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Microdevice DNA forensics by the simple tandem repeat method
Journal of Chromatography A, 1111 (2006) 206–213
Microdevice DNA forensics by the simple tandem repeat method Nils Goedecke, Brian McKenna, Sameh El-Difrawy, Elizabeth Gismondi, Abigail Swenson, Loucinda Carey, Paul Matsudaira, Daniel J. Ehrlich ∗ Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142-1479, USA Available online 22 June 2005
Abstract We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures. © 2005 Elsevier B.V. All rights reserved. Keywords: Forensic DNA analysis; Microdevice electrophoresis; Microfluidics; Short tandem repeat
1. Introduction Few practitioners would dispute that the most important recent development in human forensics has been the introduction of the simple tandem repeat (STR) method for DNA typing. However, widespread application of the method is relatively new. The Forensics Sciences Service in the UK led the way in the development of the technology and the demonstration of its power in the context of computer-searchable databases [1]. Only in the last 5 years has the USA developed a sufficient trained-operator community and a sufficiently large database to begin to achieve similar results. The success of the STR method stems from the combination of (1) the sensitivity, speed, and cost-effectiveness of a DNA assay that utilizes the polymerase chain reaction (PCR) in an efficient multiplex on hyper-variable regions of the human genome combined with (2) the power of a computer-searchable data base. However, since STR forensics is so new, the technology to support it was largely co-opted from general-purpose laboratory instrumentation without optimization for the forensics application. Over the last several years we have been developing technology specifically for STR forensics. We have chosen to use ∗
Corresponding author. Tel.: +1 617 258 7283; fax: +1 617 258 7226. E-mail address: [email protected] (D.J. Ehrlich).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.05.100
the format of microdevices for this purpose. However, interestingly, in the course of our study we have found that it is not the much-touted high-speed or compact size of microdevices that will drive the wide adoption of our technology. Rather, it appears that entirely unanticipated virtues of the microdevice, namely higher data quality and greater assay reproducibility are the most important virtues for forensics. In this paper we review our development to date of microdevice-based STR forensics. The STR method is a fairly classical genotyping assay in which PCR with labeled primers is applied (for the CODIS data base in the USA) as a 13-locus multiplex [2]. The sex marker amelogenin is thrown in. The multiplex is usually read out on a standard capillary array electrophoresis (CAE) instrument using buffer systems and sieving matrices only slightly modified from those used for DNA sequencing. Primers have been designed so as to spread the multiplex across four or five fluors within a total fragment range of less than 500 base pairs. Although the alleles of STR are most usually four-base repeats, there are several microsatellite alleles separated by a single base pair that are common in the general human population. Therefore, although the CAE separation of four-base repeats over 500 base fragments would seem to be undemanding, in fact, the microsatellites require the same single-base resolution of DNA sequencing. What is more, the forensics application is for courtroom evidence,
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with huge societal costs for errors. Hence, the allele-calling accuracy (quality score) of the forensics assay is far beyond that needed for (highly redundant) shot-gun DNA sequencing. The resolution requirement is usually set at 1.0, well above the minimum resolution of <0.3 now standard with DNA base-calling [3]. In our first publication in 1997, we showed that it was possible to use a microdevice and standard linear polyacrylamide (LPA) chemistry to separate the principle 4-base repeats of the CTTV STR multiplex in as little as 30 s [4]. This led us and others to the conjecture of a “real-time” STR microdevice. This direction will likely still be played out. However, we believe the near-term application of microdevice DNA forensics will be motivated on the factors we list in the following sections.
2. Microdevice implementations of STR forensics In a recent publication we described a microdevice electrophoresis system, comprising custom-designed four-color detector, microdevice support and data reduction software [4]. The system is designed with 16-lanes, each with a 20-cm effective length, in order to support the typical crime-scene analysis, which includes standards for positive and negative controls and a sizing ladder and must achieve highly resolved separation of the 9.3 from the 10.0 allele of the TH01 locus. Fig. 1 shows the separation device. Each of the 16 channels is 130 m wide, 60 m deep and 20 cm long. These devices are fabricated from two glass plates (Corning 1737F) each 1.1 mm thick, 43 mm wide and 250 mm long. Due to the isotropic etching the channel cross section is nearly hemispherical. The sample inlet and sample waste arms for the electrokinetic injection are both 2.5 mm long. The offset between both channels is 500 m giving a total injection volume of 3.5 nl within a classic double-T injector [5]. More
Fig. 1. The microfluidic assembly has a bonded two-layer glass body and two interface boards. The board on the anode side contains only one buffer reservoir, which can accommodate up to 2 ml of buffer. The board on the cathode side has one large buffer reservoir equivalent to the anode buffer reservoir. It also contains 32 smaller wells (70 l), which are used as sample inlets and sample wastes. All 16 lanes taper off for the scanning region, wherein they are parallel and have a total width of 3.2 mm.
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details on the fabrication procedure and device design are given in ref. [6]. A modified Hjert´en procedure is used to suppress electroosmotic flow [7]. For most of the experiments samples were prepared using the procedures outlined in the manual from Promega for PCR kit PowerPlex version 1.2 [8]. This STR kit amplifies eight loci and the sex marker amelogenin. The internal lane standard (ILS) is labeled with carboxy-X-rhodamine (CXR). The labels for the loci are fluorescein (FL), 6-carboxy-4 ,5 dichloro-2 ,7 -dimethoxy-fluorescein (JOE) and carboxytetramethylrhodamine (TMR). Each 25-l PCR reaction contained: 16.7 l nuclease free water, 2.5 l GoldSTAR 10× buffer (Promega), 2.5 l PowerPlex primer pair mix at 10× concentration, 0.8 l (4 units assuming 5 u/l) AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) and 2.5 l DNA temPlate 9947a (Promega) with a concentration of 0.8 ng/l. The amplifications were performed according to the suggested 30 cycles from the manufacturers manuals with a PCR system 9700 thermal-cycler (Perkin-Elmer, Wellesley, MA, USA). After the initial heating at 95 ◦ C for 11 min and 96 ◦ C for 1 min followed 10 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 70 ◦ C for 45 s. The next amplification step included 22 cycles of 90 ◦ C for 30 s, 60 ◦ C for 30 s and 70 ◦ C for 45 s. The procedure finishes with two hold periods: (i) 60 ◦ C for 30 min and (ii) 4 ◦ C until the product is taken out of the thermo cycler. After amplification, the PCR products were purified using a GFX spin column (Amersham Bioscience). The PCR products from four reactions were combined to gain 100 l with equalized PCR efficiency and purified with one column. This column exploits the fact that DNA clings to glass fiber using a chaotropic buffer. Excess nucleotides and salts are then washed out by use of an ethanol-based washing buffer. After the washing steps the DNA is re-suspended in 100 l nuclease-free water. The yield of this purification step is usually around 80%. The sample and standard solutions were prepared from 12 l nuclease-free water, plus 0.5 l of purified PCR product (or allelic ladder) and 0.5 l of internal lane standard (ILS). All solutions were denatured at 95 ◦ C for 3 min, then placed on crushed ice immediately for at least 5 min and used within 30 min. To carry out the separations we load fresh separation matrix (4% LPA) using a gastight 100 l glass syringe (Hamilton, Reno, NV, USA). Then the cathode, anode, and sample waste vials are filled with 1× TTE buffer and the sample vials are filled with deionized water. An electrode board is placed on top of the cathode board and the anode board to prevent evaporation and to realize electrical contact. The microdevice is placed on the instrument’s heated stage at 50 ◦ C and equilibrates in about 3 min. The run protocols are automated in our machine. Preelectrophoreses is at 3900 V for 6 min on the cross injection and 175 V for 3 min on the separation channel. Subsequently the buffer and water are replaced and the microdevice is left on the heated stage for 30 min. Shortly after that the vials are
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rinsed with water again. Then the buffer is replaced in the cathode, anode, and sample waste vials. For each lane a 13l sample is transferred into the sample vials. The sample is loaded into the microchannel using 175 V from sample inlet to sample waste vial and after 30 s an additional 390 V from cathode to anode. We call this “dual-voltage” loading mode [9]. Its purpose is to eliminate network effects that result from the fact that all channels are connected at the cathode (see Fig. 1). Next, the sample in the injector is stacked by applying a 6 s voltage impulse along the separation channel of 10 000 V [10]. Finally, the CE is started by applying the separation voltage of 3900 V giving a typical runtime of 40 min for a 600 bp fragment.
3. Data quality and reproducibility Sgueglia has thoroughly characterized the reproducibility of CAE machines for STR forensics [11]. This study was carried out in the very well controlled environment of a state forensics laboratory. Nonetheless, current CAE machines put severe constraints on environmental factors, particularly room temperature, in order to meet the required stability. This constraint is even more severe (or untenable) in a less controlled environment such as a mobile laboratory. Data reproducibility is enormously important to crime-scene forensics because of its implications to the lengthy process of real-world data analysis (see below) and also because of the “overhead” that instability implies in machine calibration. To test the data reproducibility of the microdevice we repeated much of the study of Sgueglia using our system. Fig. 2 shows a typical processed electropherogram, as read out on four photomultipliers for the Promega PowerPlex 16. In order to perform statistical analysis, data of this kind was
accumulated periodically over a period of 4 days. In each run 14 out of 16 lanes were used for the DNA temPlate 9947a and two for the allelic ladder. In this data set, 92 out of 96 lanes performed successfully. Of the four lanes (one with DNA template and three with allelic ladder) that failed, two failed due to bubbles deriving from the separation matrix in the microchannel. The other two lanes contained weak signals that could not be processed for reasons that are not explained. The values for each allele have been analyzed to determine its size in base pairs for mean, standard deviation (σ), and standard deviation bin (±3σ from mean). All of our investigations were performed using microdevices, identical to the assembly shown in Fig. 1. The quality of the signal suggests that the channel coating did not degrade during the investigation. The microdevices have been used for 7 months approximately 80 runs each. Fig. 3 represents a cutout of an electropherogram showing locus TH01. Again the sample was amplified with PowerPlex 16. The data is presented in the foreground and the background illustrates the allelic ladder. A DNA template from the female cell line 9947a has an 8 and 9.3 repeat for this locus. Most of the CODIS core loci have tetranucleotide STRs. However, there are wide spread alleles that differ only by one base pair: like TH01 9.3 and 10. Therefore, the system must be reliable enough to consistently call alleles within 0.5base-pair-accuracy for such a locus, and 1.0-base-pair for all others. To test our instrument’s consistency, we analyzed the measured base-pair values of our lanes to determine a mean and standard deviation of our samples. Fig. 4 shows the allele calling results for locus TH01 taken from our sample data. From this we calculate the window about the mean (±3σ) that can be plotted on our software over a called allele. By observing that the peak is within the window and that the windows from neighboring alleles do not overlap, we can determine to within 3σ certainty that the peak is assigned correctly.
4. Complex mixtures
Fig. 2. Electropherogram of a data lane showing DNA temPlate 9947 multiplexed with PowerPlex 16 kit. The lane data has been processed and allele called. The four segments show from top to bottom: internal lane standard (red), TMR (yellow), JOE (green) and FL (blue) labeled fragments.
Although in database building exercise (collection of samples from offenders) the DNA sample can be presumed to be from a single donor, crime-scene samples are very often mixtures of DNA from the offender and the victim [12]. Since the STR multiplex from a single donor is as many as 27 alleles, mixtures with the complexity of unknown sample ratio, genetic degeneracy, and PCR artifacts, become a tour de force in interpretation. The interpretation begins by eliminating artifacts introduced by PCR and CE, estimating the relative proportion (ratio) of donors and then considering all the possible genotype combinations [13]. The mixture ratio will be preserved throughout all loci and most commonly accepted interpretation methods use peak area and height to assign the peaks to the major and minor donor [14]. In some mixtures the process of donor profile determination is straightforward. The easiest situation occurs where both donors contribute enough to produce strong peaks but
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Fig. 3. TH01 section of an electropherogram of a data lane showing DNA temPlate 9947 multiplexed with PowerPlex 16 kit. The background shows the allelic ladder. The foreground shows the 8 and 9.3 repeats of this template. Also in the figure are the sigma bins (±3σ). The lane data has been processed and allele called Fig. 2: electropherogram for a data lane with 2:1 mixture in foreground with allelic ladder in background.
Fig. 4. Statistics of results from TH01. All repeats show a variance of less than 0.5 base pair.
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Fig. 5. Electropherogram for a data lane with 2:1 mixture in foreground with allelic ladder in background. Three alleles are visible for loci D5S818, two unbalanced alleles for D13S317, D7S820 and two balanced alleles for D16S539. The calculated relative areas (pa) of each peak confirmed the mixture ratio of 2:1. Multiple data runs for this same mixture show relative areas for each peak consistent within 3%.
at an unequal ratio that helps to distinguish between the two donors based on the allele peak height and area. Fig. 5 shows four loci for such an example. Three alleles are visible for loci D5S818-9, 10 and 13. By measuring the area under each peak and normalizing as a percentage of the loci total peak area (across the locus) we determine the relative peak area (rpa) for each (i.e., in this case rpa = 0.66, 0.17 and 0.17). The only possible interpretation for this locus is that the major donor (A) contributed a homozygous 9 while minor donor B contributed two heterozygous alleles at 10 and 13, and the mixture ratio is 2:1. Two alleles are found for loci D13S317, 11 (rpa = 0.28) and 12 (rpa = 0.72). There are two possible interpretations of this: donor A with a homozygous 12 and donor B with a homozygous 11; or donor A with a heterozygous 11, 12 and donor B with a homozygous 12. The two alleles found for D7S820, 10 (rpa = 0.33) and 12 (rpa = 0.64) offer a similar two possibilities. The two alleles are balanced for D16S539, 11 (rpa = 0.51) and 12 (rpa = 0.49), indicating that both donors are heterozygous 11, 12. Balanced alleles in mixtures are interesting in that they can exist for any mixture ratio and can easily be determined for higher ratio mixtures, but cannot be determined for mixtures that approach 1:1. In 1:1 mixtures, two equal-size peaks would provide three possible donor combinations. After evaluating 4 loci we would have four possible profiles for donors A and B. Continuing the process for all 13 CODIS loci would yield about a dozen possible donor profiles. If we identify one donor (i.e., the victim) we can then identify the other donor. Without one identified donor, we can still compare both profiles to a DNA database presenting investigators with a short list of potential suspects.
Other mixtures are more difficult to analyze. In mixtures where the mixture ratio is high, the small peaks of the minor donor will challenge the dynamic range of the instruments detection ability. For example, a mixture with a ratio of 9:1 could have one locus with one allele peak representing both donors and one locus with four peaks. The size difference between the largest peak and the smallest peak would be 20:1. For a 19:1 mixture that ratio would be 40:1. At some point minor-donor peaks will not be distinguishable above noise and/or cannot be assigned on top of a major-donor peak. The limit at which these mixtures can be interpreted will depend upon the confidence in peak area measurement, and the ability of the instrument to determine signal from noise. To test our instrument for use with mixtures we performed three series of tests to determine consistency in determining peak areas and ratios. In each test we ran mixture samples multiple times and called our raw data using our laboratorydeveloped software algorithms to assign amplicon length and peak area. To determine run-to-run variation we normalized the peak area for each allele against the sum of all measured peak areas in the locus. We then determined the average relative peak areas (rpa) and standard deviation (σ) for each allele in our mixtures. In the first series, we mixed two samples at four near-equivalent mixture ratios. In our second, we tested more extreme mixture ratios. In our last series of tests we experimented with run protocol modifications to see if we could enhance system detection for difficult mixtures. The DNA samples were prepared using the procedures outlined earlier with the PowerPlex 1.2 kit (Promega) which amplifies eight loci and the sex marker amelogenin. This
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Fig. 6. Mean relative areas for three peaks from loci CSF1PO for two DNA donors that are mixed at four different ratios. Error bars show ±3δ variance for n = 21, 18, 17 and 14, respectively.
process was done for each cell line separately. Since this process has a 20% loss of DNA, the final step is performed by eluting the DNA with 80 l of DNAse-free water. For the sample preparation, prior to the actual CE, we mixed 240 l of autoclaved, pure water with 20 l PCR product mixture and 20 l internal lane standard 60–400 base pairs (Promega). The DNA fragments of this cocktail were denatured on a hot plate for 3 min and put on ice thereafter. A sample volume of 13 l was transferred into each sample well for the subsequent injection. In our first tests we combined two cell lines 9947a and 9948 (Promega, Madison, WI, USA). We created four sample mixtures of 0.2 ml with mixture ratios of 7:3, 1:1, 3:7 and 1:9. We collected 21, 18, 17 and 14 samples, respectively, processed the raw data with our allele-calling algorithms and calculated the average relative peak area and variance for each mixture type. We found variance to be similar for all loci in our kit, with standard deviations varying from 0.006 to 0.018. Fig. 6 details the results for one locus, CSF1PO, which is located on the yellow channel in the 280–320-bp region. Our mixture provides this locus with one two-donor peak that represents the same allele from each donor, and two singledonor peaks. Looking at the four mixture ratios, CSF1PO-10 remains constant for all mixture ratios (50% of total allele area). As the amount of donor B decreases in each mixture, the relative size of CSF1P0-11 decreases and CSF1PO-12 increases. The insert into Fig. 6 shows a cutout from an electropherogram for each mixture type for this locus. The bar graph shows the average relative peak area (rpa) for each allele and sample mixture with error bars that show ±3σ. Variance is consistent throughout with a best-case rpa mea-
sured at 12.7% ± 1.8% and a worse case rpa measured at 7% ± 5.4%. In our second tests we combined two cell lines (CEPH 11881 and CEPH 12093). We created four sample mixtures of 0.2 ml with mixture ratios of 1:9, 1:14, and 1:19 concentration of cell line CEPH 11881. We collected 23, 25 and 13 samples, respectively and again processed the raw data with our software and determined the average relative peak area and variance for each mixture. Despite the discrepancy in peak heights we found variance to be similar to our first set of samples, with standard deviations varying from 0.007 to 0.016. We did not attempt mixtures greater then 1:19 as the peak areas of the minor-contributor alleles were barely above noise for this mixture. Fig. 7 details the results for one locus, CSF1PO. The mixture provides this locus with four peaks that represent two heterozygous alleles from each donor. We are particularly interested in the two peaks from the minor donor as they represent the smallest peaks we would have to identify in mixtures of this ratio. As the amount of the minor donor decreases in each mixture, the relative size of CSF1P0-10 and 13 decreases until for allele 13 in the 19:1 mixture the average relative peak area is 2.6% with a ±3σ variance of 1.8%. Note that the alleles are unbalanced with average peak area decreasing with longer alleles, a known artifact of the STR process. The results so far show that our instrument can provide a viable platform for analyzing mixed samples using the protocols and methods accepted today. Next we investigate if we can improve upon our results. As the ability to quantitate peak area with low variation is determined in part by the signal-to-noise ratio (S/N) of the electropherogram, we
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Fig. 7. Mean relative areas for four peaks from loci CSF1PO for two DNA donors that are mixed at three different ratios—9:1, 14:1 and 19:1. Error bars show ±3δ variance. Alleles 9 and 13 are from heterozygous alleles from minor donor and alleles 11 and 12 are heterozygous alleles from the major donor.
attempt to improve the S/N by varying our loading protocol for the sample. We had previously observed that by varying the loading time and/or lowering the loading voltage we could “preferentially load” our sample such that some region of the electropherogram (e.g., 100–200 bp) has an unusually large signal (see Fig. 8). Reviewing this data we
determined that for the preferentially loaded region the S/N ratio more than doubled over a standard run for a short region. This method may be useful as the mixture ratio will be conserved for all loci, and knowing this ratio to a higher degree of precision will allow for better interpretation of all loci.
Fig. 8. Electropherogram showing internal lane standard loading protocol (top) and preferential loading protocol (bottom). The standard protocol shows balanced signal over the 100–400 base pair region while preferential load shows ladder off scale at 100 bp (>20 V) and about 2 V at 400 bp. Detailed peak-height comparison shows a discrepancy in allele heights that we attribute to PCR artifacts.
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Fig. 9. Mean relative areas for four peaks from loci D5S818 for two DNA donors that are mixed at 9:1 ratio and loaded using a preferential loading protocol. Error bars show ±3δ variance growing as mixture ratio grows. Alleles 9 and 13 are from donor A and alleles 11 and 12 from donor B.
For this test we used a mixture of two cell lines K562 (Promega), and CEPH 11876 (Coriell Institute, Camden, NJ, USA) at a mixture ratio of 19:1 and total volume of 0.2 ml. We varied the loading protocol by reducing the loading voltage to 20 V using the same “dual voltage” method mentioned earlier (2 min duration). Over two 16-lane runs we gathered 25 lanes and determined the relative peak area for each peak for loci D5S818. We then calculated the mean peak area and ±3σ variance for each. The results are presented in Fig. 9. The figure shows the results for 25 lanes of data where the standard deviation of each peak varies by 0.005–0.006 (compared to 0.011–0.012 for similar-sized peaks in earlier data). The improvement is consistent with the measured improvement in S/N from the higher concentrations of short-fragment alleles that are loaded. We would see similar benefits in S/N ratio by operating the instrument in single-lane mode with longer laser dwell in data collection, and/or by actuating a dichroic beam splitter to switch to single-color detection during ratio determination. These modifications can be combined to provide cumulative benefits during a high-sensitivity ratio-determination run for difficult samples.
5. Conclusions In this paper we have reviewed our current status in implementing STR forensics in the microdevice format. In particular we have emphasized the need for single-base resolution, in some ways beyond the requirements of DNA sequencing. Because of the basic properties of the (LPA) sieving matrix, which are common to both “chip” and capillary separations, this resolution requires a microdevice channel length of a minimum of 10–15 cm. This is not difficult to achieve on the “chip” but would currently limit “real-time” assays if the data are to be used
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in court room settings. Integration of on-chip PCR with electrophoresis has already been demonstrated but would have to meet similar quality concerns before it will be admissible for current forensics [15]. We have found that one of the unanticipated major practical benefits is data quality and data stability of the microdevice compared to existing CAE instruments. Reproducibility comes, in part, because of the compact “uni-block” nature of the separation device combined with better heat sinking and thermal/mechanical stability. An intrinsic environmental robustness to uncontrolled temperature and vibration, will likely be the key factors in making microdevice systems the best choice for mobile laboratory applications. The second key advantage is signal to noise properties of the microdevice data, which cascades into very appreciable benefits in de-convolving complex mixtures and in elimination of PCR artifacts. These same benefits are critically important for low-copy-number samples, which are of increasing interest to forensics practitioners. Since forensics scientists spend far more time on expert data analysis than they do in the chemistry lab, it is these latter advantages which will propel microdevices into main stream use in the forensics laboratory.
References [1] P. Gill, A. Urquhart, E. Millican, N. Oldroyd, S. Watson, R. Sparkes, C.P. Kimpton, Int. J. Legal Med. 109 (1996) 14. [2] U. Ricci, I. Sani, S. Guarducci, C. Biondi, S. Pelagatti, V. Lazzerini, A. Brusaferri, M. Lapini, E. Andreucci, L. Giunti, M.L.G. Uzielli, Electrophoresis 21 (2000) 3564. [3] S. El-Difrawy, R. Lam, J. Aborn, M. Novotny, E. Gismondi, P. Matsudaira, B. McKenna, T. O’Neil, P. Streechon, D. Ehrlich, Rev. Sci. Instrum. 76 (7) (2005). [4] D. Schmalzing, L. Koutny, A. Adourian, P. Belgrader, P. Matsudaira, D. Ehrlich, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 10273. [5] N. Goedecke, B. McKenna, S. El-Difrawy, L. Carey, P. Matsudaira, D. Ehrlich, Electrophoresis 25 (2004) 1678. [6] L. Mitnik, L. Carey, R. Burger, S. Desmarais, L. Koutny, O. Wernet, P. Matsudaira, D. Ehrlich, Electrophoresis 23 (2002) 719. [7] S. Hjerten, J. Chromatogr. 347 (1985) 191. [8] Promega Corp., http://www.promega.com/tbs/tmd009/tmd009.html (accessed May 2005). [9] J.H. Aborn, S.A. El-Difrawy, M. Novotny, E.A. Gismondi, R. Lam, P. Matsudaira, B. McKenna, T. O’Neil, P. Streechon, D.J. Ehrlich, Lab Chip 5 (2005) 669 (accessed May 2005) www.rsc.org/ publishing/journals/article.asp?DOI=b501104c. [10] M. Vazquez, G. McKinley, L. Mitnik, S. Desmarais, P. Matsudaira, D. Ehrlich, Anal. Chem. 74 (2002) 1952. [11] J.B. Sgueglia, S. Geiger, J. Davis, Anal. Bioanal. Chem. 376 (2003) 1247. [12] Y. Torres, I. Flores, V. Prieto, M. L´opez-Soto, M. Farf´an, A. Carracedo, P. Sanz, Forensic Sci. Int. 134 (2003) 180. [13] T.M. Clayton, J.P. Whitaker, R. Sparkes, P. Gill, Forensic Sci. Int. 91 (1998) 55. [14] P. Gill, B. Sparkes, J.S. Buckleton, Forensic Sci. Int. 95 (1998) 213. [15] E.T. Lagally, C.A. Emrich, R.A. Mathies, Lab Chip 1 (2001) 102.
Microdevice DNA forensics by the simple tandem repeat method Nils Goedecke, Brian McKenna, Sameh El-Difrawy, Elizabeth Gismondi, Abigail Swenson, Loucinda Carey, Paul Matsudaira, Daniel J. Ehrlich ∗ Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142-1479, USA Available online 22 June 2005
Abstract We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures. © 2005 Elsevier B.V. All rights reserved. Keywords: Forensic DNA analysis; Microdevice electrophoresis; Microfluidics; Short tandem repeat
1. Introduction Few practitioners would dispute that the most important recent development in human forensics has been the introduction of the simple tandem repeat (STR) method for DNA typing. However, widespread application of the method is relatively new. The Forensics Sciences Service in the UK led the way in the development of the technology and the demonstration of its power in the context of computer-searchable databases [1]. Only in the last 5 years has the USA developed a sufficient trained-operator community and a sufficiently large database to begin to achieve similar results. The success of the STR method stems from the combination of (1) the sensitivity, speed, and cost-effectiveness of a DNA assay that utilizes the polymerase chain reaction (PCR) in an efficient multiplex on hyper-variable regions of the human genome combined with (2) the power of a computer-searchable data base. However, since STR forensics is so new, the technology to support it was largely co-opted from general-purpose laboratory instrumentation without optimization for the forensics application. Over the last several years we have been developing technology specifically for STR forensics. We have chosen to use ∗
Corresponding author. Tel.: +1 617 258 7283; fax: +1 617 258 7226. E-mail address: [email protected] (D.J. Ehrlich).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.05.100
the format of microdevices for this purpose. However, interestingly, in the course of our study we have found that it is not the much-touted high-speed or compact size of microdevices that will drive the wide adoption of our technology. Rather, it appears that entirely unanticipated virtues of the microdevice, namely higher data quality and greater assay reproducibility are the most important virtues for forensics. In this paper we review our development to date of microdevice-based STR forensics. The STR method is a fairly classical genotyping assay in which PCR with labeled primers is applied (for the CODIS data base in the USA) as a 13-locus multiplex [2]. The sex marker amelogenin is thrown in. The multiplex is usually read out on a standard capillary array electrophoresis (CAE) instrument using buffer systems and sieving matrices only slightly modified from those used for DNA sequencing. Primers have been designed so as to spread the multiplex across four or five fluors within a total fragment range of less than 500 base pairs. Although the alleles of STR are most usually four-base repeats, there are several microsatellite alleles separated by a single base pair that are common in the general human population. Therefore, although the CAE separation of four-base repeats over 500 base fragments would seem to be undemanding, in fact, the microsatellites require the same single-base resolution of DNA sequencing. What is more, the forensics application is for courtroom evidence,
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with huge societal costs for errors. Hence, the allele-calling accuracy (quality score) of the forensics assay is far beyond that needed for (highly redundant) shot-gun DNA sequencing. The resolution requirement is usually set at 1.0, well above the minimum resolution of <0.3 now standard with DNA base-calling [3]. In our first publication in 1997, we showed that it was possible to use a microdevice and standard linear polyacrylamide (LPA) chemistry to separate the principle 4-base repeats of the CTTV STR multiplex in as little as 30 s [4]. This led us and others to the conjecture of a “real-time” STR microdevice. This direction will likely still be played out. However, we believe the near-term application of microdevice DNA forensics will be motivated on the factors we list in the following sections.
2. Microdevice implementations of STR forensics In a recent publication we described a microdevice electrophoresis system, comprising custom-designed four-color detector, microdevice support and data reduction software [4]. The system is designed with 16-lanes, each with a 20-cm effective length, in order to support the typical crime-scene analysis, which includes standards for positive and negative controls and a sizing ladder and must achieve highly resolved separation of the 9.3 from the 10.0 allele of the TH01 locus. Fig. 1 shows the separation device. Each of the 16 channels is 130 m wide, 60 m deep and 20 cm long. These devices are fabricated from two glass plates (Corning 1737F) each 1.1 mm thick, 43 mm wide and 250 mm long. Due to the isotropic etching the channel cross section is nearly hemispherical. The sample inlet and sample waste arms for the electrokinetic injection are both 2.5 mm long. The offset between both channels is 500 m giving a total injection volume of 3.5 nl within a classic double-T injector [5]. More
Fig. 1. The microfluidic assembly has a bonded two-layer glass body and two interface boards. The board on the anode side contains only one buffer reservoir, which can accommodate up to 2 ml of buffer. The board on the cathode side has one large buffer reservoir equivalent to the anode buffer reservoir. It also contains 32 smaller wells (70 l), which are used as sample inlets and sample wastes. All 16 lanes taper off for the scanning region, wherein they are parallel and have a total width of 3.2 mm.
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details on the fabrication procedure and device design are given in ref. [6]. A modified Hjert´en procedure is used to suppress electroosmotic flow [7]. For most of the experiments samples were prepared using the procedures outlined in the manual from Promega for PCR kit PowerPlex version 1.2 [8]. This STR kit amplifies eight loci and the sex marker amelogenin. The internal lane standard (ILS) is labeled with carboxy-X-rhodamine (CXR). The labels for the loci are fluorescein (FL), 6-carboxy-4 ,5 dichloro-2 ,7 -dimethoxy-fluorescein (JOE) and carboxytetramethylrhodamine (TMR). Each 25-l PCR reaction contained: 16.7 l nuclease free water, 2.5 l GoldSTAR 10× buffer (Promega), 2.5 l PowerPlex primer pair mix at 10× concentration, 0.8 l (4 units assuming 5 u/l) AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) and 2.5 l DNA temPlate 9947a (Promega) with a concentration of 0.8 ng/l. The amplifications were performed according to the suggested 30 cycles from the manufacturers manuals with a PCR system 9700 thermal-cycler (Perkin-Elmer, Wellesley, MA, USA). After the initial heating at 95 ◦ C for 11 min and 96 ◦ C for 1 min followed 10 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 70 ◦ C for 45 s. The next amplification step included 22 cycles of 90 ◦ C for 30 s, 60 ◦ C for 30 s and 70 ◦ C for 45 s. The procedure finishes with two hold periods: (i) 60 ◦ C for 30 min and (ii) 4 ◦ C until the product is taken out of the thermo cycler. After amplification, the PCR products were purified using a GFX spin column (Amersham Bioscience). The PCR products from four reactions were combined to gain 100 l with equalized PCR efficiency and purified with one column. This column exploits the fact that DNA clings to glass fiber using a chaotropic buffer. Excess nucleotides and salts are then washed out by use of an ethanol-based washing buffer. After the washing steps the DNA is re-suspended in 100 l nuclease-free water. The yield of this purification step is usually around 80%. The sample and standard solutions were prepared from 12 l nuclease-free water, plus 0.5 l of purified PCR product (or allelic ladder) and 0.5 l of internal lane standard (ILS). All solutions were denatured at 95 ◦ C for 3 min, then placed on crushed ice immediately for at least 5 min and used within 30 min. To carry out the separations we load fresh separation matrix (4% LPA) using a gastight 100 l glass syringe (Hamilton, Reno, NV, USA). Then the cathode, anode, and sample waste vials are filled with 1× TTE buffer and the sample vials are filled with deionized water. An electrode board is placed on top of the cathode board and the anode board to prevent evaporation and to realize electrical contact. The microdevice is placed on the instrument’s heated stage at 50 ◦ C and equilibrates in about 3 min. The run protocols are automated in our machine. Preelectrophoreses is at 3900 V for 6 min on the cross injection and 175 V for 3 min on the separation channel. Subsequently the buffer and water are replaced and the microdevice is left on the heated stage for 30 min. Shortly after that the vials are
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rinsed with water again. Then the buffer is replaced in the cathode, anode, and sample waste vials. For each lane a 13l sample is transferred into the sample vials. The sample is loaded into the microchannel using 175 V from sample inlet to sample waste vial and after 30 s an additional 390 V from cathode to anode. We call this “dual-voltage” loading mode [9]. Its purpose is to eliminate network effects that result from the fact that all channels are connected at the cathode (see Fig. 1). Next, the sample in the injector is stacked by applying a 6 s voltage impulse along the separation channel of 10 000 V [10]. Finally, the CE is started by applying the separation voltage of 3900 V giving a typical runtime of 40 min for a 600 bp fragment.
3. Data quality and reproducibility Sgueglia has thoroughly characterized the reproducibility of CAE machines for STR forensics [11]. This study was carried out in the very well controlled environment of a state forensics laboratory. Nonetheless, current CAE machines put severe constraints on environmental factors, particularly room temperature, in order to meet the required stability. This constraint is even more severe (or untenable) in a less controlled environment such as a mobile laboratory. Data reproducibility is enormously important to crime-scene forensics because of its implications to the lengthy process of real-world data analysis (see below) and also because of the “overhead” that instability implies in machine calibration. To test the data reproducibility of the microdevice we repeated much of the study of Sgueglia using our system. Fig. 2 shows a typical processed electropherogram, as read out on four photomultipliers for the Promega PowerPlex 16. In order to perform statistical analysis, data of this kind was
accumulated periodically over a period of 4 days. In each run 14 out of 16 lanes were used for the DNA temPlate 9947a and two for the allelic ladder. In this data set, 92 out of 96 lanes performed successfully. Of the four lanes (one with DNA template and three with allelic ladder) that failed, two failed due to bubbles deriving from the separation matrix in the microchannel. The other two lanes contained weak signals that could not be processed for reasons that are not explained. The values for each allele have been analyzed to determine its size in base pairs for mean, standard deviation (σ), and standard deviation bin (±3σ from mean). All of our investigations were performed using microdevices, identical to the assembly shown in Fig. 1. The quality of the signal suggests that the channel coating did not degrade during the investigation. The microdevices have been used for 7 months approximately 80 runs each. Fig. 3 represents a cutout of an electropherogram showing locus TH01. Again the sample was amplified with PowerPlex 16. The data is presented in the foreground and the background illustrates the allelic ladder. A DNA template from the female cell line 9947a has an 8 and 9.3 repeat for this locus. Most of the CODIS core loci have tetranucleotide STRs. However, there are wide spread alleles that differ only by one base pair: like TH01 9.3 and 10. Therefore, the system must be reliable enough to consistently call alleles within 0.5base-pair-accuracy for such a locus, and 1.0-base-pair for all others. To test our instrument’s consistency, we analyzed the measured base-pair values of our lanes to determine a mean and standard deviation of our samples. Fig. 4 shows the allele calling results for locus TH01 taken from our sample data. From this we calculate the window about the mean (±3σ) that can be plotted on our software over a called allele. By observing that the peak is within the window and that the windows from neighboring alleles do not overlap, we can determine to within 3σ certainty that the peak is assigned correctly.
4. Complex mixtures
Fig. 2. Electropherogram of a data lane showing DNA temPlate 9947 multiplexed with PowerPlex 16 kit. The lane data has been processed and allele called. The four segments show from top to bottom: internal lane standard (red), TMR (yellow), JOE (green) and FL (blue) labeled fragments.
Although in database building exercise (collection of samples from offenders) the DNA sample can be presumed to be from a single donor, crime-scene samples are very often mixtures of DNA from the offender and the victim [12]. Since the STR multiplex from a single donor is as many as 27 alleles, mixtures with the complexity of unknown sample ratio, genetic degeneracy, and PCR artifacts, become a tour de force in interpretation. The interpretation begins by eliminating artifacts introduced by PCR and CE, estimating the relative proportion (ratio) of donors and then considering all the possible genotype combinations [13]. The mixture ratio will be preserved throughout all loci and most commonly accepted interpretation methods use peak area and height to assign the peaks to the major and minor donor [14]. In some mixtures the process of donor profile determination is straightforward. The easiest situation occurs where both donors contribute enough to produce strong peaks but
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Fig. 3. TH01 section of an electropherogram of a data lane showing DNA temPlate 9947 multiplexed with PowerPlex 16 kit. The background shows the allelic ladder. The foreground shows the 8 and 9.3 repeats of this template. Also in the figure are the sigma bins (±3σ). The lane data has been processed and allele called Fig. 2: electropherogram for a data lane with 2:1 mixture in foreground with allelic ladder in background.
Fig. 4. Statistics of results from TH01. All repeats show a variance of less than 0.5 base pair.
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Fig. 5. Electropherogram for a data lane with 2:1 mixture in foreground with allelic ladder in background. Three alleles are visible for loci D5S818, two unbalanced alleles for D13S317, D7S820 and two balanced alleles for D16S539. The calculated relative areas (pa) of each peak confirmed the mixture ratio of 2:1. Multiple data runs for this same mixture show relative areas for each peak consistent within 3%.
at an unequal ratio that helps to distinguish between the two donors based on the allele peak height and area. Fig. 5 shows four loci for such an example. Three alleles are visible for loci D5S818-9, 10 and 13. By measuring the area under each peak and normalizing as a percentage of the loci total peak area (across the locus) we determine the relative peak area (rpa) for each (i.e., in this case rpa = 0.66, 0.17 and 0.17). The only possible interpretation for this locus is that the major donor (A) contributed a homozygous 9 while minor donor B contributed two heterozygous alleles at 10 and 13, and the mixture ratio is 2:1. Two alleles are found for loci D13S317, 11 (rpa = 0.28) and 12 (rpa = 0.72). There are two possible interpretations of this: donor A with a homozygous 12 and donor B with a homozygous 11; or donor A with a heterozygous 11, 12 and donor B with a homozygous 12. The two alleles found for D7S820, 10 (rpa = 0.33) and 12 (rpa = 0.64) offer a similar two possibilities. The two alleles are balanced for D16S539, 11 (rpa = 0.51) and 12 (rpa = 0.49), indicating that both donors are heterozygous 11, 12. Balanced alleles in mixtures are interesting in that they can exist for any mixture ratio and can easily be determined for higher ratio mixtures, but cannot be determined for mixtures that approach 1:1. In 1:1 mixtures, two equal-size peaks would provide three possible donor combinations. After evaluating 4 loci we would have four possible profiles for donors A and B. Continuing the process for all 13 CODIS loci would yield about a dozen possible donor profiles. If we identify one donor (i.e., the victim) we can then identify the other donor. Without one identified donor, we can still compare both profiles to a DNA database presenting investigators with a short list of potential suspects.
Other mixtures are more difficult to analyze. In mixtures where the mixture ratio is high, the small peaks of the minor donor will challenge the dynamic range of the instruments detection ability. For example, a mixture with a ratio of 9:1 could have one locus with one allele peak representing both donors and one locus with four peaks. The size difference between the largest peak and the smallest peak would be 20:1. For a 19:1 mixture that ratio would be 40:1. At some point minor-donor peaks will not be distinguishable above noise and/or cannot be assigned on top of a major-donor peak. The limit at which these mixtures can be interpreted will depend upon the confidence in peak area measurement, and the ability of the instrument to determine signal from noise. To test our instrument for use with mixtures we performed three series of tests to determine consistency in determining peak areas and ratios. In each test we ran mixture samples multiple times and called our raw data using our laboratorydeveloped software algorithms to assign amplicon length and peak area. To determine run-to-run variation we normalized the peak area for each allele against the sum of all measured peak areas in the locus. We then determined the average relative peak areas (rpa) and standard deviation (σ) for each allele in our mixtures. In the first series, we mixed two samples at four near-equivalent mixture ratios. In our second, we tested more extreme mixture ratios. In our last series of tests we experimented with run protocol modifications to see if we could enhance system detection for difficult mixtures. The DNA samples were prepared using the procedures outlined earlier with the PowerPlex 1.2 kit (Promega) which amplifies eight loci and the sex marker amelogenin. This
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Fig. 6. Mean relative areas for three peaks from loci CSF1PO for two DNA donors that are mixed at four different ratios. Error bars show ±3δ variance for n = 21, 18, 17 and 14, respectively.
process was done for each cell line separately. Since this process has a 20% loss of DNA, the final step is performed by eluting the DNA with 80 l of DNAse-free water. For the sample preparation, prior to the actual CE, we mixed 240 l of autoclaved, pure water with 20 l PCR product mixture and 20 l internal lane standard 60–400 base pairs (Promega). The DNA fragments of this cocktail were denatured on a hot plate for 3 min and put on ice thereafter. A sample volume of 13 l was transferred into each sample well for the subsequent injection. In our first tests we combined two cell lines 9947a and 9948 (Promega, Madison, WI, USA). We created four sample mixtures of 0.2 ml with mixture ratios of 7:3, 1:1, 3:7 and 1:9. We collected 21, 18, 17 and 14 samples, respectively, processed the raw data with our allele-calling algorithms and calculated the average relative peak area and variance for each mixture type. We found variance to be similar for all loci in our kit, with standard deviations varying from 0.006 to 0.018. Fig. 6 details the results for one locus, CSF1PO, which is located on the yellow channel in the 280–320-bp region. Our mixture provides this locus with one two-donor peak that represents the same allele from each donor, and two singledonor peaks. Looking at the four mixture ratios, CSF1PO-10 remains constant for all mixture ratios (50% of total allele area). As the amount of donor B decreases in each mixture, the relative size of CSF1P0-11 decreases and CSF1PO-12 increases. The insert into Fig. 6 shows a cutout from an electropherogram for each mixture type for this locus. The bar graph shows the average relative peak area (rpa) for each allele and sample mixture with error bars that show ±3σ. Variance is consistent throughout with a best-case rpa mea-
sured at 12.7% ± 1.8% and a worse case rpa measured at 7% ± 5.4%. In our second tests we combined two cell lines (CEPH 11881 and CEPH 12093). We created four sample mixtures of 0.2 ml with mixture ratios of 1:9, 1:14, and 1:19 concentration of cell line CEPH 11881. We collected 23, 25 and 13 samples, respectively and again processed the raw data with our software and determined the average relative peak area and variance for each mixture. Despite the discrepancy in peak heights we found variance to be similar to our first set of samples, with standard deviations varying from 0.007 to 0.016. We did not attempt mixtures greater then 1:19 as the peak areas of the minor-contributor alleles were barely above noise for this mixture. Fig. 7 details the results for one locus, CSF1PO. The mixture provides this locus with four peaks that represent two heterozygous alleles from each donor. We are particularly interested in the two peaks from the minor donor as they represent the smallest peaks we would have to identify in mixtures of this ratio. As the amount of the minor donor decreases in each mixture, the relative size of CSF1P0-10 and 13 decreases until for allele 13 in the 19:1 mixture the average relative peak area is 2.6% with a ±3σ variance of 1.8%. Note that the alleles are unbalanced with average peak area decreasing with longer alleles, a known artifact of the STR process. The results so far show that our instrument can provide a viable platform for analyzing mixed samples using the protocols and methods accepted today. Next we investigate if we can improve upon our results. As the ability to quantitate peak area with low variation is determined in part by the signal-to-noise ratio (S/N) of the electropherogram, we
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Fig. 7. Mean relative areas for four peaks from loci CSF1PO for two DNA donors that are mixed at three different ratios—9:1, 14:1 and 19:1. Error bars show ±3δ variance. Alleles 9 and 13 are from heterozygous alleles from minor donor and alleles 11 and 12 are heterozygous alleles from the major donor.
attempt to improve the S/N by varying our loading protocol for the sample. We had previously observed that by varying the loading time and/or lowering the loading voltage we could “preferentially load” our sample such that some region of the electropherogram (e.g., 100–200 bp) has an unusually large signal (see Fig. 8). Reviewing this data we
determined that for the preferentially loaded region the S/N ratio more than doubled over a standard run for a short region. This method may be useful as the mixture ratio will be conserved for all loci, and knowing this ratio to a higher degree of precision will allow for better interpretation of all loci.
Fig. 8. Electropherogram showing internal lane standard loading protocol (top) and preferential loading protocol (bottom). The standard protocol shows balanced signal over the 100–400 base pair region while preferential load shows ladder off scale at 100 bp (>20 V) and about 2 V at 400 bp. Detailed peak-height comparison shows a discrepancy in allele heights that we attribute to PCR artifacts.
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Fig. 9. Mean relative areas for four peaks from loci D5S818 for two DNA donors that are mixed at 9:1 ratio and loaded using a preferential loading protocol. Error bars show ±3δ variance growing as mixture ratio grows. Alleles 9 and 13 are from donor A and alleles 11 and 12 from donor B.
For this test we used a mixture of two cell lines K562 (Promega), and CEPH 11876 (Coriell Institute, Camden, NJ, USA) at a mixture ratio of 19:1 and total volume of 0.2 ml. We varied the loading protocol by reducing the loading voltage to 20 V using the same “dual voltage” method mentioned earlier (2 min duration). Over two 16-lane runs we gathered 25 lanes and determined the relative peak area for each peak for loci D5S818. We then calculated the mean peak area and ±3σ variance for each. The results are presented in Fig. 9. The figure shows the results for 25 lanes of data where the standard deviation of each peak varies by 0.005–0.006 (compared to 0.011–0.012 for similar-sized peaks in earlier data). The improvement is consistent with the measured improvement in S/N from the higher concentrations of short-fragment alleles that are loaded. We would see similar benefits in S/N ratio by operating the instrument in single-lane mode with longer laser dwell in data collection, and/or by actuating a dichroic beam splitter to switch to single-color detection during ratio determination. These modifications can be combined to provide cumulative benefits during a high-sensitivity ratio-determination run for difficult samples.
5. Conclusions In this paper we have reviewed our current status in implementing STR forensics in the microdevice format. In particular we have emphasized the need for single-base resolution, in some ways beyond the requirements of DNA sequencing. Because of the basic properties of the (LPA) sieving matrix, which are common to both “chip” and capillary separations, this resolution requires a microdevice channel length of a minimum of 10–15 cm. This is not difficult to achieve on the “chip” but would currently limit “real-time” assays if the data are to be used
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in court room settings. Integration of on-chip PCR with electrophoresis has already been demonstrated but would have to meet similar quality concerns before it will be admissible for current forensics [15]. We have found that one of the unanticipated major practical benefits is data quality and data stability of the microdevice compared to existing CAE instruments. Reproducibility comes, in part, because of the compact “uni-block” nature of the separation device combined with better heat sinking and thermal/mechanical stability. An intrinsic environmental robustness to uncontrolled temperature and vibration, will likely be the key factors in making microdevice systems the best choice for mobile laboratory applications. The second key advantage is signal to noise properties of the microdevice data, which cascades into very appreciable benefits in de-convolving complex mixtures and in elimination of PCR artifacts. These same benefits are critically important for low-copy-number samples, which are of increasing interest to forensics practitioners. Since forensics scientists spend far more time on expert data analysis than they do in the chemistry lab, it is these latter advantages which will propel microdevices into main stream use in the forensics laboratory.
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