The Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) has validated hybridization capture methods paired with massively parallel sequencing to generate genetic data to support identification of fallen U.S. servicemembers.  Hybridization capture is an effective enrichment approach for targets within the degraded DNA of these decades-old remains.  Two bait sets are currently used for capture enrichment in casework: one panel targets the whole mtDNA genome (mtG), and the other targets ~95,000 nuclear SNPs (95K).  Due to the low coverage (1X) analysis of the 95K data, libraries are first captured with the mtG baits to sort samples as well as ensure libraries are single-source and indicate 95K capture may be successful.  Based on the mtG analyses, select libraries are then recaptured with 95K baits.  

Generating both 95K and mtG data from one capture event, using a combination of 95K baits with mtG baits spiked in, would unlock major gains in turnaround time and cost-benefit analysis.  Other bait panels are designed to target both nuclear SNPs and the mtG.  Therefore, the objective became evaluating this potential efficiency gain of a combined 95K/mtG panel.  

This study assessed the feasibility of obtaining sufficient SNP and mtG data through simultaneous capture enrichment with a combined 95K/mtG panel and determine the optimal bait ratio.  Libraries of various sample types and quality which had undergone separate mtG and 95K captures were recaptured using the combined bait set.  Prior optimization tested 95K:mtG bait ratios of 500:1, 400:1, and 300:1 (by volume).  The 500:1 ratio was selected for subsequent testing of 29 libraries representative of casework.  In these sets, the 500:1 bait ratio generated slightly more balanced data, with significantly improved mtG coverage for some samples without sacrificing SNP recovery.  The 500:1 spike-in capture data yielded the same kinship inferences as 95K-only capture data, with strong statistical support.  Additional testing of the spike-in approach with high-quality reference samples was also successful.

This spike-in approach proved to be a valid strategy for processing all but the lowest-quality samples.  With the flexibility of interchanging bait sets in hybridization capture, this approach could incorporate mtG targets into any higher-density SNP panel to optimize processing and data generation.

Disclaimer

The opinions or assertions presented hereafter are the private views of the speaker(s) and should not be construed as official or as reflecting the views of the Department of Defense, its branches, the Defense Health Agency, or the AFMES.

Authors

• Katelyn Kjelland (AFMES-AFDIL, United States of America)
• Jacqueline (Tyler) Thomas (AFMES-AFDIL, United States of America)
• Kimberly Andreaggi (AFMES-AFDIL, United States of America)