Characterization of a modified amplification approach for improved STR recovery from severely degraded skeletal elements

Forensic Science International: Genetics 6 (2012) 578–587

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Characterization of a modified amplification approach for improved STR recovery from severely degraded skeletal elements Jodi A. Irwin *, Rebecca S. Just, Odile M. Loreille, Thomas J. Parsons 1 Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Rockville, MD 20850, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 November 2011 Accepted 31 January 2012

Degraded skeletal remains generally contain limited quantities of genetic material and thus DNA-based identification efforts often target the mitochondrial DNA (mtDNA) control region due to the relative abundance of intact mtDNA as compared to nuclear DNA. In many missing person cases, however, the discriminatory power of mtDNA is inadequate to permit identification when associated anthropological, odontological, or contextual evidence is also limited, and/or the event involves a large number of individuals. In situations such as these, more aggressive amplification protocols which can permit recovery of STR data are badly needed as they may represent the last hope for conclusive identification. We have previously demonstrated the potential of a modified Promega PowerPlex 16 amplification strategy for the recovery of autosomal STR data from severely degraded skeletal elements. Here, we further characterize the results obtained under these modified parameters on a variety of sample types including pristine control DNA and representative case work specimens. Not only is the amplification approach evaluated here sensitive to extremely low authentic DNA input quantities (6 pg), but when the method was applied to thirty-one challenging casework specimens, nine or more alleles were reproducibly recovered from 69% of the samples tested. Moreover, when we independently considered bone samples extracted with a protocol that includes complete demineralization of the bone matrix, the percentage of samples yielding nine or more reproducible alleles increased to 95% with the modified amplification parameters. Overall, direct comparisons between the modified amplification protocol and the standard amplification protocol demonstrated that allele recovery was significantly greater using the aggressive parameters, with only a minimal associated increase in artifactual data. Published by Elsevier Ireland Ltd.

Keywords: Degraded skeletal remains Missing persons Short tandem repeats

1. Introduction The potential to use genetic information for missing persons’ identifications is largely dependent on two things: the quality of the evidentiary specimens (which itself depends heavily on the environmental conditions to which the samples were exposed) and the availability of direct and/or family reference samples for comparison. Under ideal circumstances, evidence specimens are of high enough quality that commercial, off-the-shelf kits targeting autosomal short tandem repeats (STRs) may be used. The resulting STR data can then be directly compared to autosomal STR profiles from a direct, or family, reference for the presumed deceased individual. However, since many missing persons cases deviate from this idealized scenario, particularly in terms of evidence specimen quality, mitochondrial DNA (mtDNA) is often the marker

* Corresponding author. Tel.: +1 301 319 0244; fax: +1 301 295 5932. E-mail address: [email protected] (J.A. Irwin). 1 Current address: International Commission on Missing Persons, Alipasˇina 45a, 71000 Sarajevo, Bosnia and Herzegovina. 1872-4973/$ – see front matter . Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigen.2012.01.010

of choice for the most difficult material because of its relative abundance in samples that harbor little or no nuclear DNA and its uniparental mode of inheritance which allows for distant maternal relatives to serve as references [1–3]. Unfortunately, the limited discriminatory power of mtDNA means that it is useful in the identification process only in combination with substantial nonDNA evidence, such as anthropological, odontological or circumstantial data. In cases where the non-DNA evidence is limited, the addition of mtDNA matching data may not be sufficient to confidently determine identity; and in cases involving large numbers of individuals or open events, mtDNA may be of quite limited utility. As a result, and despite the difficulty of recovering nuclear DNA markers from degraded material, efforts are continuously underway to improve the success of STR recovery in these cases. To date, efforts to improve DNA profiling success from degraded skeletal elements have primarily focused on the retrieval of more (and higher quality) DNA at the extraction step [4–7] and increased sensitivity at the amplification stage via smaller amplicons or modified PCR parameters [8–16]. These types of protocol optimizations have led to nuclear DNA recovery in cases of

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historical interest [17–19], various proof of concept studies demonstrating the potential of STR typing for the purposes of missing persons identifications [20–24], and disaster victim identification in the World Trade Center attack [25]. Nuclear STR testing from degraded skeletal remains has been used with great success in the world’s largest missing persons identification project [26], where STR profiles have been obtained from over 36,000 bone and tooth samples recovered from victims of the 1990s conflicts in the former Yugoslavia, resulting in the DNA identification of over 16,000 individuals. In that case, STR recovery rates from skeletal elements recovered three to twenty years after death from a wide range of environmental sites were high, with over 76% of the specimens producing 12 loci or more. These previous studies, however, have been either limited by sample size, or restricted to a single geographic region and/or historical event. As a result, and despite significant sample-tosample variability in even these situations (which itself offers some insight into the highly unpredictable nature of DNA preservation and DNA typing success), the general potential of STR recovery from a broader range of sample quality is somewhat difficult to assess from the available literature. The survival of DNA in postmortem samples is understood to be a function of both specimen age and environmental exposure [27]; but on shorter time scales relevant to many investigations, variables such as temperature, temperature fluctuations, oxygen, humidity, UV irradiation and microorganisms are the primary factors in determining DNA degradation or survival [28]. In an effort to gain further insight into STR profiling success from a broad range of skeletal sample quality, we examine STR recovery rates from typical missing persons specimens encountered at the Armed Forces DNA Identification Laboratory (AFDIL). The cases targeted here span the major 20th century U.S. military conflicts and include samples of variable postmortem age, exposed to a variety of environmental conditions, and subject to different thermal and seasonal regimes. As with other studies, we employ aggressive amplification parameters to increase sensitivity to the low quality/quantity endogenous DNA and as a result, the STR profiles produced tend to exhibit characteristics similar to those encountered in so-called ‘‘low-template’’ or ‘‘low-copy number’’ DNA testing (allelic dropout, allelic drop-in and elevated stutter) [29–31]. However, and quite different from other low-template DNA applications, the specimens commonly received in these missing persons cases are generally available in quantities large enough for multiple samplings and/or extractions, and they are nearly universally single-source. Here, we further characterize the features of this modified amplification approach on the severely degraded skeletal elements routinely encountered in missing persons casework at the AFDIL. 2. Materials and methods All amplifications were conducted using the PowerPlexTM 16 (PP16) System (Promega Corporation, Madison, WI). Standard amplifications were performed according to the manufacturer’s recommendations. For the modified amplifications, thermal cycling temperatures and times were maintained, but twice the manufacturer’s recommended concentration of Taq polymerase (a total of 8 units of Taq in a 25 ml reaction) and an additional six cycles (36 cycles total) were used. PCR products were separated on an Applied Biosystems 3100 Genetic Analyzer using standard parameters and a 10 s injection, and data were analyzed using Genescan software version 3.7 (Applied Biosystems, Foster City, CA). Genotyper version 3.7 (Applied Biosystems) was used to assign allele calls to electropherograms using the allelic ladder provided with the kit as the reference. A relative fluorescent unit (RFU) cut-off of 100 was used

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to designate authentic alleles in the finalized profile for any given amplification, whether they were observed in homozygous or heterozygous state. For the portions of the study that assessed the practical application of the modified approach on degraded material (as opposed to the average allele recovery on a per amplification basis) consensus STR profiles were built from at least two, but generally three, amplification replicates. Alleles reported in the final consensus profiles were reproduced in the majority (either 2/2 or 2/3) of the amplifications conducted. 2.1. Assay sensitivity Sensitivity of the modified approach was assessed using a dilution series of DNA standard 9947A (Promega Corporation). The DNA standard was first quantified in a range within the detection limits of the Quantifiler1 Human DNA Quantification Kit (Applied Biosystems) and then serially diluted to eight concentrations. Based on those dilutions, five replicates at each of the following quantities were amplified with both the manufacturer’s recommended PP16 protocol and the modified protocol: 100 pg, 50 pg, 25 pg, 12.5 pg, 6.25 pg, 1 pg, 0.5 pg and 0.25 pg. Based on the number of alleles expected in the 9947A PP16 profile, sensitivity of the standard and modified approaches was assessed by (1) averaging the number of alleles recovered over the five amplification replicates at each DNA concentration, and (2) duplication of alleles in three amplifications (i.e. consensus profiles generated from all ten possible triplicate combinations from the five amplification replicates). Since the STR profile of the DNA standard was known, the resulting sensitivity data were also used to evaluate stochastic effects of the modified approach, including allelic (or locus) dropout and allelic drop-in due to elevated stutter peaks and amplification of random, low level contaminants. 2.2. Artificially degraded samples To evaluate the effectiveness of the modified approach on known degraded samples, blood and saliva samples that had undergone artificial degradation were tested. These samples were received as part of a collaborative study and had been degraded in a humidifier for the time frames specified in Dixon et al. [32]. When typed using PP16, Individual One (Indiv One, from here on) was expected to exhibit twenty-eight different alleles, while Individual Two (Indiv Two) was expected to exhibit twenty-nine alleles. The total number of alleles expected on a per amplification basis was used to assess allele recovery from both the standard and modified amplifications. In terms of the final sums of alleles recovered, spurious non-authentic alleles were considered separately from recovered authentic alleles. For this portion of the study, due to limited extract volume, artificially degraded samples were each amplified twice under both the standard and modified PP16 conditions. Alleles observed in both amplifications from the respective protocols were included in the final profile for each sample under each protocol. Since the complete genotypes of the artificially degraded samples were known, these data were used to ascertain average values for allelic drop-in and allelic dropout with both protocols. 2.3. Degraded skeletal elements To assess the utility of the modified amplification on authentic case material, thirty-one skeletal elements, represented by nearly seventy extracts, were tested. The skeletal remains ranged in age from 30 to 80 years post mortem and were recovered from an extremely wide variety of environments including Western Europe, Southeast Asia, Korea, and the South Pacific. Remains were extracted with either a standard phenol/chloroform protocol

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as described in Edson et al. [33] or a modified extraction protocol that includes complete demineralization of the bone [4]. For the purposes of this study, and to stay consistent with the validated procedures employed in the AFDIL laboratory for mitochondrial DNA case work, extracts were not further purified or concentrated. In fact, the additional Taq used in the modified protocol was included specifically to overcome inhibition resulting from large volumes of these skeletal remains extracts. In all cases, extract volume was maximized in an effort to also maximize allele sampling and recovery. Standard PP16 reactions were not included in this portion of the study since, in most cases, limited extract volumes did not permit a direct comparison between the two protocols with the desired number of replicates for each. Furthermore, the limited number of direct comparisons that were conducted during early pilot studies clearly demonstrated the benefits of the modified protocol over the standard protocol (Fig. 1). The bone specimen amplifications were evaluated from the standpoints of quantifiable DNA, allelic drop-in and allelic dropout. For each of these three items, specific caveats must be noted. First, nuclear DNA quantities were only measured on a subset of the samples, due to the fact that quantitation methods sensitive enough for the low DNA quantities present in these remains were not available at the time many of the samples were tested. Second, because the bone specimens used for this portion of the study represented authentic case material, the complete genotypes of the individuals were unknown. Strictly speaking, ‘‘known’’ samples of aged, degraded skeletal elements (from the standpoint of autosomal STR profiles) were unavailable. Thus, measures of allelic dropout and drop-in were based on the profile inferred for a particular individual or sample using the data recovered from the modified amplification replicates. This could only be done in those cases for which the extraction and amplification replicates provided enough data to infer a complete profile. As a result, the total number of samples used for these analyses was reduced from the overall total tested with the modified protocol. 2.4. Statistical analyses Statistical analyses of the data were conducted in Excel and GraphPad Prism v. 4.02 (GraphPad Software, Inc., La Jolla, CA). [(Fig._1)TD$IG]

Simple linear regressions were performed to investigate the relationship between allelic dropout and median allele size for the experimental series investigating sensitivity on control DNA and artificially degraded samples amplified using the standard and modified protocols. In GraphPad, Grubb’s test was used to identify outliers, linearity was assessed by runs tests, and consistency with a normal distribution was evaluated using the D’Agostino and Pearson omnibus normality test. For the degraded skeletal extracts, Fisher’s exact test was used to compare the incidence of allelic drop-in among different sample sets, and a modified Wald approximation [34] was used to calculate 95% confidence intervals for the proportion of amplifications with drop-in. 3. Results and discussion 3.1. Sensitivity data and artificially degraded samples 3.1.1. Allele recovery Average results of the five replicates from each of eight quantities of pristine DNA (Fig. 2A and B) highlight the improvement in authentic allele recovery when the modified protocol was used. A comparison between the two protocols of average allele recoveries revealed that the modified approach produced more than twice as much data as the standard approach across all DNA inputs. In addition, the modified approach produced authentic data from as little as 0.5 pg of input DNA. These measures of allelic recovery also, of course, reflect the proportion of allelic dropout observed in the sensitivity data. Using the manufacturer recommended amplification protocol, allelic dropout was observed at all DNA input quantities. This result was expected given that the recommended DNA input is one nanogram. Using the modified approach, however, allelic dropout was only observed in amplifications of 50 pg or less of input DNA. At 12.5 pg, an average of 77% of the alleles were recovered and at 6.25 pg, nearly 50% of the alleles were recovered. At these input quantities with the standard amplification protocol, only a handful of alleles could be recovered among the five amplification replicates (see Fig. 2A and B). These data indicate that, on average, when twice the recommended Taq concentration and six additional cycles are used with this particular STR kit (PP16) roughly 50% of the targeted alleles can be recovered from a DNA quantity equivalent to that

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Fig. 1. Direct comparison of the modified versus standard amplification approach on aged, degraded skeletal case material. The standard and modified PP16 amplification protocols were applied to two extracts sampled from the same set of aged, degraded remains for which references were available. With the first extract, a complete profile was recovered using the modified protocol, yet only about half of these alleles were recovered using the standard approach. No replicated alleles were recovered when the standard protocol was applied to the second extract, yet the modified approach produced more than 50% of the profile.

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Fig. 2. Box and whisker plots of two measures of allele recovery from the sensitivity data. Panels A and B show the average fraction of alleles recovered from five replicate amplifications of a 9947A DNA dilution series, ranging from 100 to 0.25 pg, using the standard (A) and modified (B) PP16 protocols. Panels C and D show the average fraction of the profile recovered among all possible sets of triplicate amplifications (derived from the five replicate amplifications) for the standard (C) and modified (D) amplification conditions, where allelic recovery is defined by duplication of the allele in at least two of the three replicates.

present in a single cell [35]. Interestingly, these results also suggest that adjustments to the assay seem to mitigate stochastic effects that are observed under manufacturer recommended parameters at lower DNA inputs. In other words, with DNA input quantities lower than the manufacturer recommendation, the stochastic issues are actually exacerbated by performing the assay under standard parameters. To assess the practical utility of the modified approach based on duplication of alleles in three amplifications (instead of an average allele recovery), all ten possible triplicate combinations from the five amplification replicates were evaluated for profile recovery based on allele reproducibility (Fig. 2C and D). When this consensus approach was used, the modified protocol produced, on average, nearly complete profiles (more than 90% of alleles reproducibly recovered) at DNA inputs of 25 pg or more, and 73% of the profile with as little as 12.5 pg of input DNA. In contrast, 25 pg and 12.5 pg of input DNA resulted in profile recoveries of 30% and 4% respectively under standard conditions. Generally speaking, the modified parameters required four times less input DNA to reproduce PP16 profiles generated under standard conditions. Using the sensitivity data, we investigated allele recovery based on amplicon size to determine whether or not this played a part in the amplification success of particular loci when pristine DNA was in low quantity. Fig. 3 shows a linear regression analysis of average allelic dropout by median locus size for both standard and

modified amplification conditions across all sensitivity replicates and DNA input quantities tested. One locus, TH01, exhibited an extremely high rate of dropout (98.75%) under standard amplification conditions and was removed from the analysis as an outlier. The remaining data suggest a slightly negative but nonsignificant correlation between allele size and dropout for both amplification protocols (correlation coefficients were 0.28 and 0.35 for the standard and modified protocols respectively, and in both cases p  0.05). This was the expected result given that the DNA present was in low quantity but not degraded. Although the sensitivity data provided an indication of how little input DNA could be detected with the modified amplification, the pristine templates used for this portion of the study did not adequately model the highly degraded templates likely to be encountered in the missing persons and mass disaster case work for which these specific modified parameters were intended. As a result, artificially degraded samples were also tested to better assess the performance of the modified protocol on samples for which the DNA was both degraded and in low quantity. For these samples, total allele recovery decreased rapidly for both the standard and modified protocols when less than 1 ng of DNA (as determined with Quantifiler) was used in the reaction (Fig. 4). However, the modified approach performed significantly better at the lower DNA input quantities. With 400 pg of input DNA, the standard reaction recovered less than 10% of the profile

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Average Percentage Dropout (of total possible alleles at a locus)

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Locus (smallest to largest) Fig. 3. Allelic dropout by locus size in sensitivity data. The scatter plot reflects the average percentage of alleles that did not amplify in 40 standard protocol amplifications and 40 modified protocol amplifications (80 amplifications in total) from a 9947A dilution series. Loci are ordered by 9947A median allele size; from left to right, Amelogenin, D3S1358, D5S818, vWA, TH01, D13S317, D8S1179 and D21S11 (median allele sizes are the same), D7S820, TPOX, D16S539, D18S51, CSF1PO, FGA, Penta D, and Penta E. The standard amplification data point identified as an outlier, TH01, is included on the graph (as a hollow diamond) but was not included in the regression analysis.

on average, whereas the modified amplification recovered 70% of the profile. In terms of allelic reproducibility across replicate amplifications, similar patterns emerged. When no data were reproducible under standard conditions, at least one third of the profile was reproduced with the modified parameters. Overall, the

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modified approach produced nearly twice as much reproducible data from samples that yielded only a handful of reproducible alleles under standard conditions. Unlike the sensitivity data, which indicated that approximately 50% of a profile can be recovered from 6.25 pg of input DNA under modified amplification conditions, allele recovery with the degraded samples was reduced to 50% of the profile with as much as 300 pg of input DNA – a nearly 200-fold difference. The reason for this enormous discrepancy in data recovery between the sensitivity samples and the artificially degraded samples is no doubt the method underlying the Quantifiler assay used for DNA quantitation. Because the Quantifiler amplicon (62 bp) is smaller than even the smallest PP16 locus (Amelogenin, at approximately 120 bp), Quantifiler tends to overestimate the quantity of amplifiable DNA templates in degraded extracts. Clearly, samples that quantify well will only amplify well with standard-sized STR amplicons when the DNA is not significantly degraded. As we had done with the pristine low-template samples, we evaluated allelic dropout in the artificially degraded samples from the perspective of amplicon size (Fig. 5). Given that many of these samples were highly degraded and given that the quantification results, when considered along with the overall allele recovery data, indicated the presence of a substantial number of smaller templates in the extracts, we expected to see an increase in allelic dropout with the larger loci. This was the trend observed among profiles produced using the modified amplification protocol, but the pattern was not as obvious as expected among the samples amplified using the standard protocol. The positive correlation was highly significant (p = 0.001) for profiles generated under modified parameters, but nonsignificant when the standard protocol was applied (p  0.05). Similarly, the R2 values suggest that while very little of the observed pattern of allelic dropout can be explained by amplicon size in the standard amplifications (R2 = 0.035), the regression line for the modified protocol exhibited a higher goodness of fit with 54.5% of the variance in dropout related to amplicon size. The difference between the standard and modified protocols in correlation between successful amplification and amplicon size with these degraded samples is surprising. We suspect that this is because there is simply too little DNA to observe/resolve the

[(Fig._5)TD$IG] Average Percentage Dropout (of total possible alleles at a locus)

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Locus (smallest to largest) Fig. 5. Allelic dropout by locus size in artificially degraded blood and saliva samples. Scatter plot depicting the average percentage of alleles that did not amplify in 24 standard amplifications and 24 modified amplifications from artificially degraded samples with known profiles. Profiles of those samples which were not subject to degradation (see ‘‘Blood 0 Days’’ and ‘‘Saliva 0 Days’’ in Fig. 3) were excluded from the analysis. Loci are ordered by median allele size for the two known profiles; from left to right, Amelogenin, D3S1358, D5S818, vWA, TH01, D13S317, D8S1179, D21S11, D7S820, TPOX, D16S539, D18S51, FGA, CSF1PO, Penta D, and Penta E.

expected pattern of dropout as a function of allele size when these low quantity templates are amplified using the standard, lesssensitive parameters. In these situations, the amplification of particular alleles/loci appears to be dictated more by chance than the characteristics of the endogenous templates (e.g. fragment size). 3.1.2. Allelic drop-in The greater sensitivity of the modified amplification approach to authentic DNA was also reflected in greater sensitivity to socalled ‘drop-in’ alleles. To evaluate the occurrence of allelic drop-in with the modified approach, we tabulated the number of drop in alleles observed in the sensitivity and artificially degraded sample sets. For the sensitivity data, drop-ins were tabulated over the five amplification replicates, for both amplification protocols, at all DNA input quantities except 100 pg (due to excessively high fluorescent signal in some reactions). In total, across both amplification methods, drop-in alleles were observed in four of seventy amplifications: two standard and two modified amplifications (5.7% of standard amplifications and 5.7% of modified amplifications). Turning to the artificially degraded samples, under standard conditions, allelic drop-in was found in three of thirty-two amplifications (9.4%). Under the modified conditions, spurious alleles were observed in five out of thirty-two amplifications (15.6%). Thus, for the artificially degraded samples, the modified approach produced about twice as many spurious alleles as the standard PP16 protocol. When data from the sensitivity samples and artificially degraded samples were considered together as a set of known profiles (134 amplifications total), 71.4% of all drop-in alleles observed occurred in stutter positions. Generally speaking, and particularly when loci are heterozygous and replicate amplifications are performed, high stutter versus true contamination is obvious. Yet, elevated stutter has been included in the total drop-in allele count to provide the most conservative estimate. In all cases, however, the true spurious alleles were easily recognized and in no case were they reproduced in replicate amplifications of the same sample. Therefore the slight increase for drop in alleles in single amplifications is more than offset by the advantages in recovering additional authentic alleles.

3.1.3. Heterozygote balance Results from sensitivity and artificially degraded samples were combined to evaluate differences in heterozygote balance between the standard and modified amplification protocols. Results from the same known samples and the same DNA input quantities were used for the comparisons, and the ratio of minor to major peak (in terms of peak height) was evaluated for both sets of amplification protocols. In total, sixty-seven amplifications were evaluated with each amplification approach, for a total data set of 134 reactions. Only potentially heterozygous loci (those at which at least one of the two expected alleles was present) were investigated. Fig. 6A summarizes the data for the sixty-seven standard amplifications. Of the 358 heterozygous loci investigated, the average relative fluorescent unit (RFU) value of the major peak was 709, with a median of 344. Of these, seventy-four (20.7%) exhibited complete dropout of one allele. For the loci at which no dropout occurred, the average minor to major peak ratio was 0.785 with a standard deviation of 0.153. No other loci in this dataset exhibited peak imbalance in the lower range that is generally considered for stutter peaks (<15%, when standard amplification conditions and recommended DNA input quantities are used [36]). Fig. 6B reflects the data for the sixty-seven modified amplifications. Under these conditions, 24.3% of the heterozygous loci exhibited complete dropout of one allele, 3.6% more than under standard conditions. In addition, average peak height values of the major allele were three and a half times greater than major peak heights observed under standard conditions (mean 2436; median 1992) and peak height ratios for the modified amplifications were distributed much more broadly (mean 0.683; standard deviation 0.235). It should also be noted that in the modified amplification data, 5.0% of data points gave ratios that were equal to or less than 0.2, suggesting that on an amplification by amplification basis, greater caution should be exercised with the modified protocol when interpreting alleles at stutter positions. Clearly, in some cases, these minor peaks reflect authentic alleles rather than stutter artifacts. As with other stochastic artifacts observed with the modified approach, this type of imbalance is generally clarified upon multiple amplifications. In those cases where the distinction between stutter and authenticity is not clarified, it would be conservative to assume that allele dropout has occurred.

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A. Standard protocol amplificaons Major peak height (RFUs)

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Rao of Minor to Major peak Fig. 6. Heterozygote balance in known profiles. Scatter plots of the minor peak to major peak ratio versus the major peak height (in RFUs) using the standard (A) and modified (B) PP16 protocols. The data examined included all replicate sensitivity amplifications at all DNA input levels except 100 pg (due to excessive signal in some amplifications) and all artificially degraded sample amplifications, for a total of sixty-seven profiles per amplification approach. Specific loci evaluated include only those for which at least one of two expected alleles was present in a given profile.

Broad-scale comparisons between the plots from the standard and modified amplifications highlight the dramatic differences in heterozygote peak balance that can be expected with the different amplification strategies. While the scatter plot points for the standard amplifications cluster in the lower right quadrant, reflecting generally good peak balance and RFU values less than 1000, the points in the modified amplification plot are distributed much more widely, indicating that both the peak height of the major allele and the balance between peaks vary greatly under the modified conditions. The large distribution of data points along the y-axis in the modified amplification plot reflects the notable increase in peak heights that results from the additional Taq and cycles. In fact, even when the RFUs of the major peak reach 8000, dropout of the second allele may still be observed. In the standard amplifications, dropout was only observed when the major peak (remaining peak, in this case) was present at 500 RFUs or less. Peak imbalance is more pronounced with this modified protocol than with other described protocols [30,31,37]. More than likely, the additional Taq polymerase employed in our assay exacerbates peak imbalance issues, particularly when one allele of a heterozygote is preferentially amplified in an early cycle of the PCR. 3.1.4. Overall performance on known samples An overview of the authentic allele recovery and allelic drop-in results from the standard and modified amplifications of known samples is shown in Table 1. The results suggest that despite the slightly greater incidence of drop-in alleles and increased peak height imbalance with the modified protocol, as described in the previous sections, total recovery of authentic alleles is significantly greater in the modified amplifications. In addition, we have found that the allelic dropout or dramatic peak imbalance observed in

any one amplification is generally clarified by the other amplification replicates. Although each replicate is equally likely to exhibit random artifacts, the artifacts (by their very nature) are not reproducible. So, overall, the stochastic effects observed in any particular amplification are offset by total allele recovery across multiple amplifications. 3.1.5. Degraded skeletal extracts 3.1.5.1. Allele recovery. To determine if modified protocol PP16 amplification success could be at all predicted by Quantifilermeasured DNA quantities, the PP16 results from thirty-six degraded skeletal extracts for which the complete STR profile could be accurately inferred were evaluated with respect to the input DNA quantities. The results are shown in Fig. 7 and while the data suggest highly variable allele recoveries in the lower DNA input ranges (<100 pg), the plot also illustrates that complete, or nearly complete profiles can often be recovered when more than

Table 1 Overall performance of standard and modified PP16 protocols on known samples. The table combines data from all 144 profiles generated from a dilution series replicates of DNA standard 9947A (40 profiles per amplification approach) and artificially degraded blood and saliva samples (32 profiles per amplification approach) to assess the overall performance of each protocol in terms of allele recovery and allelic drop-in. The number of spurious alleles listed includes those in stutter positions.

# of alleles recovered (1912 expected) % of authentic alleles recovered # of spurious alleles Spurious allele noise

Standard PP16

Modified PP16

933 48.8% 5 0.3%

1365 71.3% 9 0.5%

[(Fig._7)TD$IG]

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Fig. 7. Predictive value of Quantifiler-measured DNA quantities in PP16 typing success on degraded skeletal extracts. The scatter plot depicts PP16 profile recovery with the modified amplification strategy from degraded skeletal remains at various Quantifiler-measured DNA input quantities. Data points represent thirty-six skeletal extracts for which a complete profile could be inferred. The data suggest that allele recovery is variable when low quantities of DNA are detected by Quantifiler, but that complete or nearly complete profiles are recoverable when the modified protocol is applied to 200 pg or greater of Quantifiler-measured DNA. These results are consistent with profile recovery from degraded skeletal remains using a modified Yfiler amplification approach [22].

200 pg of Quantifiler-measured DNA are amplified under the modified conditions. These results are quite similar to those observed with a modified Yfiler (Applied Biosystems) assay [22] applied to a different (but similar in terms of origin, age and quality) set of skeletal remains, and further support the observation that when DNA concentrations of more than 20–25 pg/ml are detected by Quantifiler in significantly degraded samples/extracts, there is a good chance of recovering a highly informative autosomal STR profile. Interestingly, the plot also suggests that even when no DNA is detected by real-time PCR quantitation, in some cases data can still be recovered with the modified amplification strategy – up to 40% of the profile in one particular amplification. In these cases, we have verified that the real-time amplifications were not inhibited and it is possible that the quantities are simply below the lower dynamic range of the assay (23 pg/ml) – where the quantification data may be unreliable, but the modified protocol sensitivity data suggest allele recovery is possible. Overall, these data suggest that the Quantifiler-determined quantities were a relatively useful predictor of success when the detected quantities were large. The opposite was not true however. When the quant values were low, the PP16 results were not necessarily poor.

[(Fig._8)TD$IG]

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3.1.5.2. Allelic drop-in. Nearly 200 amplifications from degraded skeletal remains were evaluated along with their associated negative controls for the presence of spurious alleles. Overall, twenty-nine amplifications exhibited spurious alleles, resulting in a 15.0% chance of drop-in per amplification. However, one particular set of skeletal remains that exhibited reproducible artifacts at D5S818 accounted for over one-tenth of all observed drop-in alleles and 55% of drop-in alleles observed at the D5 locus. For this set of skeletal remains (and no other set), the observed incidence of drop-in at D5 was significantly greater (p < 0.0001) than expected based on the incidence of drop-in at D5 among negative control amplifications. Given that the artifacts were observed in multiple independent extractions of different skeletal elements, these surplus alleles are suspected to result from nonspecific amplification facilitated by inefficient binding of the primer to the target template. Binding site mutations at D5 with the PP16 primers have been previously reported [38–40] and while we cannot establish whether or not the D5 primers are actually responsible for these artifacts, the peaks appear to reflect erroneous amplification of non-specific targets from the human genome, or perhaps microbial contaminants. Though not regularly encountered in this study, non-specific amplification of bacterial or fungal contaminants have been shown to manifest on occasion in degraded skeletal elements such as those tested here [41]. In terms of drop-in, if this single set of skeletal remains is removed as an outlier, the chance of observing a drop-in allele reduces to approximately 11.0% (95% confidence interval is 6.2– 15.9%). As with the sensitivity and artificially degraded datasets, a large proportion of these drop-in alleles occurred in stutter positions (42% in the non-probative case samples). Furthermore, when only the data obtained from samples extracted with the Loreille et al. [4] protocol were considered, the drop-in percentage decreased to just 5% (95% confidence interval is 1.1–9.9%). In these cases, it is likely that the recovery of more, and higher quality, authentic DNA with the Loreille et al. extraction facilitates STR data recovery while minimizing artifacts. 3.1.5.3. Overall success with aged, degraded skeletal elements. To assess the practical utility of the modified amplification protocol on representative casework material, consensus profiles based on multiple amplifications were generated for thirty-one skeletal elements. The overall success with the modified amplification approach on these remains is summarized in Fig. 8. Sixty-nine percent of the skeletal remains tested resulted in between nine and thirty-two alleles in the consensus profiles generated from two or

Fig. 8. PP16 profile recovery from skeletal remains extracts. Pie charts summarizing consensus profile allele recovery from 30 to 80 year old skeletal elements using the modified amplification. The samples were extracted as described in Edson et al. [33] and Loreille et al. [4]. (A) Reflects allele recovery from all 67 extracts, while (B) represents allele recovery from the 20 samples extracted using the Loreille et al. protocol only. The ‘Full/Nearly Full’ category encompasses consensus profiles of twenty-five or more alleles; ‘Partial’ refers to the recovery of nine to twenty-four alleles; and ‘Poor/No’ recovery indicates that eight or fewer alleles were reflected in the consensus profile.

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three amplifications. The proportion of successful amplifications (partial or full/nearly full profiles) increased when an extraction protocol including decalcification of the bone matrix was combined with the amplification approach. Similar results were observed by Loreille et al. [4]; and when the results from both of these studies are combined, the observed differences in allele recovery between the two extraction protocols are statistically significant (p = 0.01). The extraction of skeletal remains is now regularly performed in the AFDIL laboratory using the protocol of Loreille et al. [4] and while additional data would be desirable to assess the general utility of the modified PP16 approach on these extracts, our preliminary results suggest that nine or more alleles will likely be reproducible from the majority of aged, degraded skeletal samples typically encountered in our laboratory. 4. Conclusions We have characterized an aggressive amplification protocol for the typing of autosomal STRs from degraded skeletal remains. Our results demonstrate that overall allele recovery is significantly greater with the modified protocol than with the manufacturer recommended amplification conditions, with a comparatively small associated increase in the recovery of non-authentic alleles. Our data also exhibit the stochastic effects related to allele sampling from the extract and preferential amplification during early cycles of the PCR – which, in this study – manifested in comparatively skewed peak heights at heterozygous loci, and slightly increased rates of drop-in alleles. Amplification replicates adequately mitigated these issues; and when applied to actual case specimens, the modified amplification approach produced nine or more reproducible alleles in approximately 69% of the specimens tested. Despite the reaction and/or kit specific features observed in these amplifications of degraded and low quantity templates, the overall results from this study are remarkably consistent with those from a similar Y chromosome STR assay (2 Taq, 36 cycles) tested on a comparable set of samples [22]. The sensitivities of the two assays are nearly identical in terms of profile recovery at given DNA input quantities (compare Fig. 2 here to Fig. 1 in Sturk et al. [22]). Also, the chances of an amplification exhibiting stochastic drop-in alleles among the pristine, low quantity templates are similar (11% in Yfiler compared to 7% in PP16). Most encouraging though is the coherent picture that emerges from both the modified PP16 and Yfiler amplifications of authentic missing persons’ case material. With both assays, at least one third of the respective profile (Yfiler or PP16) was reproducibly recovered from more than 70% of the specimens tested. With both assays, Quantifiler measured input quantities of 250 pg or more produced complete or nearly complete profiles despite the highly degraded state of the templates. And finally, with both assays, drop-in was observed in approximately 15% of the authentic case work material. Taken together, the data from these two studies demonstrate that, despite the inherently stochastic nature of any single amplification – due to kit specific features, the quantity/ quality of the target template, and the extreme sample-to-sample variability among the specimens tested – consistent and reproducible results can be obtained for some of the most challenging skeletal elements. The lingering issues with the approach/application investigated here pertain primarily to a better understanding of stutter, heterozygote balance, and allelic dropout, specifically as they relate to our confidence in determining homozygosity. Experience indicates that replicate amplifications generally clarify specific issues. For instance, very high stutter peaks tend to be difficult to reproduce because of the random nature of their occurrence in the

first place. For stutter peaks to be interpreted as an authentic allele in a final profile, the stutter template must be generated in one of the first cycles of the amplification (so that the stutter allele can be amplified in high proportion relative to the authentic allele), it must be reproducible in the replicate amplifications and, it must generally happen at an otherwise homozygous locus (a reproducible stutter peak at a heterozygous locus would be evident in replicate amplifications as a third allele). While possible, this particular scenario was not encountered in the data generated in this study. Nevertheless, further in-depth analyses are warranted to define the data parameters under which we may assign homozygous autosomal loci, considering stutter averages and extremes alongside patterns of heterozygote balance. The authentic case material to which these amplification parameters will be regularly applied in our laboratory is nearly universally single-source, and samples are generally available to us in a quantity sufficient to allow for multiple samplings and/or extractions. In these types of situations where multiple samplings/ extractions are possible for clarification of unusual artifacts, heterozygote imbalance and allelic dropout, we are in favor of performing additional amplifications if (1) more data may resolve the question at hand and (2) the risks associated with generating those data (replication of spurious alleles, for instance) are minimal in comparison to the additional information that could be gained. Thus far, an approach based on allele replication is being followed for establishing reproducibility and authenticity [30,31,42,43]. Given that a conservative estimate of spurious allele duplication in even five amplifications is <0.0001 on average based on these data (this calculation does not take into account allele frequency, which would reduce this probability even further) there is, generally speaking, little risk in conducting additional amplifications and/or samplings. Clearly, in those cases for which the data are poor, background signal is high and spurious alleles are ubiquitous, caution should be exercised. But in general, this uncertainty would be reflected in weak or inconclusive results anyway. As DNA typing methods are increasingly employed on evermore small, damaged and degraded specimens and as DNA-based missing persons and mass fatality initiatives expand, the need for alternate means of sample re-association and identification will also become increasingly important. Although mtDNA data has largely been fit for purpose in most of the decades old missing persons cases encountered in our laboratory, it is also true that particular sets of commingled elements will never be accurately re-associated based on mtDNA data alone. Likewise, maternal references required for identification are not always, and may never be, available for many missing individuals. Thus, the recovery of STR data from a broad range of highly damaged and degraded skeletal specimens, using straightforward modifications to standard commercially available 16/17 locus STR kits, together with highly efficient DNA extraction methods, is an extremely promising step towards a greater number of identifications in large-scale missing persons’ efforts.

Acknowledgements The authors would like to thank LTC Louis Finelli, James Canik, Brion Smith, James Ross, Lanelle Chisolm, the Armed Forces Medical Examiner System and the American Registry of Pathology for their help in making this work possible. We are also grateful to Kimberly Andreaggi, Demris Lee and Suzanne Barritt for valuable discussion. The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or as views of the US Department of Defense or the US Department of the Army.

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References [1] M. Stoneking, D. Hedgecock, R.G. Higuchi, L. Vigilant, H.A. Erlich, Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes, Am. J. Hum. Genet. 48 (1991) 370–382. [2] M.M. Holland, D.L. Fisher, L.G. Mitchell, W.C. Rodriguez, J.J. Canik, C.R. Merril, et al., Mitochondrial DNA sequence analysis of human skeletal remains: Identification of remains from the Vietnam war, J. Forensic Sci. 38 (1993) 542–553. [3] M.M. Holland, T.J. Parsons, Mitochondrial DNA sequence analysis – validation and use for forensic casework, Forensic Sci. Rev. 11 (1999). [4] O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1 (2007) 191–195. [5] S. Amory, R. Huel, A. Bilic´, O. Loreille, T.J. Parsons, Automatable full demineralization DNA extraction procedure from degraded skeletal remaina, Forensic Sci. Int. Genet. (2011), doi:10.1016/j.fsigen.2011.08.004. [6] N. Rohland, M. Hofreiter, Ancient DNA extraction from bones and teeth, Nat. Protoc. 2 (2007) 1756–1762. [7] S.B. Seo, A. Zhang, H.Y. Kim, J.A. Yi, H.Y. Lee, D.H. Shin, et al., Technical note: Efficiency of total demineralization and ion-exchange column for DNA extraction from bone, Am. J. Phys. Anthropol. 141 (2010) 158–162. [8] P. Wiegand, M. Kleiber, Less is more – length reduction of STR amplicons using redesigned primers, Int. J. Legal Med. 114 (2001) 285–287. [9] H. Ohtaki, T. Yamamoto, T. Yoshimoto, R. Uchihi, C. Ooshima, Y. Katsumata, et al., A powerful, novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations, Electrophoresis 23 (2002) 3332–3340. [10] J.M. Butler, Y. Shen, B.R. McCord, The development of reduced size STR amplicons as tools for analysis of degraded DNA, J. Forensic Sci. 48 (2003) 1054–1064. [11] M.D. Coble, J.M. Butler, Characterization of new miniSTR loci to aid analysis of degraded DNA, J. Forensic Sci. 50 (2005) 43–53. [12] P. Grubwieser, R. Muhlmann, B. Berger, H. Niederstatter, M. Pavlic, W. Parson, A new ‘‘miniSTR-multiplex’’ displaying reduced amplicon lengths for the analysis of degraded DNA, Int. J. Legal Med. 120 (2006) 115–120. [13] T.J. Parsons, R. Huel, J. Davoren, C. Katzmarzyk, A. Milos, A. Selmanovic, et al., Application of novel ‘‘mini-amplicon’’ STR multiplexes to high volume casework on degraded skeletal remains, Forensic Sci. Int. Genet. 1 (2007) 175–179. [14] J.J. Mulero, C.W. Chang, R.E. Lagace, D.Y. Wang, J.L. Bas, T.P. McMahon, et al., Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA, J. Forensic Sci. 53 (2008) 838–852. [15] Applied, Biosystems, Improved recovery of interpretable results from challenging samples using the Identifiler1 Plus Kit – an overview of test site data, Forensic News (2009). [16] A. Piccinini, S. Coco, W. Parson, C. Cattaneo, D. Gaudio, R. Barbazza, et al., World War One Italian and Austrian soldier identification project: DNA results of the first case, Forensic Sci. Int. Genet. 4 (2010) 329–333. [17] J.A. Irwin, S.M. Edson, O. Loreille, R.S. Just, S.M. Barritt, D.A. Lee, et al., DNA identification of ‘‘Earthquake McGoon’’ 50 years postmortem, J. Forensic Sci. 52 (2007) 1115–1118. [18] W. Parson, A. Brandstatter, H. Niederstatter, P. Grubwieser, R. Scheithauer, Unravelling the mystery of Nanga Parbat, Int. J. Legal Med. 121 (2007) 309–310. [19] M.D. Coble, O.M. Loreille, M.J. Wadhams, S.M. Edson, K. Maynard, C.E. Meyer, et al., Mystery solved: the identification of the two missing Romanov children using DNA analysis, PLoS One 4 (2009) e4838. [20] J.A. Irwin, M.D. Leney, O. Loreille, S.M. Barritt, A.F. Christensen, T.D. Holland, et al., Application of low copy number STR typing to the identification of aged, degraded skeletal remains, J. Forensic Sci. 52 (2007) 1322–1327. [21] D. Marjanovic, A. Durmic-Pasic, N. Bakal, S. Haveric, B. Kalamujic, L. Kovacevic, et al., DNA identification of skeletal remains from the World War II mass graves uncovered in Slovenia, Croat. Med. J. 48 (2007) 513–519. [22] K.A. Sturk, M.D. Coble, S.M. Barritt, J.A. Irwin, Evaluation of modified Yfiler amplification strategy for compromised samples, Croat. Med. J. 50 (2009) 228–238.

587

[23] H.Y. Lee, M.J. Park, N.Y. Kim, J.E. Sim, W.I. Yang, K.J. Shin, Simple and highly effective DNA extraction methods from old skeletal remains using silica columns, Forensic Sci. Int. Genet. 4 (2010) 275–280. [24] M.T. Putkonen, J.U. Palo, J.M. Cano, M. Hedman, A. Sajantila, Factors affecting the STR amplification success in poorly preserved bone samples, Investig. Genet. 1 (2010) 9. [25] M.M. Holland, C.A. Cave, C.A. Holland, T.W. Bille, Development of a Quality, High Throughput DNA Analysis Procedure for Skeletal Samples to Assist with the Identification of Victims from the World Trade Center Attacks, Success rates of nuclear short tandem repeat typing from different skeletal elements, Croat. Med. J. 44 (2003) 264–272. [26] A. Milos, A. Selmanovic, L. Smajlovic, R.L. Huel, C. Katzmarzyk, A. Rizvic, et al., Success rates of nuclear short tandem repeat typing from different skeletal elements, Croat. Med. J. 48 (2007) 486–493. [27] C.I. Smith, A.T. Chamberlain, M.S. Riley, C. Stringer, M.J. Collins, The thermal history of human fossils and the likelihood of successful DNA amplification, J. Hum. Evol. 45 (2003) 203–217. [28] M. Hofreiter, D. Serre, H.N. Poinar, M. Kuch, S. Paabo, Ancient DNA, Nat. Rev. Genet. 2 (2001) 353–359. [29] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40. [30] J.P. Whitaker, E.A. Cotton, P. Gill, A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis, Forensic Sci. Int. 123 (2001) 215–223. [31] T. Caragine, R. Mikulasovich, J. Tamariz, E. Bajda, J. Sebestyen, H. Baum, et al., Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler, Croat. Med. J. 50 (2009) 250–267. [32] L.A. Dixon, A.E. Dobbins, H.K. Pulker, J.M. Butler, P.M. Vallone, M.D. Coble, et al., Analysis of artificially degraded DNA using STRs and SNPs – results of a collaborative European (EDNAP) exercise, Forensic Sci. Int. 164 (2006) 33–44. [33] S.M. Edson, J.P. Ross, M.D. Coble, T.J. Parsons, S.M. Barritt, Naming the dead confronting the realities of rapid identification of degraded skeletal remains, Forensic Sci. Rev. 16 (2004) 63–90. [34] A. Agresti, B.A. Coull, Approximate is better than exact for interval estimation of binomial proportions, Am. Stat. 52 (1998) 119–126. [35] I. Findlay, A. Taylor, P. Quirke, R. Frazier, A. Urquhart, DNA fingerprinting from single cells, Nature 389 (1997) 555–556. [36] J. Butler, Forensic DNA Typing; Chapter 6: Biology of STR, Elsevier Academic Press, Burlington, MA, 2005, p. 126. [37] S. Petricevic, J. Whitaker, J. Buckleton, S. Vintiner, J. Patel, P. Simon, et al., Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex, Forensic Sci. Int. Genet. 4 (2010) 305–310. [38] C. Alves, L. Gusmao, L. Pereira, A. Amorim, in: B. Brinkmann, A. Carracedo (Eds.), Multiplex STR Genotyping: Comparison Study, Population Data and New Sequence Information in Progress in Forensic Genetics, vol. 9, Elsevier, Amsterdam, 2003, pp. 131–135. [39] M. Delamoye, C. Duverneuil, K. Riva, M. Leterreux, S. Taieb, P. De Mazancourt, False homozygosities at various loci revealed by discrepancies between commercial kits: implications for genetic databases, Forensic Sci. Int. 143 (2004) 47–52. [40] R.L. Huel, L. Basic, K. Madacki-Todorovic, L. Smajlovic, I. Eminovic, I. Berbic, et al., Variant alleles, triallelic patterns, and point mutations observed in nuclear short tandem repeat typing of populations in Bosnia and Serbia, Croat. Med. J. 48 (2007) 494–502. [41] A. Alonso, S. Andelinovic, P. Martin, D. Sutlovic, I. Erceg, E. Huffine, et al., DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples, Croat. Med. J. 42 (2001) 260–266. [42] A. Cooper, H.N. Poinar, Ancient DNA: do it right or not at all, Science 289 (2000) 1139. [43] M.T. Gilbert, H.J. Bandelt, M. Hofreiter, I. Barnes, Assessing ancient DNA studies, Trends Ecol. Evol. 20 (2005) 541–544.