 6 shows an example of peak heights of alleles from one contributor at one locus.

Peak heights do not considerably differ between the single source condition and the 1:1

mixture condition. This corroborates the findings in Figure 5 and suggests DNA amplifies

independently when more than one contributor is present, or at least RFU values can be

treated independently.

D21S11 Identifiler

-500

0

500

1000

1500

2000

0.25 0.5 1 2

Nominal Target (ng)

Pe

ak

He

igh

t (r

fu)

Allele 30 in 1:1

Allele 30 Single Source

Allele 31 in 1:1

Allele 31 Single Source

D21S11 Minifiler

-500

0

500

1000

1500

0.063 0.125 0.25 0.5 1

Nominal Target (ng)

Pe

ak

He

igh

t (r

fu)

Allele 30 in 1:1

Allele 30 Single Source

Allele 31 in 1:1

Allele 31 Single Source

Figure 6. An example of peak height comparisons of each allele at heterozygote locus D21S11 from one contributor at varying nominal targets.

Figure 7 shows the peak height ratios for the major and minor contributors at

1:2, 1:4, and 1:9 mixture ratios for the D21S11 locus for both amplification kits. At the

0.25 ng target for Identifiler® only the major contributor is represented due to one or

both alleles of the minor contributor dropping out. Table 10 shows the resulting peak

height ratios for mixture ratios of 1:1, 1:2, 1:4, 1:9 and 1:19 at varying target amounts.

Significant drop out of one or both alleles was evident for the minor contributor at

ratios of 1:9 and 1:19 for both kits. Both kits demonstrated successful amplification of

the minor at the 1:2 mixture ratios for targets equal to or greater than 0.5 ng. Minifiler®

50

was more inconsistent than Identifiler®; for example, the peak height ratio for the minor

at D16S539 for the 1:1 and 1:2 mixtures ranged from 0.26 to 0.83, while Identifier®’s

range was 0.58 to 0.89 for the same locus and mixture ratio. However, the minor

contributor for Minifiler® at that loci was present at the 0.0625 ng target for both ratios,

1:1 and 1:2, and was present at the 0.125 ng target for the 1:1 mixture ratio, while the

minor was absent at those targets and ratios for Identifiler®, suggesting that Minifiler®

has a lower limit of detection but not necessarily as reproducible as Identifiler®.

In the Identifiler® kit the minor contributor maintains a fairly constant peak

height ratio at 0.5, 1, 2, and 4 ng in the 1:2 and 1:4, shown in Table 10. The minor’s

peak height ratio of the 1:9 mixture is maintained at 1, 2, and 4 ng. The minor

contributor did not fare as well in the Minifiler® kit. The minor’s presence varies at

different ratios and target amounts of DNA; for example, the minor at the 1:4 mixture is

present at 0.125 and 0.25 ng is below threshold at 0.5 and 1 ng, but then reappears at 2

ng.

D21S11 Identifiler

0

0.2

0.4

0.6

0.8

1

1.2

0.25 0.5 1 2 4

Target (ng)

Pe

ak

He

igh

Ra

tio Major in 1:2

Minor in 1:2

Major in 1:4

Minor in 1:4

Major in 1:9

Minor in 1:9

D21S11 Minifiler

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.125 0.25 0.5 1 2

Target (ng)

Pe

ak

He

igh

t R

ati

o Major in 1:2

Minor in 1:2

Major in 1:4

Minor in 1:4

Major in 1:9

Minor in 1:9

Figure 7. An example of peak height ratios for the major and minor contributor in 1:2, 1:4, and 1:9 mixture ratios at varying targets.

51

Using AmpFℓSTR® Profiler Plus® and Cofiler® PCR amplification kits, DNA samples

from two donors mixed in ratios ranging from 1:20 to 1:1 to 20:1 showed that the minor

could be reliably detected when present at 10% of the major component at a 2 ng total

template amount. At 5%, the minor component, at some samples was detected but at

times not typable (28). Validation of the Identifiler® kit demonstrated similar results,

analysis of 1:10 mixtures using a 1 ng target showed robust results and the minor

component alleles when not in a stutter position were reliably detected. The minors’

genotype in a 1:20 mixture was detectable but often times fell below the 50 RFU

threshold (7). A Minifiler® mixture of 1:10 also showed complete and reproducible

amplification of the minor contributor at a 1 ng total target amount (minor contribution

of 0.091 ng). Mixture ratios greater than 1:10 resulted in partial profiles for the minor

contributor in a sample (30).

Table 10. Mixture ratios and resulting peak height ratios at varying target amounts for loci D16S539 and D21S11.

A blank cell indicates 1 or 2 alleles dropped out

Mixture Ratios for Locus D16S539 Minifiler® Total 1:1 1:2 1:4 1:9 1:19

Amount of DNA (ng)

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

0.0625 0.44 0.58 0.75 0.50 0.125 0.41 0.26 0.56 0.85 0.59 0.25 0.95 0.95 0.60 0.39 0.76 0.86 0.79 0.71 0.5 0.66 0.54 0.78 0.66 0.88 0.85 0.78 0.94 1 0.52 0.78 0.89 0.64 0.61 0.73 0.83

2 0.77 0.83 0.55 0.73 0.96 0.32 0.86 0.89

52

Mixture Ratios for Locus D16S539 Identifiler® Total 1:1 1:2 1:4 1:9 1:19

Amount of DNA (ng)

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

0.0625 0.61 0.125 0.82 0.70 0.89 0.87

0.25 0.72 0.96 0.88 0.60 0.64

0.5 0.70 0.77 0.78 0.82 0.70 0.60 0.82 1 0.75 0.75 0.66 0.69 0.83 0.98 0.59 0.75

2 0.99 0.89 0.99 0.67 0.90 0.77 0.92 0.83 0.61

4 0.81 0.81 0.89 0.58 0.96 0.91 0.72 0.58 0.93

A blank cell indicates 1 or 2 alleles dropped out.

Mixture Ratios for Locus D21S11 Minifiler® Total 1:1 1:2 1:4 1:9 1:19

Amount of DNA (ng)

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

0.0625 0.52 0.89 0.125 0.70 0.84 0.56 0.48 0.22 0.72 0.83 0.18 0.25 0.77 0.52 0.54 0.92 0.65 0.35 0.69 0.53 0.5 0.94 0.63 0.95 0.50 0.95 0.94 0.67 1 0.82 0.61 0.94 0.64 0.73 0.95 0.45 0.93 2 0.98 0.74 0.82 0.83 0.87 0.77 0.86 0.54 0.79

A blank cell indicates 1 or 2 alleles dropped out.

Mixture Ratios for Locus D21S11 Identifiler® Total 1:1 1:2 1:4 1:9 1:19

Amount of DNA (ng)

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

Major PHR

Minor PHR

0.0625 0.125 0.99 0.87 0.25 0.83 0.99 0.73 0.67 0.64 0.5 0.76 0.86 0.79 0.73 0.80 0.94 0.83 0.82 1 0.81 0.96 0.67 0.72 0.90 0.75 0.99 0.77 0.75 2 0.84 0.76 0.55 0.74 0.82 0.70 0.73 0.64 0.83 0.61 4 0.73 0.99 0.98 0.77 0.84 0.85 0.84 0.79 0.93

A blank cell indicates 1 or 2 alleles dropped out.

CONCLUSIONS

Minifiler® has a larger average peak height at a given target than Identifiler®,

albeit is less sensitive, whereby the calibration sensitivities are significantly different

from analytical sensitivities at 0.5 ng. This suggests that Identifiler® is the

recommended kit for obtaining a full DNA profile from non-compromised samples even

53

for samples which contain limited DNA quantities. Both the Identifiler® and Minifiler®

kits demonstrated increased peak height variance with a decreasing target amount;

however, Minifiler® showed more variance across all targets when compared to

Identifiler®. Peak height ratios significantly differed at low targets for both kits. Hence

analysts attempting to deduce profiles from a contributor where the target is less than

0.5 ng in Identifiler® and 0.25 ng in Minifiler® should be cautious and recognize that

these ratios may not be representative of those obtained in typical validation studies.

Peak heights and their ratios did not vary between mixture and single source

conditions, suggesting peak height ratios obtained through single source validation

studies are appropriate to use for mixture deconvolution. The probability of deducing

the minor contributor in a mixture depends on both the amount of target DNA being

amplified and the minor to major ratio. At targets less than 0.5 ng and a ratio 1:9 or

greater the likelihood of genotyping the minor contributor becomes increasingly

difficult.

54

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