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Split DNA over replicates or perform one amplification?
Forensic Science International: Genetics Supplement Series 5 (2015) e532–e533
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Split DNA over replicates or perform one amplification? Corina C.G. Benschop* , Seong Yeon Yoo1, Titia Sijen Department of Human Biological Traces (R&D), Netherlands Forensic Institute, The Hague, The Netherlands
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2015 Accepted 22 September 2015 Available online 25 September 2015
We assessed various approaches for DNA profiling using the same total amount of DNA. The choice of profiling approach affects genotyping success and may in addition affect the likelihood ratio (LR). ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Low-template DNA Replicate analyses Ethanol precipitation Likelihood ratios
1. Introduction Ideally, one PCR analysis with sufficient DNA is performed. But with casework, low amounts of DNA may reside in a volume that is too large for one PCR analysis. To obtain informative DNA profiling results, the PCR product can be split over multiple replicates, possibly using an enhanced interrogation technique. Alternatively, one may choose to concentrate the DNA extract and perform one PCR. In this study, we assessed the performance of both approaches. 2. Materials and methods Thirty two-person mixtures were prepared comprising ten different donor ratios (1:2, 1:3, 1:4, 1:5, 1:6, 2:1, 3:1, 4:1, 5:1 and 6:1) for three donor couples that had low, high or moderate allele sharing. The DNA concentration of the minor component in each mixture was 10 pg/ml (thus the major component had up to 60 pg/ ml). Five different procedures were followed to generate NGM DNA profiles: (1) one amplification with sufficient DNA for which 10 ml of each mixture was used (thus 100 pg for the minor and up to 600 pg for a major component); (2) one amplification for which 10 ml of the mixture was subjected to standard ethanol precipitation and re-dissolved in 10 ml to mimic concentration of a low concentration DNA extract (theoretically the amplification has
* Corresponding author at: Laan van Ypenburg 6, The Hague 2497 GB, The Netherlands. Fax: +31 708886555. E-mail address: c.benschop@nfi.minvenj.nl (C.C.G. Benschop). 1 Present address: DNA Analysis Division, Seoul Institute, National Forensic Service, Republic of Korea. http://dx.doi.org/10.1016/j.fsigss.2015.09.210 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
100 pg for the minor and up to 600 pg for a major component); (3) four amplifications each using 2.5 ml of each mixture (thus 25 pg for the minor and up to 150 pg for a major component) with PCR products analyzed using standard 3kV5s capillary electrophoresis (CE) injection settings; (4) the amplifications of procedure 3 now analyzed using enhanced 9kV10s CE injection settings as described in Ref. [1] except no filtration of PCR product; (5) the amplifications of procedure 3 now submitted to five additional amplification rounds [2] (making a total of 34 cycles). The profiles were examined for drop-out percentages (shared alleles included and homozygous alleles counted as two), drop-in proportion (number drop-in alleles/ the total number of alleles) and peak height. In addition, 300 likelihood ratios (LRs) were computed using LRmix Studio v1 as described in Ref. [1], using either of the two true donors in the mixture as the person of interest (POI) under the prosecution hypothesis (Hp). The drop-out rate used the calculated rate for the amplification (or average rate when four amplifications were performed and a minimum rate was set at 0.10), the drop-in rate was set at 0.05 and the theta correction at zero. 3. Results and discussion When examining the drop-out rates for the individual profiles with focus on the minor component (Fig. 1A), we see the lowest drop-out rates for one amplification with sufficient DNA (which represents the ideal situation); ethanol precipitation shows a slightly higher percentage of drop-out. When the DNA extract is split over four amplifications (note that individual profiles are regarded so data for the four profiles are not combined into for instance a consensus profile), standard analysis results in high drop-out rates that decrease substantially when enhanced
C.C.G. Benschop et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e532–e533
e533
Fig. 1. Percentages of drop-out for the minor donor (A) and avg. peak height (B) for NGM profiles generated using five different approaches.
Fig. 2. Log10(LR)s computed using the minor (dots) or major contributor (plusses) as the POI under Hp. For each mixture, the log10(LR) for 1 amp. full input is plotted on the Yaxis with on the X-axes the corresponding log10(LR) s for 1 amp. full input preceded by ethanol precipitation (A), 4 amps. 1/4 input (B), 4 amps. 1/4 input & enhanced CE (C) or 4 amps. 1/4 input & increased cycling (D).
interrogation (enhanced CE/increased cycling) are applied (Fig. 1A). When peak heights are regarded, ethanol precipitation appears to give slightly lower peaks. Profiles from four-fold lower inputs have substantially lower peaks when analysed with standard injection settings, but higher peaks when enhanced interrogation techniques are applied (Fig. 1B). Also the peak height variation increases when these techniques are applied (Fig. 1B). The proportion of drop-in alleles was low when standard injection settings were applied (for one amplification on average (avg) 0.7% and maximum (max) 4.9%, after ethanol precipitation avg 0.2% and max 2.6% and with 1/4 input 0%), but reached up to 7.5% (avg 2.1%) and 9.8% (avg 2.2%) for enhanced CE and increased cycling, respectively. Next, we calculated LRs for which all replicates were included when the DNA was split over four amplifications. When the minor contributor was the POI, false exclusions (LR in favour of the defence hypothesis, Hd) were obtained regularly with the split amplifications and standard CE injection (Fig. 2B, red dots), which represents the least sensitive method (Fig. 1). This derives from the high levels of drop-out for these minor contributors. When the major contributor was used as the POI, LRs were generally larger, as there are less possible genotype combinations under Hd (Fig. 2B, red plusses). There was one occasion in which increased cycling profiles yielded an LR in favour of Hd (Fig. 2D), which was not observed when using a larger drop-in rate (0.10), which appears more appropriate for such profiles.
4. Concluding remarks The choice of profiling approach depends (among others) on the workflow within a forensic laboratory. This approach, however, does affect the loss of information and thus may also affect the LR, specifically when a LT minor contributor is the POI under Hp. In this study, largest differences were observed when comparing the split amplifications and standard CE injection to any of the other approaches tested. Conflict of interest None. Acknowledgement We thank prof. Peter de Knijff for kindly providing DNA extracts that were used in this study. References [1] C.C.G. Benschop, H. Haned, S. Yoo, T. Sijen, Evaluation of samples comprising minute amounts of DNA, Sci. Justice (2015) , doi:http://dx.doi.org/10.1016/j. scijus.2015.05.002. [2] A.A. Westen, L.J.W. Grol, J. Harteveld, A.S. Matai, P. de Knijff, T. Sijen, Assessment of the stochastic threshold, back- and forward stutter filters and low template techniques for NGM, Forensic Sci. Int. Genet. 6 (2012) 708–715.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Split DNA over replicates or perform one amplification? Corina C.G. Benschop* , Seong Yeon Yoo1, Titia Sijen Department of Human Biological Traces (R&D), Netherlands Forensic Institute, The Hague, The Netherlands
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2015 Accepted 22 September 2015 Available online 25 September 2015
We assessed various approaches for DNA profiling using the same total amount of DNA. The choice of profiling approach affects genotyping success and may in addition affect the likelihood ratio (LR). ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Low-template DNA Replicate analyses Ethanol precipitation Likelihood ratios
1. Introduction Ideally, one PCR analysis with sufficient DNA is performed. But with casework, low amounts of DNA may reside in a volume that is too large for one PCR analysis. To obtain informative DNA profiling results, the PCR product can be split over multiple replicates, possibly using an enhanced interrogation technique. Alternatively, one may choose to concentrate the DNA extract and perform one PCR. In this study, we assessed the performance of both approaches. 2. Materials and methods Thirty two-person mixtures were prepared comprising ten different donor ratios (1:2, 1:3, 1:4, 1:5, 1:6, 2:1, 3:1, 4:1, 5:1 and 6:1) for three donor couples that had low, high or moderate allele sharing. The DNA concentration of the minor component in each mixture was 10 pg/ml (thus the major component had up to 60 pg/ ml). Five different procedures were followed to generate NGM DNA profiles: (1) one amplification with sufficient DNA for which 10 ml of each mixture was used (thus 100 pg for the minor and up to 600 pg for a major component); (2) one amplification for which 10 ml of the mixture was subjected to standard ethanol precipitation and re-dissolved in 10 ml to mimic concentration of a low concentration DNA extract (theoretically the amplification has
* Corresponding author at: Laan van Ypenburg 6, The Hague 2497 GB, The Netherlands. Fax: +31 708886555. E-mail address: c.benschop@nfi.minvenj.nl (C.C.G. Benschop). 1 Present address: DNA Analysis Division, Seoul Institute, National Forensic Service, Republic of Korea. http://dx.doi.org/10.1016/j.fsigss.2015.09.210 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
100 pg for the minor and up to 600 pg for a major component); (3) four amplifications each using 2.5 ml of each mixture (thus 25 pg for the minor and up to 150 pg for a major component) with PCR products analyzed using standard 3kV5s capillary electrophoresis (CE) injection settings; (4) the amplifications of procedure 3 now analyzed using enhanced 9kV10s CE injection settings as described in Ref. [1] except no filtration of PCR product; (5) the amplifications of procedure 3 now submitted to five additional amplification rounds [2] (making a total of 34 cycles). The profiles were examined for drop-out percentages (shared alleles included and homozygous alleles counted as two), drop-in proportion (number drop-in alleles/ the total number of alleles) and peak height. In addition, 300 likelihood ratios (LRs) were computed using LRmix Studio v1 as described in Ref. [1], using either of the two true donors in the mixture as the person of interest (POI) under the prosecution hypothesis (Hp). The drop-out rate used the calculated rate for the amplification (or average rate when four amplifications were performed and a minimum rate was set at 0.10), the drop-in rate was set at 0.05 and the theta correction at zero. 3. Results and discussion When examining the drop-out rates for the individual profiles with focus on the minor component (Fig. 1A), we see the lowest drop-out rates for one amplification with sufficient DNA (which represents the ideal situation); ethanol precipitation shows a slightly higher percentage of drop-out. When the DNA extract is split over four amplifications (note that individual profiles are regarded so data for the four profiles are not combined into for instance a consensus profile), standard analysis results in high drop-out rates that decrease substantially when enhanced
C.C.G. Benschop et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e532–e533
e533
Fig. 1. Percentages of drop-out for the minor donor (A) and avg. peak height (B) for NGM profiles generated using five different approaches.
Fig. 2. Log10(LR)s computed using the minor (dots) or major contributor (plusses) as the POI under Hp. For each mixture, the log10(LR) for 1 amp. full input is plotted on the Yaxis with on the X-axes the corresponding log10(LR) s for 1 amp. full input preceded by ethanol precipitation (A), 4 amps. 1/4 input (B), 4 amps. 1/4 input & enhanced CE (C) or 4 amps. 1/4 input & increased cycling (D).
interrogation (enhanced CE/increased cycling) are applied (Fig. 1A). When peak heights are regarded, ethanol precipitation appears to give slightly lower peaks. Profiles from four-fold lower inputs have substantially lower peaks when analysed with standard injection settings, but higher peaks when enhanced interrogation techniques are applied (Fig. 1B). Also the peak height variation increases when these techniques are applied (Fig. 1B). The proportion of drop-in alleles was low when standard injection settings were applied (for one amplification on average (avg) 0.7% and maximum (max) 4.9%, after ethanol precipitation avg 0.2% and max 2.6% and with 1/4 input 0%), but reached up to 7.5% (avg 2.1%) and 9.8% (avg 2.2%) for enhanced CE and increased cycling, respectively. Next, we calculated LRs for which all replicates were included when the DNA was split over four amplifications. When the minor contributor was the POI, false exclusions (LR in favour of the defence hypothesis, Hd) were obtained regularly with the split amplifications and standard CE injection (Fig. 2B, red dots), which represents the least sensitive method (Fig. 1). This derives from the high levels of drop-out for these minor contributors. When the major contributor was used as the POI, LRs were generally larger, as there are less possible genotype combinations under Hd (Fig. 2B, red plusses). There was one occasion in which increased cycling profiles yielded an LR in favour of Hd (Fig. 2D), which was not observed when using a larger drop-in rate (0.10), which appears more appropriate for such profiles.
4. Concluding remarks The choice of profiling approach depends (among others) on the workflow within a forensic laboratory. This approach, however, does affect the loss of information and thus may also affect the LR, specifically when a LT minor contributor is the POI under Hp. In this study, largest differences were observed when comparing the split amplifications and standard CE injection to any of the other approaches tested. Conflict of interest None. Acknowledgement We thank prof. Peter de Knijff for kindly providing DNA extracts that were used in this study. References [1] C.C.G. Benschop, H. Haned, S. Yoo, T. Sijen, Evaluation of samples comprising minute amounts of DNA, Sci. Justice (2015) , doi:http://dx.doi.org/10.1016/j. scijus.2015.05.002. [2] A.A. Westen, L.J.W. Grol, J. Harteveld, A.S. Matai, P. de Knijff, T. Sijen, Assessment of the stochastic threshold, back- and forward stutter filters and low template techniques for NGM, Forensic Sci. Int. Genet. 6 (2012) 708–715.