Taking the Bait: Utilization of Probe-Capture Enrichment in Human Remains Identification

Purpose: Human remains are frequently encountered in forensic laboratories, coming from crime scenes, mass graves, historical samples, mass disasters, and military conflicts. Short tandem repeat (STR) markers evaluated via capillary electrophoresis (CE) are the gold standard for human remains identification (HRID) in forensic investigations due to their high variability and robust database of comparative samples. However, CE excludes valuable sequence-level information both within and around STRs and is often unsuccessful when used on challenged and degraded samples. The problem forensic laboratories face is choosing between depleting sample volumes to repeat individualizing STR analysis or perform costly, time-consuming, and less discriminatory mitochondrial DNA analysis. New DNA sequencing methodologies combined with novel enrichment techniques may provide a more effective platform that overcomes the most common challenges associated with HRID. Our goal for this portion of the project was to design a RNA-baiting assay to capture forensically relevant STRs.

Methods: A custom myBaits panel targeting forensically relevant regions of interest was designed with Arbor Biosciences. DNA extract from whole blood samples was used. Samples were barcoded with Illumina-compatible barcode adapters, pooled, and then probe-captured, resulting in single stranded DNA covering our targeted regions. Fill-in PCR was performed on the captured single stranded DNA using Illumina primers to create DS DNA, which was then sequenced on the MiSeq. Our data was analyzed using our custom script, STRspy, which uses minimap 2 or BWA-mem to align the data to the designated reference database. STRspy then evaluates the reads aligning to each designated loci (forensically relevant STRs in this case), classifying the allele calls as well as the SNPs in the flanking region to create sequence-based genetic profiles. These calls also include the length-based allele designation for each sequence-based allele, therefore allowing the results to be compatible with current CODIS data.

Results: We found that our RNA-baiting assay was successful in capturing our targeted regions of interest. The STR regions targeted for enrichment showed significantly enhanced coverage compared to the coverage across the entire genome.

Conclusions: Our results indicate that probe-capture is a viable enrichment technique for DNA samples.