FINAL by: OSAC Program Office; Version 2, January 13, 2020 1 

OSAC RESEARCH NEEDS ASSESSMENT FORM 

Title of research need: Characterization, optimization, and comparison of DNA sequencing methods

Describe

the need:

DNA sequencing is a considered a major technological advancement in the field of forensic

DNA profiling. As opposed to Whole Genome Sequencing (WGS), the forensics field tends

to focus on select regions of the genome to avoid potential ethical pitfalls in generating too

much data, in addition to dealing with the significantly lower DNA quality and quantity that

is often observed in casework. To date, there are two commercial companies that have

produced benchtop sequencers that fit the need of most forensic laboratories in terms of

cost and throughput. With the advent of Massive Parallel sequencing (MPS), genetic data

being generated using commercial sequencing kits and commercial software for Short

Tandem Repeats (STRs), in addition to intelligence‐based methodologies such as

ancestry/phenotype prediction and genetic genealogy that require SNP data input, there is

a need to 1. compare the performance of data generated from STR CE analyses and SNP

arrays versus commercial sequencing platforms using MPS (SNP and/or STR). 2. Compare

the use of MPS pipelines, both commercial and non‐commercial, for their efficacy in

alignment and genotype calling between platforms/methods. 3. In terms of the physical

chemistry and newly developing methods/approaches to sequencing, (i.e., hybridization,

amplicon sequencing etc.), a comparison of these methodologies for low input DNA and

mixture samples in particular. Integrating MPS sequencing into casework will be an

extreme benefit to the field of DNA profiling and intelligence, however a full assessment of

what’s available/possible including performances is necessary.

Keyword(s): SNPs, STRs, Massive Parallel Sequencing, pipelines, software

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)

Yes, in the use of MPS for casework. 

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, but not enough in detail. 
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3. Key bibliographic references relating to this research need: 

1) Alonso, A., Barrio, P.A., Müller, P., Köcher, S., Berger, B., Martin, P., Bodner, M., Willuweit, S., Parson, W., 

Roewer, L. and Budowle, B. Current state-of-art of STR sequencing in forensic genetics. Electrophoresis. 

2018;39: 2655-2668. 

2) Peter de Knijff. From next generation sequencing to now generation sequencing in forensics. Forensic Sci Int

Genet. 2019;38:175-180 

3) Ballard, D., Winkler-Galicki, J. & Wesoły, J. Massive parallel sequencing in forensics: advantages, issues, 

technicalities, and prospects. Int J Legal Med. 2020; 134:1291–1303. 

4) Tytgat, O.; Gansemans, Y.; Weymaere, J.; Rubben, K.; Deforce, D.; Van Nieuwerburgh, F. Nanopore Sequencing 

of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling. Genes. 2020;11:381. 

5) Wang, D., Tao, R., Li, Z. et al. STRsearch: a new pipeline for targeted profiling of short tandem repeats in 

massively parallel sequencing data. Hereditas. 2020; 157: 8. 

6) Jonathan L. King, August E. Woerner, Sammed N. Mandape, Kapema Bupe Kapema, Rodrigo Soares MouraNeto, Rosane Silva, Bruce Budowle. STRait Razor Online: An enhanced user interface to facilitate 

interpretation of MPS data. Forensic Sci Int Genet. 2021; 52:102463. 

7) Nam Nhut Phan, Amrita Chattopadhyay, Tsui-Ting Lee, Hsiang-I Yin, Tzu-Pin Lu, Liang-Chuan Lai, Hsiao-Lin 

Hwa, Mong-Hsun Tsai, Eric Y Chuang. High-performance deep learning pipeline predicts individuals in 

mixtures of DNA using sequencing data. Briefings in Bioinformatics. 2021. 

8) Erin M. Gorden, Kimberly Sturk-Andreaggi, Charla Marshall. Capture enrichment and massively parallel 

sequencing for human identification. Forensic Sci Int Genet. 2021;102496. 

9) Michael Hofreiter, Jiri Sneberger, Martin Pospisek, Daniel Vanek. Progress in forensic bone DNA 

analysis:Lessons learned from ancient DNA. Forensic Sci Int Genet. 2021;54. 

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?

Yes, “Better ways to enrich or target genomic areas of forensic DNA interest as opposed to a traditional PCRbased approach”. 

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

Must compare and contrast commercial and non-commercial methods/software/lab techniques used in 

sequencing particular regions of the genome for STR/SNP genotype calling. Need to generate comparison 

information on this data, min read count, algorithms used etc. so that future standards may be implemented. At 

present consolidating all types of chemistries, pipelines, sample preparation, sample performance and 

concordance etc. used for sequencing protocols in forensic casework (in particular low input and mixtures) will 

be extremely valuable, especially with regards STR data for identification and SNP data for intelligence purposes. 

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

subcommittee(s)? 

Better exploration of the caveats of bad samples/data and minimum requirements for accurate genotyping calls 

due to their importance in downstream identifying and intelligence information. 

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

It will provide an overview of the current climate in terms of commercial entities, types of methods, kit 

performance, including new approaches (i.e. hybridization v PCR) pre sequencing, and more importantly a 
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significant exploration of the algorithms utilized for variant calling, from STRs to SNPs. This step is vital for 

correct input of data into statistical and modelling tools/programs downstream. Sequencing alignment 

methodologies and thresholds for genotyping calling need exploration and comparison within commercial and 

non-commercial software/tools. 

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.