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OSAC RESEARCH NEEDS ASSESSMENT FORM 

Title of research need: Characterization, development and validation of methods in single cell

isolation and analysis

Describe

the need:

Single cell research has grown over the last decade as a result of technological innovations

and is now commonplace in the greater biological research community. Consequently, the

applications and benefits of the single cell analysis technologies have crossed over into the

forensic DNA analysis field. The technology and methods to make this possible have

become more accessible, easier to use, more diverse, and less expensive than they were

decades ago. The primary benefit of single cell analysis is the ability to avoid or simplify the

interpretation of DNA mixtures – a single cell is by definition contributed by a single donor.  

This analysis is versatile, being appropriate for use with standard forensic DNA typing

methods, such as DNA fragment analysis and capillary electrophoresis, and DNA

sequencing technologies. The potential benefits extend beyond that of mixture analysis

including the ability to identify the tissue source of the DNA profile that was generated and

through characterizing the behavior and response of single cells in the forensic workflow,

can reveal more about the dynamics of stochastic effects such as inter and intra locus peak

or read balance, allele dropout, and detection thresholds. This type of analysis is in its 

infancy, and, with proper research support, these benefits can come to fruition.

Keyword(s): DNA mixture, single‐cell analysis, mixture deconvolution, low‐template DNA

Submitting subcommittee(s): Human Biology Date Approved: 10/05/2021

(If SAC review identifies additional subcommittees, add them to the box above.)

Background Information:

1. Does this research need address a gap(s) in a current or planned standard? (ex.: Field identification system 

for on scene opioid detection and confirmation)

No 

2. Are you aware of any ongoing research that may address this research need that has not yet been published 

(e.g., research presented in conference proceedings, studies that you or a colleague have participated in but 

have yet to be published)?

Yes, these details, however, are not available at this point. 

3. Key bibliographic references relating to this research need: 

1) Gill, P. et al. Genotyping and interpretation of STR-DNA: Low-template, mixtures and database matches - 

Twenty years of research and development. Forensic Sci. Int. Genet. (2015). 

doi:10.1016/j.fsigen.2015.03.014 
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2) Benschop, C. C. G., Haned, H., de Blaeij, T. J. P., Meulenbroek, A. J. & Sijen, T. Assessment of mock cases 

involving complex low template DNA mixtures: A descriptive study. Forensic Sci. Int. Genet. 6, 697–707 

(2012). 9. 

3) Watkins, D. R. L., Myers, D., Xavier, H. E. & Marciano, M. A. Revisiting single cell analysis in forensic science. 

Sci. Reports 2021 111 11, 1–12 (2021). 

4) Findlay, I., Taylor, A., Quirke, P., Frazier, R. & Urquhart, A. DNA fingerprinting from single cells. Nature 389, 

555–556 (1997). 

5) Verdon, T. J., Mitchell, R. J., Chen, W., Xiao, K. & Van Oorschot, R. A. H. FACS separation of non-compromised 

forensically relevant biological mixtures. Forensic Sci. Int. Genet. 14, 194–200 (2015). 

6) Vandewoestyne, M. & Deforce, D. Laser capture microdissection in forensic research: a review. Int. J. Legal 

Med. 124, 513–521 (2010). 

7) Fontana, F. et al. Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision 

of a digital approach. Forensic Sci. Int. Genet. 29, 225–241 (2017). 

8) Williamson, V. R., Laris, T. M., Romano, R. & Marciano, M. A. Enhanced DNA Mixture Deconvolution of Sexual 

Offense Samples Using the DEPArray System. Forensic Sci. Int. Genet. 34, 265–276 (2018). 

9) Anslinger, K., Bayer, B. & von Máriássy, D. Application of DEPArray technology for the isolation of white 

blood cells from cell mixtures in chimerism analysis. Rechtsmedizin 1–4 (2017). doi:10.1007/s00194-017-

0221-7 

10)Anslinger, K., Graw, M. & Bayer, B. Deconvolution of blood-blood mixtures using DEPArray TM separated 

single cell STR profiling. Rechtsmedizin 29, 30–40 (2019). 

11)Harrel, M., Gangitano, D. & Hughes-Stamm, S. The effects of extra PCR cycles when amplifying skeletal 

samples with the GlobalFiler® PCR Amplification Kit. Int. J. Legal Med. 133, 745–750 (2019). 

12) Pfeifer, C. M., Klein-Unseld, R., Klintschar, M. & Wiegand, P. Comparison of different interpretation strategies 

for low template DNA mixtures. Forensic Sci. Int. Genet. 6, 716–722 (2012). 

13) Grisedale, K. S. & van Daal, A. Comparison of STR profiling from low template DNA extracts with and without 

the consensus profiling method. Investig. Genet. 3, 1 (2012). 

14)Weiler, N. E. C., Matai, A. S. & Sijen, T. Extended PCR conditions to reduce drop-out frequencies in low 

template STR typing including unequal mixtures. doi:10.1016/j.fsigen.2011.03.002 

15)Duijs, F., Van De Merwe, L., Sijen, T. & Benschop, C. C. G. Low-template methods yield limited extra 

information for PowerPlex® Fusion 6C profiling. (2018). doi:10.1016/j.legalmed.2018.06.001 

16) Gill, P., Whitaker, J., Flaxman, C., Brown, N. & Buckleton, J. An investigation of the rigor of interpretation rules 

for STRs derived from less than 100 pg of DNA. Forensic Science International 112, (2000). 

17)Kloosterman, A. D. & Kersbergen, P. Efficacy and limits of genotyping low copy number DNA samples by 

multiplex PCR of STR loci. 

18) Butler, J. M. & Hill, C. R. Scientific Issues with Analysis of Low Amounts of DNA. [Internet] (2010). Available 

at: https://www.promega.com/resources/profiles-in-dna/2010/scientific-issues-with-analysis-of-lowamounts-of-dna/ . (Accessed: 10th April 2020) 

19) Budowle, B., Eisenberg, A. J. & van Daal, A. Validity of Low Copy Number Typing and Applications to Forensic 

Science. Croat. Med. J. 50, 207–217 (2009). 

20) Bessekri, M. W., Aggoune, A., Lazreg, S., Bucht, R. & Fuller, V. Comparative study on the effects of reduced PCR 

reaction volumes and increased cycle number, on the sensitivity and the stochastic threshold of the 

AmpFlSTR Identifiler 1 Plus kit. Forensic Sci. Int. Genet. Suppl. Ser. 4, PE306-E307 (2013). 

21) Butler, J. M. Advanced Topics in Forensic DNA Typing. Advanced Topics in Forensic DNA Typing: 

Methodology (2012). doi:10.1016/B978-0-12-374513-2.00017-8 
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4. Review the annual operational/research needs published by the National Institute of Justice (NIJ) at 

https://nij.ojp.gov/topics/articles/forensic-science-research-and-development-technology-working-groupoperational#latest? Is your research need identified by NIJ?

Indirectly yes, “The ability to differentiate, physically separate, and selectively analyze DNA and/or cells from

multiple donors or multiple tissue/cell types contributing to mixtures, with minimal or no sample loss”

5. In what ways would the research results improve current laboratory capabilities? 

Research into the use of single cells will permit less reliance on mixture analysis. This may lead to less complex, 

high confidence interpretation and conclusions. In addition, this will permit the correlation between the DNA 

profile generated and the tissue source used to generate the profile. 

6. In what ways would the research results improve understanding of the scientific basis for the 

subcommittee(s)? 

Gaining knowledge on the dynamics of low template analyses using PCR-based fragment analysis and DNA 

sequencing including, stochastic effects and detection thresholds will enable the committee members to make 

more informed recommendations for aspects of forensic analyses that involve these topics. 

7. In what ways would the research results improve services to the criminal justice system? 

Research into the use of single cells will permit less reliance on mixture analysis. This will lead to less complex, 

higher confidence interpretations conclusions. This type of analysis is in its infancy, and, with proper research 

support, these benefits can come to fruition. 

8. Status assessment (I, II, III, or IV): II

Major gap in 

current 

knowledge 

Minor gap in 

current 

knowledge 

 

 No or limited

current research is 

being conducted I III 

 Existing current 

research is being 

conducted II IV 

This research need has been identified by one or more subcommittees of OSAC and is being provided as an

informational resource to the community.