Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure

Abstract

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory's (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months. The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control.

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth

“…The protocol originally described in Marshall et al. used a Qiagen MinElute ® Kit for extract purification. As NGS use is further expanded in use at the laboratory, EP#4 continues to be modified and may involve an organic purification.…”

“…Next Generation Sequencing was performed using an in‐house developed capture protocol . Samples were run on the Illumina MiSeq using the v2 kit (Illumina, San Diego, CA).…”

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

“…Mitochondrial DNA (mtDNA) testing in forensic casework is generally performed with amplicons targeting the non-coding control region (CR) and capillary electrophoresis-based Sanger sequencing. However, over the past five or so years, next generation sequencing (NGS) has been shown to be an equally robust technology for the development of forensic quality mitochondrial DNA sequence data [1][2][3][4][5][6][7][8][9][10][11]. NGS not only offers the capability to reduce workflows, but it also allows for the generation of larger, more informative genetic data sets at higher throughput and overall lower cost per nucleotide than capillary electrophoresis-based methods [12,13].…”

Peer Review #1 of "Mitochondrial DNA in human identification: a review (v0.1)"

“…Manuscript to be reviewed consistent estimation of mtDNA haplotypes. Many other studies on the use of NGS technologies for forensic genetics and mtDNA analysis have been published (Chaitanya et al, 2015;Churchill, Stoljarova, King, & Budowle, 2018;Hollard et al, 2017;Just, Irwin, & Parson, 2015;Just, Scheible, Fast, Sturk-Andreaggi, Higginbotham, et al, 2014;Just, Scheible, Fast, Sturk-Andreaggi, Röck, et al, 2014;Lopopolo, Børsting, Pereira, & Morling, 2016;Ma et al, 2018;Marshall et al, 2017;Ovchinnikov, Malek, Kjelland, & Drees, 2016;Park et al, 2017;Templeton et al, 2013;Young, King, Budowle, & Armogida, 2017). However, further validation studies and specialized software functionality tailored to forensic practice should be produced in order to facilitate the incorporation of NGS processing into standard casework applications (Amorim & Pinto, 2018;Peck et al, 2016).…”