Technical Focus. Being Direct: Development and Optimization of a Direct Amplification Workflow for Single-Source Samples

Technical Focus Being Direct: Development and Optimization of a Direct Amplification Workflow for Single-Source Samples Forensic News 2012 Nicola Oldroyd, Jeff Sailus, Jennifer Schroeder, Lori Hennessy, Dennis Wang, Andrea Carbonaro Life Technologies Corporation Short Tandem Repeat (STR) multiplex PCR kits have formed the basis of forensic DNA testing for the last 15 years. Until recently, kits were generally designed to handle all types of samples as part of a single processing workflow. This approach has several advantages, not least minimizing the number of chemistries and protocols in operation in the laboratory. However, it does not reflect the very different nature of the samples involved or the interpretation demands associated with each sample type. Casework samples are often complex and unpredictable in nature, present on highly variable substrates, yield low-quantity and/or low-quality DNA, are aged or environmentally exposed and can contain more than one contributor. Database or casework reference samples in contrast are far more predictable, collected on standardized substrates (usually paper or swabs), yield high-quality DNA, are generally processed close to the time of collection and are collected from a single individual. Processing these very different types of samples according to the traditional workflow involving extraction, quantitation, amplification, electrophoresis and data analysis has been the norm and is manageable where sample numbers are not overwhelming. As sample numbers increase however, the differences in the samples hold the key to developing more efficient workflows that more closely reflect the specific demands of the samples involved. The number of single-source samples processed by laboratories has increased rapidly in recent years due to the success of national DNA databases. Expansion of legislation to permit sample collection from more individuals and for more crimes, coupled with the associated increase in reference samples for an expanded number of casework investigations has led laboratories to look for ways to simplify and streamline the processing of these more straightforward samples. Automation of the existing workflow is part of the solution but even greater efficiencies can be achieved by removing rate-limiting steps within the workflow and optimizing the remaining steps to ensure bottlenecks in the process are not simply shifted from one part of the workflow to another. STR amplification directly from single-source samples (direct amplification) offers the ideal route by which the need for extraction and quantitation can be removed and the workflow streamlined. In this article we will describe the evolution of Life Technologies chemistries for direct amplification and evaluate the various means by which both the quality of the results and the efficiency of the workflow as a whole can be maximized. Development of direct amplification STR kits Recent advances in extraction technology such as the PrepFiler Express and PrepFiler Express BTA kits have helped to significantly reduce the amount of time required to extract even difficult samples. However, extraction and quantitation still remain two of the most time consuming aspects of DNA sample processing. Both are vitally important for casework samples to help analysts understand the nature of the sample and determine the best way to process it such that the chance of a result is maximized. For single-source samples however, far fewer questions surround the sample, which provides the opportunity to eliminate these processes altogether through direct amplification. Achieving direct amplification is part of the process, but equally important is the ability to generate results comparable to those obtained using aqueous extraction to prevent issues with data interpretation downstream in the workflow. Knowing clearly what constitutes a high-quality profile for any particular sample type can assist in evaluating whether or not your choice of chemistry is delivering the desired results. High-quality results for single-source samples can be defined as follows: Well-defined peaks demonstrating good resolution with minimum occurrence of allelic drop-out or off-scale data Well-balanced profiles within each dye color (intracolor balance) Well-balanced alleles within a heterozygote (PHR) Clean baselines with minimal interference from artifacts Data meeting all of these criteria are more amenable to analysis using Expert System software, ensuring that time savings generated by removal of bottlenecks at the start of the workflow are consolidated by more efficient analysis downstream. Our goal therefore was to develop Direct Amplification PCR kits that could deliver data meeting these criteria and enable laboratories to realize the maximum benefits of the direct amplification workflow. 1

First attempts Preliminary investigations involving direct amplification from samples on FTA paper yielded highly variable and generally low-quality results, as STR kits developed to handle extracted DNA were simply not capable of overcoming the type and concentration of inhibitors commonly found in unpurified samples (Figure 1). [e] Figure 1 Figure 1. Examples of direct amplification of 1.2 mm punches taken from blood deposited on FTA Classic card using the Identifiler kit (panels b-e) compared to the quality of profile obtained from the Control DNA 007 (a). Figure 2 Manipulation of the punch size of the paper added to the reaction improved the performance to a certain extent, with smaller punch sizes generating more reproducible results (Figure 2), but we were still unable to generate the level of reproducibility required if direct amplification was to become a viable option for operational forensic laboratories. Clearly, a complete redevelopment of our existing chemistry was necessary to achieve successful direct amplification. Direct success the Identifiler Direct kit Using a design of experiments (DOE) approach, we developed an entirely new buffer system and amplification parameters engineered specifically to overcome the inhibitors present in unpurified blood and buccal samples on FTA Classic and Indicating cards respectively. This buffer was incorporated into the Identifiler Direct kit, which generates the data quality expected from purified samples on FTA cards without the need for purification and quantitation (Figure 3). A large number of FTA samples were tested as part of the developmental validation of the Identifiler Direct kit to confirm that the quality and reproducibility of the results were sufficient to fully support the use of direct amplification in an operational forensic environment. Of particular note was data generated from an extensive population study that demonstrated high levels of heterozygote and intracolor balance (Figure 4). These metrics are an effective measure of data quality and are of particular importance if laboratories are to maximize the efficiency of single-source sample data analysis (optimization of data analysis software to support the direct amplification workflow will be discussed in more detail later in this article). Figure 3 Figure 2. Examples of profiles obtained from a blood sample on FTA Classic card using punch sizes of 1.2 mm (b) 0.75 mm (c) and 0.5 mm (d) compared to the quality of profile obtained from the Control DNA 007 (a). All amplifications performed using the Identifiler kit. Figure 3. Examples of profiles obtained from two purified blood samples using the Identifiler kit (a,b) and unpurified 1.2 mm punches of the same blood samples on FTA Classic card using the Identifiler Direct kit (c,d). The profiles from purified and unpurified samples are indistinguishable. 2

Figure 4 Intracolor balance (%) Intracolor balance (%) Figure 4. Peak height ratio (a) and intracolor balance (b) results from a population study conducted as part of the Identifiler Direct kit developmental validation study. All results from 1.2 mm punches of blood samples on FTA Classic card. New directions expanding to non-fta substrates Focusing our initial direct amplification efforts on a single substrate type allowed us to gain a comprehensive understanding of the nature of direct amplification and the associated challenges. However, FTA card represents only one of several different substrates used to collect single-source samples for database or casework reference use, and our next goal was to find a way to expand the use of direct amplification to alternative substrates. One of the benefits of treated paper substrates such as FTA is that samples are lysed on contact with the paper, thus releasing [e] [f] [g] Figure 5 Figure 5. Examples of profiles obtained from 1.2 mm punches taken from buccal samples on Bode Buccal DNA Collector (b-d) and buccal samples on 3 different swab types: Copan Flocked Swab (e), Whatman Omniswab (f) and Puritan Cotton Swab (g). A profile from the Control DNA 9947A is included for comparison (a). All samples were amplified with the Identifiler Direct kit. the DNA and making it available to the PCR reagents for amplification. Two common substrates used for sample collection are untreated filter paper and swabs, neither of which lyse the sample. Therefore, for these substrates, a means of lysing the sample is required that releases the DNA for amplification but does not add significantly to the number of steps or time required for the overall workflow. Our solution to this problem is the Prep-n-Go Buffer which lyses DNA collected on untreated paper or swab substrates quickly, without the need for heated incubation. This workflow generates the same high-quality data already demonstrated on FTA (Figure 5). Fast and direct enabling direct amplification on new marker sets The Identifiler Direct kit project allowed us to evaluate options for successful direct amplification from multiple substrates, but, in order to meet the loci needs of international jurisdictions, our next goal was to extend this capability to alternative marker sets. The NGM SElect kit contains all the markers specified in the expanded European Standard Set of loci and is the most widely used of our next-generation kits in Europe. Leveraging our experiences with the Identifiler Direct kit, we have now developed an enhanced system based on the NGM SElect kit that can not only perform direct amplification from treated paper, untreated paper, and swab substrates, but can also complete the amplification in under an hour, reducing still further the time required to process single-source samples. An updated buffer system and the inclusion of a new fast-cycling enzyme allow the NGM SElect Express kit to generate profiles with all the necessary quality hallmarks in the shortest cycling time possible. For more information on the NGM SElect Express kit see the article in this issue of Forensic News entitled What s New: NGM SElect Express PCR Amplification Kit Now Available! 3

Figure 6 Figure 6. Examples of direct amplification of 1.2 mm punches taken from a blood sample on FTA Classic card (a) and a buccal sample on FTA indicating card (b) using the NGM kit. TH01 in particular may show lower than expected levels of performance in buccal samples when amplified under direct amplification conditions. We have also conducted experiments to determine the capability of our other next-generation STR kits to perform direct amplification, should either the NGM SElect or Identifiler kit configurations not suit the needs of particular laboratories. The NGM kit has been shown to be capable of performing direct amplification on the substrate types already mentioned using very similar workflows. However, the lack of specific optimization of this kit for direct amplification does mean that result quality is not as reproducible across all sample types as compared to the Identifiler Direct and NGM SElect Express kits. Blood samples produce higher-quality results than buccal samples, particularly in terms of balance within each dye color (Figure 6). Laboratories may find that the use of Prep-n-Go Buffer with samples on FTA card improves the balance of the profile, and for all sample types a slight adjustment to the recommended PCR parameters of an increase from 10 to 25 minutes of the final extension step to ensure complete addition is required. Individual laboratories will need to evaluate whether the data quality of kits not designed specifically for direct amplification meets their requirements. Life Technologies will continue to evaluate further opportunities for development of new direct amplification systems to accommodate users specific locus requirements. Maximizing the quality of direct amplification results Regardless of the direct amplification system being used, there are a number of measures each laboratory can take to maximize the quality of data obtained and increase the efficiency of the amplification and any downstream analysis processes. Figure 7 Average peakk heights / sample (RFU) Figure 7. Mean peak height analysis of Identifiler Direct kit amplifications of 1.2 mm punches taken from blood samples on FTA Classic card (red) and buccal samples on FTA Indicating card (blue). Data generated from internal testing and external customer test site collaborations. Buccal sample data demonstrate a wider range of peak heights than blood samples due to the lower homogeneity of the cell composition in this sample type (majority data range indicated by dashed lines to aid comparison). Choice of sample type Many jurisdictions have already settled on preferred substrates and sample types for the collection of database and/or casework reference samples, however laboratories are constantly exploring ways to improve success rates and, here, sample type plays a crucial role. Also with more and more countries passing database legislation and determining the logistics of how these databases will 4

operate, considering the most appropriate sample type/substrate combination to facilitate future processing is an important aspect of maximizing throughput potential. Paper substrates are inherently easier to automate than swab substrates due to the availability of automated punching systems, but swab substrates may be more cost effective if budgets are under pressure. Blood samples generally produce more consistent results than buccal samples due to the greater homogeneity of the body fluid (Figure 7) and therefore deliver slightly higher first-pass success rates (Table 1). Buccal samples are, however, logistically and, in some cases ethically, easier to collect, therefore usage of this sample type is expanding. Effective transfer of buccal samples from swab to paper substrates For storage on paper substrates, buccal samples may be collected using swabs and then transferred to the paper. The type of swab used and the method of transfer can have a significant effect on the ultimate success of the amplification. Our studies have shown devices designed specifically to facilitate sample transfer such as the Whatman Easicollect system produce reliable and consistent results (data not shown) when used correctly. Alternatives such as individual foam paddle swabs can also work well provided laboratories optimize the transfer process and evaluate transfer success rates through internal validation studies. Poor transfer of buccal cells from the swab to the paper will ultimately lead to low amplification rates and impact the efficiency of the direct amplification workflow. Using the optimum punch size and position for paper substrates Our investigations indicate that the size and the position of the punch can make a significant difference to the signal intensity and reproducibility of the profile. Table 1 VTS Study CTS Study Sample Type PCR Success Rate (50 RFU) Range of Success Rates Mean Success Rate Interpretation Success Rate (150 RFU) Range of Success Rates Mean Success Rate Number of Samples Tested Blood 99.4% 99.4% 95.7 98.8% 97.3% 414 Buccal 91.8 99.4% 97.1% 84.2 95.5% 90.9% 653 Blood 100% 100% 98.8 100% 99.8% 437 Buccal 98.7 100% 99.0% 91.7 100% 94.7% 703 Table 1. Success rate analysis of the results generated by our external customer test sites during verification (VTS) and confirmation (CTS) testing of the Identifiler Direct kit. Blood samples demonstrate higher overall first-pass success rates due to the greater homogeneity of the sample type but both samples types still deliver average success rates in excess of 90%. The smaller the punch size, the lower the level of inhibitor added to the reaction, which generally maximizes the performance potential of the assay. However, very small punch sizes (<1 mm diameter) can be difficult to handle due to static issues, may be difficult to visualize in the well, are more difficult to automate due to the limited availability of small automated punch heads, and, for buccal samples, may not contain enough DNA for successful amplification. Larger punch sizes, though easier to handle, generally contain higher levels of inhibitor that can have a detrimental effect on the performance of the assay. Both the Identifiler Direct and the NGM SElect Express kits are optimized for a 1.2 mm punch size, which represents a balance between minimizing the inhibitor level and maximizing ease of handling. Another factor influencing the effect of punch size is the reaction volume of the assay. Performance on the 1.2 mm punch size has been validated using the recommended reaction volume of 25 µl. Reducing the reaction volume may have an effect similar to that observed when the punch size Average Peak Height (RFU) Figure 8 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 A Rep #1 1 2 3 4 5 A Rep #2 Figure 8. Mean peak height results demonstrating the effect of punch position on the signal intensity of the profile. 80 µl of three different blood samples was spotted onto FTA Classic cards and 5 x 1.2 mm punches taken at intervals from the center to the edge of the sample deposition area (a). Punches were taken in duplicate for each sample and amplified using the Identifiler Direct kit. Signal intensity decreases as distance from the center of the sample deposition increases (b). B Rep #1 Sample B Rep #2 80 µl-1 80 µl-2 80 µl-3 80 µl-4 80 µl-5 C Rep #1 C Rep #2 5

Figure 9 Table 2 Sample Type Substrate Treated Paper Untreated Paper Swabs Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A Buccal 26, 27, 28 cycles 26, 27, 28 cycles 25, 26, 27 cycles Figure 9. An example of the Bode Buccal DNA Collector (a) showing untreated collection paper housed within a plastic receptacle. The exposed end can be used to collect buccal cells directly from the inside of the mouth, and our studies indicate that taking the punch from the tip of the collector yields the most reproducible results (b). is increased: the performance of the assay may become compromised due to reagents being present in insufficient quantities. With regard to the position of the punch, our studies indicate that, for paper substrates where the blood has been spotted or the buccal sample deposited via transfer from a swab, as the punch position moves away from the center of the sample deposition area, the signal intensity decreases (Figure 8). Our recommendation is therefore to punch the sample as close to the center of the sample as possible to maximize signal intensity and reproducibility. For paper substrates used directly as a buccal swab (e.g., the Bode Buccal DNA Collector ), our studies and those of our collaborating test site UNT Center for Human Identification, University of North Texas Health Science Center indicate sampling close to the edge of the swab will generate the best results (Figure 9). Optimization of cycle number The unpurified nature of the samples and the inability to quantitate paper substrates mean that laboratories evaluating the use of direct amplification should expect to see greater variation in peak height from sample to sample than is generally observed with purified samples. To account for sample-to-sample and instrument-to-instrument variation, each laboratory is recommended to conduct an initial sensitivity study for each sample type/substrate combination to determine the optimum cycle number for their particular environment. Most laboratories should only need to evaluate three different cycle Table 2. Suggested cycle number ranges for determination of optimum cycle number for the sample types/substrates supported for use with AmpFlSTR Identifiler Direct and NGM SElect Express kits. Buccal samples in general require at least one cycle number higher than blood samples. Our testing has not included blood samples on swab substrates, as these are not a frequently encountered database or reference sample type. Prep-n-Go Buffer is required for amplification of samples on untreated paper and swab substrates. Laboratories may choose to evaluate alternative cycle numbers if the ranges suggested do not yield optimal results. numbers to identify their particular optimum, with the cycle numbers recommended depending on the sample/substrate combination (Table 2). The optimum PCR cycle number should generate profiles with heterozygote peak heights of 1,000 to 3,000 RFU on a 31XX Genetic Analyzer or 3,000 to 12,000 on a 3500 Series Genetic Analyzer with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. More details on conducting the sensitivity study can be found in the Identifiler Direct and NGM SElect Express Kit User Guides. Optimization of software parameters When evaluating direct amplification as an option in the laboratory, the temptation is to focus only on the kit itself and the parameters that directly affect its performance. However, to truly maximize the impact and efficiency of the direct amplification workflow, it is beneficial to optimize the software settings used to analyze the profile. This will help to ensure that the processing bottlenecks removed at the beginning of the workflow are not negated by increases in analysis time. The goal of a direct amplification workflow in data analysis terms is to maximize the number of samples passing through the software without the need for extensive, time-consuming, error-prone manual edits by the user. Data quality is a key factor in determining the ease with which data can be analyzed hence our attention to all the performance aspects of the chemistry during development. The better the data quality, the more suitable it becomes for processing with Expert Systems such as GeneMapper ID-X Software. Whether you wish to take advantage 6

of the full Expert System workflow, where samples passed by the software need no manual review, or you are simply looking to optimize your software system to minimize the level of manual editing required, particular attention paid to optimizing the following analysis parameters can help achieve the desired result: 1. Allele label filter settings The default settings in the GeneMapper and GeneMapper ID-X Software analysis methods employ locus-specific stutter filters in order to remove labels only from stutter peaks that fall within the expected range. All other peaks are left labeled, as these settings are more tailored to casework evidence samples where all extraneous peaks need to be evaluated and assessed as potential indicators of a mixed profile. For known single-source database and casework reference samples, such conservative analysis may not be required and the settings can be adjusted accordingly (Figure 10). For single-source samples, users may prefer the use of a global filter which removes labels from all peaks falling below a defined percentage threshold of the highest peak in any particular dye color. This would cover stutter peaks, pull-up, spikes, artifacts, and any other sources of baseline noise, leaving only the alleles remaining. As a starting point users may consider evaluation of a 20% filter but each laboratory can refine this percentage according to the results of internal studies. These settings would minimize the number of extraneous peaks labeled thereby reducing the number of labels that would need to be removed manually by the analyst. 2. Peak Quality settings As with the Allele settings, the default Peak Quality settings are also geared towards the analysis of casework evidence samples and the detection of potential mixtures. We recommend all laboratories determine the most appropriate settings for this tab for casework evidence data based on internal studies, and most laboratories would employ exactly the same process for single-source samples. In general, you would expect to see differences in the values used for four key parameters between analysis settings for evidence and single-source samples (Figure 11). Homozygous and heterozygous peak height minimum values are designed to alert the user to the presence of low-intensity data and therefore the risk of allelic dropout. As discussed earlier in this article, users of direct amplification kits are encouraged to choose amplification conditions that effectively eliminate instances of allelic dropout, dramatically reducing Figure 11 Figure 10 Figure 10. Comparison of suggested GeneMapper ID-X Software analysis parameter Allele settings for casework evidence sample using the NGM SElect kit (a) and single-source database and casework reference samples using the NGM SElect Express kit (b). For single-source samples, marker-specific stutter filters are deselected in favor of the more general global filter. Figure 11. Default analysis method Peak Quality settings in GeneMapper ID-X Software. Fields indicated by red arrows can accommodate more lenient values to avoid flagging successful single-source profiles unnecessarily during analysis. 7

the risk of low-signal data for these types of samples. These settings can therefore be set more leniently. Likewise the Peak Height Ratio settings are designed to detect peak height ratio imbalance, which can be an indicator of the presence of a mixture. Database and casework reference samples are expected to be single-source, permitting this parameter to be set more generously to avoid flagging samples suffering only from inherent amplification variation. Other parameters, such as allele number, will detect more general contamination events so the user does not need to rely on the more subtle measures of profile quality. Due to the wider range of peak height expected in data produced by direct amplification, users may wish to evaluate increasing the Maximum Peak Height setting. This setting is designed to alert the user when data is approaching off-scale and thereby becoming more difficult to interpret. For casework evidence data, this is an important factor. However, users may feel that for single-source samples, higher signal data that has not gone off-scale can still be interpreted successfully without the need for reinjection or reamplification. Here we have dealt only with the analysis parameters that have particular bearing on the efficiency of single-source data analysis. Laboratories should however evaluate all the analysis parameters used for single-source samples as rigorously as they would for casework evidence, and ensure samples are flagged only if they suffer from issues affecting the genotyping accuracy of the profile (Figure 12). Removing a few alleles manually may not seem like a huge task but the process can be prone to error and, as sample numbers increase, editing can become a substantial Figure 12 Figure 12. Demonstration of the impact of combining high-quality Direct Amplification kit data with optimized analysis parameters. Analysis of Identifiler Direct Kit Test Site blood and buccal sample data (amplified at 25 and 27 cycles respectively) using default GeneMapper ID-X Software analysis parameters results in a project where the majority of samples require manual review (a) and extensive editing to remove extraneous labels (b). Analysis of the same data set using parameters adjusted to better support analysis of single-source samples results in a project where the majority of samples require no manual review, having met all specified thresholds (c) and no editing (d). Project C would require significantly less analysis time than Project A. The only differences in the analysis parameters used were: use of a Partial Analysis Range instead of Full Range; use of a 20% Global filter instead of marker-specific stutter, increased Maximum Peak Height setting from 5,000 to 7,000 RFU, and reduction of Peak Height Ratio setting from 0.7 to 0.6. 8

burden. Analysis of extended data sets including those generated by our collaborating test sites demonstrates clearly the value of optimizing analysis parameters such that samples are passed by the software whenever it is appropriate. This practice significantly improves the efficiency of the data analysis process and consolidates the time savings offered by direct amplification STR kits (Figure 12). Being direct Over the last three years Life Technologies has conducted extensive scientific investigations to develop innovative STR chemistries capable of amplifying samples directly from their substrates without the need for extraction or purification. This process is known as direct amplification but in our opinion, the term represents more than just a choice of kit. It reflects an entire workflow through which laboratories can significantly reduce the time taken to generate results from single-source samples. The combination of a kit designed to produce high-quality data and an optimized analysis system enables laboratories to realize maximum time and labor savings, whilst also matching or improving their success rates with existing methods. How to Cite This Article N. Oldroyd, J. Sailus, J. Schroeder, L. Hennessy, D. Wang, A. Carbonaro, Being Direct: Development and Optimization of a Direct Amplification Workflow for Single-Source Samples. Forensic News February; 2012. [cited: year, month, date]; Available from: http://www.appliedbiosystems.com/forensicnews 9