Rootless hair shafts are often collected at crime scenes. However, the DNA in rootless hair fails in conventional forensic DNA assays.
Astrea Forensics has developed a breakthrough approach for DNA recovery and analysis from rootless hairs. This approach enables forensic genetic genealogy, yielding actionable leads.
- Astrea Forensics has adapted paleogenetic methods (technology for recovering and sequencing DNA from remains that are often tens of thousands of years old) for DNA based forensics. These methods are ideal for extracting and sequencing the ultrashort DNA fragments in rootless hair and other degraded samples
- Rootless hair contains billions of nuclear and mitochondrial DNA fragments that are mostly less than 75 base pairs in length – too short for amplifying CODIS markers, including miniSTRs
- With our approach, these billions of ultrashort DNA fragments can be recovered with nextgeneration sequencing (NGS) to reconstruct the full mitochondrial genome and generate nuclear genome data suitable for forensic genetic genealogy.
- From a single hair, Astrea Forensics can often produce a genotype file ready for upload to genealogy databases like GEDmatch or FamilyTreeDNA.
In just a few short years, forensic genetic genealogy has proven to be a revolutionary approach to human identification. From the Golden State Killer to the victims of the Bear Brook murders, law enforcement agencies can access an entirely new path towards solving violent crime cases and unidentified remains. This powerful new genotyping approach requires tens to hundreds of nanograms of high-quality DNA. Until now.
Many forensic samples lack enough recoverable, high-quality DNA to use in traditional genotyping assays, like genotype arrays, even when STR amplification is successful. This limitation is especially pronounced for samples with highly fragmented DNA, like rootless hair. A single rootless hair yields only picograms of ultrashort DNA. These samples often fail traditional forensic testing because the DNA fragments are mostly less than 75 base pairs long. These DNA fragments are too short to amplify using the widely used CODIS markers (Figure 1), resulting in profiles that are hopeless for solving crimes.
Figure 1: Amplicon size ranges for CODIS STR and miniSTR markers¹. In red, the average range of DNA fragment lengths in hair.
Fear not! A sample containing only 100 picograms of short DNA still contains over 1 billion unique DNA fragments! How then to recover these data for use in genetic genealogy?
First, our paleogenetic-inspired methods are designed to recover and sequence as many unique fragments as possible, even when the DNA fragments are damaged, e.g. nicked, single stranded, jagged ends. Second, we sequence all recovered DNA fragments to generate a comprehensive view of the DNA from the sample using whole genome sequencing. Finally, we use a specially developed algorithm to convert this raw sequence data into a genotype file suitable for forensic genetic genealogy. This workflow is shown in Figure 2.
Because we can observe or impute sites across the entire human genome, we can choose from hundreds of millions of informative markers to include in the genotype file.
Figure 2: Astrea Forensics general laboratory workflowᶧ
Advantages of hair for genetic genealogy
Rootless hair has unique advantages in forensic contexts. Hair is insoluble, preserving DNA over centuries (Figure 3). Because its exterior can be decontaminated with mild bleach, the extracted DNA is less susceptible to microbial contamination, unlike bone and tooth samples. Importantly, each single hair is a discrete biological unit that necessarily derives from a single individual. Thus, sample mixtures – a common obstacle in DNA-based forensics assays – in not an issue for DNA derived from single hairs.
Many investigations have collected hair samples with little luck in human identification. Our methods use rootless hairs 5 cm in length but have had success with smaller strands.
Figure 3: DNA fragment lengths observed in three rootless hairs samples from two individuals collected at different time points. Samples 1a and 1b derive from the same individual, collected 23 years and 0 years ago (i.e. present day), respectively. Sample 2 is from a second individual, approximately 140 years old. Hairs were processed following laboratory methods described above.
Phase I deliverable
With as little as 100 mg of bone powder or a few centimeters of hair, our paleogenetic-trained scientists prepare samples for whole genome sequencing²,³. After shallow NGS sequencing for quality-control analysis, we measure the amount of usable human DNA present in each sample. If enough DNA fragments are observed, we report the chromosomal sex estimation and mitochondrial haplotype of each sample. Because each cell contains many mitochondrial genome copies, we can often reconstruct the entire mitochondrial genome for a sample, even when very few autosomal DNA fragments are observed.
The quality-control sequencing also allows us to estimate the amount of sequencing necessary to achieve a high quality genotype/SNP profile. This Phase I quality-control analysis allows us to determine the feasibility and cost of generating the SNP profile.
Phase II deliverable
Based on quality-control analysis, we deeply sequence approximately 300-400 million read pairs. The Astrea Forensics pipeline works by analyzing DNA sequence data to generate a genotype file in a format suitable for upload to GEDmatch, FamilyTreeDNA, or other databases used for forensic genetic genealogy.
Once the genotype file is delivered, we are committed to supporting the work of law enforcement and forensic genetic genealogists until the case is solved. To see examples where Astrea Forensics’ technology was used to solve cold cases, visit https://www.astreaforensics.com/in-the-news.
Astrea Forensics, LLC is a biotechnology company focused on applying the most advanced DNA technology to forensics casework. Using the company’s unparalleled expertise in ancient DNA research and next-generation-sequencing, Astrea uses proprietary methods for improving the recovery of DNA data from the most degraded of samples, including rootless hair. Astrea offers services to law enforcement and related agencies for recovering, sequencing, and genotyping DNA from samples that fail conventional testing. Astrea has both laboratory and computational innovations that directly address this problem. The product provides a genomewide solution for investigative genetic genealogy.
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- Butler, John M., Yin Shen, and Bruce R. McCord. "The development of reduced size STR amplicons as tools for analysis of degraded DNA." Journal of forensic sciences 48.5 (2003): 1054-1064.
- Dabney, Jesse, et al. "Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments." Proceedings of the National Academy of Sciences 110.39 (2013): 15758-1576
- Troll, Christopher J., et al. "A ligation-based single-stranded library preparation method to analyze cellfree DNA and synthetic oligos." BMC genomics 20.1 (2019): 1-14
† figure created with BioRender.com