Information about the web application & Profile Files A,B,C & D
This web site provides data and information related to various aspects of DNA data analysis and interpretation as it applies to routine forensic DNA testing procedures using PCR amplification of STR loci. There are two major components to this web site:
1) Profiles: Electronic data in .fsa format available for download from single source and two-, three- and four-person mixed DNA samples, and
2) Lessons: Descriptive text and problems for review.
This Introduction section contains information regarding the two components of this web site and suggestions on how each may be used. Possible applications include training evaluation of protocols or software and comparison to other laboratory or published data. Information on the Materials and Methods used to generate the data and the grant funding to support this web site is also detailed here.
While the kits, instrumentation and/or software used in the generation of the profiles for this web site may not be the same as those used in a specific DNA testing laboratory, the general scientific principles and foundational information provided here may still be instructional and provide helpful guidance to DNA analysts. The basic concepts presented are generally constant regardless of the amplification kits and extraction, quantitation and electrophoresis procedures used. These basic concepts are useful for providing training and understanding of the principles of PCR amplification, injection, stochastic effects, allele sharing, data interpretation, etc., which have general significance and applications to all practices of DNA testing, analysis and interpretation.
High quality human DNA samples from unrelated individuals were used in the generation of the data provided on this website. While individual profiles provided here may not be representative of all types of profiles encountered in the analysis of routine casework in a crime laboratory, it is likely that some aspects of the DNA profiles generated over a wide range of conditions and kits for this website reflect the types of interpretational issues that result from analyzing DNA profiles of less than optimal quality. Some typical casework profiles may require the understanding of profiles generated under both optimal and suboptimal conditions. For example, a profile from a two-person mixed DNA sample where the DNA from at least one of the contributors had some degree of degradation may have qualities of profiles from both optimal conditions of amplification (e.g., on the left side of the electropherogram where the profile is consistent with the amplification of adequate amounts of reasonably intact high quality DNA) as well as from the amplification of low template DNA where significant stochastic effects might be expected (e.g., on the right side of the electropherogram where only a very small amount of suitable length intact DNA template was available for copying into longer DNA products due to degradation). Similarly, the DNA profile from a two-person mixture containing a high amount of DNA from one contributor and a small amount of DNA from the second contributor will likely require the combination of interpretational analyses of both optimal amounts of high quality DNA as well as small amounts, or low template amounts, of DNA.
Electronic DNA profile data generated according to the Materials and Methods section for four single source samples, two sets of two-person mixed DNA samples, two sets of three-person mixed DNA samples and one set of four-person mixed DNA samples are available as .fsa files for download in the Profiles Section. The samples were amplified using varying amounts of DNA with the PowerPlex® 16 HS System, the AmpFlSTR® Identifiler® PCR Amplification Kit, the AmpFlSTR® MiniFiler® PCR Amplification Kit and/or the AmpFlSTR® Yfiler® PCR Amplification Kit and injected for 2, 5 or 10 seconds on a 3130 Genetic Analyzer.
These profiles may be used by DNA testing laboratories in a number of different ways, even if the amplification kits differ from the kits in use by the laboratory. Some suggested uses are:
* As additional training samples for understanding the effects of varying parameters in the DNA testing assay, such as varying the:
- amount of DNA in an amplification reaction;
- injection time on the Genetic Analyzer;
- ratio of the DNA in mixed DNA samples;
- sources of contributing DNA in mixed DNA samples;
- number of contributors to mixed DNA samples;
- STR amplification kit used, where the kits differ by:
* Amplification of autosomal vs. Y STR sequences;
* Amplification of longer vs. miniSTR sequences.
* For the validation of DNA interpretation procedures used in the laboratory through the use of profiles from known contributor(s) with known genotypes at each locus;
* As training samples for interpretation exercises of single source and mixed DNA profiles generated over a wide range of conditions;
* For the assessment and critique of interpretation procedures or software packages where knowledge of the true contributor(s) to the DNA profile is needed for evaluation.
A series of lessons regarding DNA profile interpretation are included in this section. The lessons start with basic information regarding DNA profile interpretation of single source samples and proceed through discussions regarding topics relevant to the interpretation of two-person mixtures and more complex three- and four-person mixtures. The variations observed in DNA profiles resulting from simultaneous replicate amplifications of DNA, from amplifying varying amounts of DNA and from injecting amplified product for varying lengths of time are highlighted in these lessons.
While high quality single source DNA samples are generally simple to interpret, it is critical to appreciate the limitations and nuances of single source DNA profile interpretation, especially when the profiles obtained are not of the highest quality. The variables associated with single source DNA sample profile generation and interpretation form the foundation for the interpretation of mixed DNA samples as well as demonstrating the myriad uncertainties that exist with more complex DNA mixtures.
The lessons are divided into several sections that may be accessed by clicking on the tabs at the top of the page within each lesson or by clicking on the “Next section” tab at the bottom of each page. Profiles and figures provided in the lessons can be accessed according to the instructions in the “Help - How to use the web application” section.
Each lesson contains questions and/or problems that provide additional exercises for furthering the understanding of the lesson. The answers to the problems may be saved as documentation of completion of the lesson or for reference at a later time or may be printed. Relevant references for the lessons are provided under the Reference tab in each lesson.
It is suggested that the lessons be reviewed in order, especially if they are part of a laboratory training program. However, the lessons may be opened and reviewed in any order at any time.
Materials and Methods
Sample preparation and quantitation
Four human whole blood samples from unrelated individuals, identified as Samples A, B, C and D, were purchased from Bioreclamation (Westbury, NY). Samples A, C and D are from males and Sample B is from a female. High molecular weight DNA was extracted from multiple 1 ml aliquots of the whole blood after lysis in Gill Buffer (10mMTris pH 8.0, 10mM EDTA, 0.1M NaCl, 2% (v/v) SDS and 300 µg/mLProteinase K) using standard procedures for phenol/chloroform extraction and ethanol precipitation (1). After air drying, the DNA was dissolved in 20 µl of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) at 56° C, the multiple aliquots were pooled for each sample, and quantitated in quadruplicate using UV-VIS spectroscopy (Genesys 10S, Thermoscientific) after making 1:99 dilutions. After subtraction of the blank value tested prior to each sample, a mean concentration of DNA was generated for each sample. The concentrations for the four samples ranged from 477 ng/µl to 2411 ng/µl. The average and two standard deviation values of A260/A280 over the four measurements for all four samples was 1.8 (±0.2) indicating that the DNA samples were sufficiently purified and essentially protein-free.
Amplification – Single Source Samples
The following four kits were used for amplification of the single source samples:
1) PowerPlex® 16 HS System from Promega Corporation, Madison, WI;
2) AmpFlSTR® Identifiler® PCR Amplification Kit, Life Technologies, CA;
3) AmpFlSTR® MiniFiler® PCR Amplification Kit, Life Technologies, CA;
4) AmpFlSTR® Yfiler® PCR Amplification Kit, Life Technologies, CA.
A 1 ng/µl DNA stock was prepared for each of the four DNA samples and from this, 0.1 ng/µl and 0.01 ng/µl DNA stocks were prepared by dilution. A bulk DNA + TE + master mix stock for each sample at each targeted DNA amount for each kit was made in sufficient amounts for four replicate amplifications according to the manufacturer’s recommended protocol (2-5). A volume of 25 µl of the bulk master mix + DNA at each DNA concentration for each kit was added to four wells in the amplification plate, resulting in the quadruplicate amplifications of the following total amounts of DNA for each sample:
1) 4 ng;
2) 2 ng;
3) 1 ng;
4) 0.5 ng;
5) 0.25 ng;
6) 0.125 ng; and
7) 0.0625 ng.
Since Sample B is from a female, no Yfiler amplification was performed for that sample. The samples amplified with the PowerPlex 16 HS kit were amplified once rather than in quadruplicate.
Please note: Sample B has a silent 10 allele at the D7 locus in Identifiler.
Amplification – Mixed DNA Samples
Two-person (Samples A+B and Samples C+D), three-person (Samples A+B+C and A+C+D) and four-person (Samples A+B+C+D) mixture stocks were made at each of the various ratios according to Table 1. Each of the mixed DNA samples was amplified at each of the targeted amounts of DNA as shown in Table 1. The two-person mixtures were amplified with each of the amplification kits listed above for the single source samples according to the manufacturer’s recommended protocol, except the A+B mixture was not amplified with the Yfiler kit since Sample B is from a female. The ACD and BAC three-person mixtures were amplified with the Identifiler kit, the ACD mixtures (three males) were amplified with the Yfiler kit and three ratios of the BAC mixtures were amplified with the PowerPlex 16 HS kit (see Table 1). The four-person mixtures were amplified with the Identifiler kit only.
The samples were amplified for 28 cycles (Identifiler kit), 30 cycles (Yfiler and MiniFiler kits), or 32 cycles (PowerPlex 16 HS kit) using an Applied Biosystems GeneAmp® PCR 9700 System using 9600 emulation mode according to the manufacturer’s recommended protocol, with an additional 30 minute extension at the end of the thermal cycling. Positive and negative amplification control samples were amplified concurrently.
Table 1. Mixed DNA Sample Combinations, Mixture Ratios and the Targeted Amount of DNA Amplified
|Mixtures||Combined Samples||Mixture Ratio||Target Amount of DNA for Amplification (ng)|
|1:19||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|1:9||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|1:4||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|1:2||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|1:1||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|2:1||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|4:1||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|9:1||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|19:1||4, 2, 1, 0.5, 0.25, 0.125, 0.0625|
|Three-Person Mixtures||B+A+C||1.5:3:1*||7, 4, 2, 1, 0.5, 0.25|
|3:3:1*||7, 4, 2, 1, 0.5, 0.2|
|1.5:6:1*||9, 4, 2, 1, 0.5, 0.3|
|1.5:3:2||7, 3, 1.5, 0.8, 0.4, 0.2|
|6:3:1||7, 3.5, 1.7, 0.8, 0.4, 0.2|
|1.6:12:1||10, 5, 2.5, 1.2, 0.6, 0.3|
|1.6:3:4||6, 3, 1.5, 0.7, 0.4, 0.2|
|A+C+D||3:1:1||7, 3, 1.7, 0.8, 0.4, 0.2|
|6:1:1||8, 4, 2, 1, 0.5, 0.25|
|3:2:1||6, 3, 1.5, 0.7, 0.4, 0.2|
|3:1:2||6, 3, 1.5, 0.7, 0.4, 0.2|
|12:1:1||9, 5, 2.5, 1.2, 0.6, 0.3|
|3:4:1||5, 2.5, 1.3, 0.7, 0.3, 0.2|
|3:1:4||5, 2.5, 1.3, 0.7, 0.3, 0.2|
|Four-Person Mixtures||B+A+C+D||1.6:3:1:1||7, 3.3, 1.7, 0.8, 0.4, 0.2|
|3:3:1:1||7, 3.3, 1.6, 0.8, 0.4, 0.2|
|1.6:6:1:1||8, 4, 2, 1, 0.5, 0.25|
|1.6:3:2:1||6, 3, 1.5, 0.8, 0.4, 0.2|
|1.6:3:1:2||6, 3, 1.5, 0.8, 0.4, 0.2|
|3:6:1:1||7.5, 4, 2, 1, 0.5,0.2|
|1.6:6:2:1||7, 3.5, 2, 1, 0.5, 0.2|
|1.6:3:2:2||6, 3, 1.5, 0.7, 0.4, 0.2|
*The three mixture ratios amplified with the PowerPlex 16 HS kit.
AmpFlSTR® amplified product samples were prepared for electrophoresis by combining 8.3 µl of highly deionized formamide (Applied Biosystems) and 0.7 µl of GeneScan™ 600 LIZ™ Size Standard (Applied Biosystems) with 1 µl of amplified product or allelic ladder in the appropriate wells of the run plate. PowerPlex® 16 HS amplicons were prepared using the same protocol, except the volumes were 9.5 µl of Hi-Di formamide and 0.5 µl of the Internal Lane Standard provided with the amplification kit. After heating to 95° C for 3 min on a heating block and snap-cooling for 3 min in a metal block in a -20° C freezer, the prepared samples were injected sequentially onto an Applied Biosystems’ 3130 Genetic Analyzer at 3 kV for 2, 5 and 10 seconds (6). Data were collected using Data Collection Software version 3.0. The raw data files stored as .fsa files are available for download under “Profile Files” on this website.
Profiles used in the “Lessons” section of this website were generated by analyzing the data using GeneMapper ID-X software version 1.1 with a peak amplitude threshold of 30 RFU and the default stutter filter settings (7). Any edits made during analysis will be observed on the profiles. All positive and negative amplification controls gave expected results for all amplifications with all kits.
Some data tables in the lessons contain averaged RFU values. The average RFU values were calculated by summing the peak heights in RFUs for all true allele peaks present for the color stated or for the entire profile, as appropriate, and dividing by the number of allele peaks observed. Remember that a single peak representing a homozygous genotype counts as two alleles for calculating averages.
Sample Naming Convention Used
Each sample is named according to the following convention with each segment of information separated by an underscore:
Thus, a sample name of “ID_2_SCD_NG0.25_R4,1_A1_V1.3” indicates that the Identifiler kit was used to amplify a two-person mixture comprised of Samples C and D, with a target amount of amplified DNA of 0.25 ng, mixed in a ratio of 4 parts Sample C (0.2 ng) + 1 part Sample D (0.05 ng) in the first amplification (and possibly only) tube for that composition, with 1 µl of product used in the sample preparation for injection and with an injection time of 10 seconds.
1. Gill, P., Jeffreys, A. J., and Werret, D. J., Forensic Applications of DNA "Fingerprints," Nature, Vol. 318, Dec. 1985, pp. 577-579.
2. PowerPlex® 16 HS Technical Manual
3. AmpFlSTR® Identifiler® PCR Amplification Kit User Guide
4. AmpFlSTR® MiniFiler™ PCR Amplification Kit User Guide
5. AmpFlSTR® Yfiler® PCR Amplification Kit User Guide
6. Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide
7. GeneMapper® ID-X Software Version 1.0/1.1 Installation Guide
CCD charge-coupled device camera
CE capillary electrophoresis instrument
GMID-X GeneMapper ID-X (LifeTechnologies)
Identifiler AmpFlSTR® Identifiler® PCR Amplification Kit
MiniFiler AmpFlSTR® MiniFiler™ PCR Amplification Kit
PCR Polymerase Chain Reaction
PowerPlex 16 HS PowerPlex® 16 HS System (Promega)
RFU relative fluorescent units
STR short tandem repeat
Yfiler AmpFlSTR® Yfiler® PCR Amplification Kit
Glossary and Definitions
A comprehensive list of terms and their definitions can be found in Section 2 of the Quality Assurance Standards for Forensic DNA Testing Laboratories.
Additional information regarding DNA analysis and interpretation of DNA, including mixed DNA samples, can be found in numerous journal articles published in various publications around the world with some emphasis on forensic sciences, for example, Journal of Forensic Sciences, Forensic Science International, Forensic Science International:Genetics, Legal Medicine, Journal of Forensic and Legal Medicine, Science and Justice, and the Croatian Journal of Medicine, among others.
Textbooks that may be helpful include:
Buckleton, J.S., Triggs, C.M., and Walsh, S.J. (2005) Forensic DNA Evidence
Interpretation. CRC Press, 552 pages
Butler, J.M. (2010) Fundamentals of Forensic DNA Typing. Elsevier Academic
Press, 520 pages
Butler, J.M. (2012) Advanced Topics in Forensic DNA Typing: Methodology.
Elsevier Academic Press, 704 pages
Evett, I.W. and Weir, B.S. (1998) Interpreting DNA Evidence: Statistical Genetics
for Forensic Scientists. Sinauer Associates, 278 pages
Fung, W.K. and Hu, Y.Q. (2008) Statistical DNA Forensics: Theory, Methods and
Computation. John Wiley & Sons Ltd., 262 pages
The National Institute of Standards and Technology STRbase website can be found at http://www.cstl.nist.gov/strbase/. This site, which is frequently updated, contains an extensive reference list and various helpful sections with information on a wide variety of topics related to forensic DNA analysis, and includes links to many publications and presentations. The “Mixture Interpretation” section may be of special interest to readers of this website.
Links to other helpful websites are provided below:
American Academy of Forensic Sciences
American Society of Human Genetics
Federal Bureau of Investigation/Combined DNA Index System (FBI/CODIS)
Federal Bureau of Investigation Quality Assurance Standards (FBI QAS) Documents
International Society of Forensic Genetics
Scientific Working Group on DNA Analysis Methods (SWGDAM)
Funding and Disclaimer
This project was supported by Award No. 2008-DN-BX-K158 awarded by the National Institute of Justice, Office of Justice Programs, U. S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice. The information on the website and the opinions of the authors are current and accurate at the time of writing, however, are subject to change as new information and relevant scientific studies become available. Future updates to the web site are not planned at this time, but may occur as needed as time and funding are available.
The above-mentioned grant was awarded to Boston University School of Medicine, Principle Investigator Robin W. Cotton, Ph.D., Associate Professor Department of Anatomy and Neurobiology, Division of Graduate Medical Sciences, Boston University School of Medicine.
This website was built by Boston University Information Services & Technology. It took considerable cleverness and patience on their part to make the features of the website come together as we had envisioned.
The following individuals have made substantial contributions to the development of the web site, generation of DNA profiles and/or participated in the writing of the lessons:
Robin W. Cotton, Ph.D., Boston University School of Medicine
Catherine Grgicak, Ph.D., Boston University School of Medicine
Margaret Terrill, M.S.F.S., Consultant, East Amherst, NY
Charlotte J. Word, Ph.D., Consultant, Gaithersburg, MD
Sarah Cortes, Inman Techonologies IT
Special thanks to the following individuals for lively discussions, helpful comments and otherwise intellectual interactions regarding the various issues of DNA testing and interpretation:
Boston University Students from the Biomedical Forensic Sciences Program, Department of Anatomy and Neurobiology, Division of Graduate Medical Sciences, Boston University School of Medicine.
Todd Bille, Ph.D.
John M. Butler, Ph.D.
Michael D. Coble, Ph.D.
This website is dedicated to DNA analysts throughout the world who generate DNA profiles daily from the plethora of evidence provided to them, while maintaining neutrality and providing scientifically accurate interpretations and conclusions. We benefit from their efforts and dedication, as the law enforcement and legal communities use the data they obtain for the purpose of seeking justice for victims, the wrongfully accused, the guilty and the general public.