Introduction
Forensic DNA analysis often comprises challenging samples caused by partial DNA degradation, presence of PCR inhibitors, low DNA quantities, and/or mixtures of multiple contributors. These variables can require iterative rounds of time-consuming testing to generate sufficient data for comparisons and identifications. Dental remains (i.e.
, teeth samples) are commonly subjected to these variables and can be the sole DNA source when viable nuclear DNA are not available due to soft tissue decomposition [
1,
2]. Improved upstream sample collection and cellular recovery, in combination with a highly informative quantification assay and deep, targeted massively parallel sequencing (MPS) analysis collectively maximize the genetic information potential from dental remains. Application of an optimized workflow that combines extraction, quantification, and MPS protocols was evaluated to assess performance on forensic dental samples.
The Dental Forensic Kit (DFK
MR) was specifically designed to optimize dental tissue retrieval in routine forensic nuclear DNA and/or mitochondrial DNA genotyping and forensic histopathology applications [
1‐
3]. By conducting dental tissue preparation (dental pulp, root cement) in physiological conditions, the DFK method maximizes DNA retrieval and thus genetic information potential from challenging samples. Attributes of the DFK method that make it a feasible alternative to other methodologies for hard tissues [
4,
5] include faster processing time to obtain two distinct tissue samples (dental pulp, root cement), decreased contamination (grinding not required), and tooth preservation for further re-extraction or additional testing, if needed. Additionally, the method’s non-destructive aspect has cultural and/or religious implications in that what may be the sole remains for burial can be handled in the most sensitive manner. After tissue retrieval with DFK, the QuickExtract™ FFPE DNA Extraction Kit (Lucigen®) was used to effectively extract nuclear DNA [
6,
7].
Quantity and quality of total human and total male DNA can be assessed using the InnoQuant® HY Kit (InnoGenomics Technologies) [
8,
9]. Based on real-time qPCR quantification of high copy number Alu and SVA retrotransposable elements (> 1000 copies per genome), this method assesses autosomal targets of two lengths (short and long) to enhance sensitivity and reproducibility of DNA quantitation and degradation detection in forensic samples [
8,
9]. Quality information includes detection of polymerase chain reaction (PCR) inhibitors and determination of a degradation index (DI), which correlates strongly to amplification and genotyping success [
8,
9]. The InnoQuant HY method was integrated as the second portion of the three-part workflow.
The last portion of the comprehensive workflow is massively parallel sequencing (MPS), also known as next-generation sequencing (NGS), which was assessed for ability to relieve limitations of amplicon length-based STR allele calling after capillary electrophoresis (CE). These limitations include the inability to analyze different types of genetic polymorphisms in a single reaction and workflow, low-resolution genotyping, loss of data in degraded DNA samples, and incomplete resolution of complex DNA mixtures [
10].
Massively parallel sequencing (MPS) technology has replaced Sanger sequencing in basic science and in medical and diagnostic fields [
11‐
21]. Advantages of MPS in forensic applications include high-throughput processing, simultaneous detection of STRs on autosomes and sex chromosomes, analysis of identity informative SNPs (iiSNPs) and SNPs related to biogeographical ancestry (aiSNPs) and physical traits (phenotypes; piSNPs), and ability to distinguish between alleles that are identical by CE-based sizing. MPS advantages broaden the possibilities of analyses and may provide critical information in challenging samples for criminal cases, missing person cases, and mass disaster tragedies [
22,
23].
This report explores the application of MPS using sequencing by synthesis (SBS) [
24] on the MiSeq FGx® sequencer (Verogen, Inc.) to analyze dental remains. The MiSeq FGx sequencer, ForenSeq® DNA Signature Prep Kit, and ForenSeq Universal Analysis Software were developed and forensically validated [
25,
26] for human identification and generation of investigative leads [
27‐
29]. The ForenSeq DNA Signature Prep Kit contains two PCR primer set options: (1) DNA Primer Mix A (DPMA) targets Amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iSNPs and (2) DNA Primer Mix B (DPMB) targets all loci in DPMA, as well as 56 aiSNPs and 22 piSNPs for biogeographical ancestry and phenotype (hair and eye color) estimation [
30‐
34]. Previous evaluations of MiSeq FGx System’s performance and genotyping concordance have been reported [
35‐
41].
Majority of the amplicons in the ForenSeq kit are shorter in base pair (bp) length compared to CE assays, providing a significant advantage when analyzing degraded DNA samples. This is possible because MPS is not limited by the spectral overlap of fluorescent dyes as is fragment sizing-based CE detection. Of the 231 markers targeted with ForenSeq DPMB, 191 total STR and SNP markers have
maximum amplicon sizes less than 200 bp in length [
27]. As a comparison, GlobalFiler®, PowerPlex® Fusion 6c, and MiniFiler® have approximately nine, eight, and six
maximum STR amplicon sizes that are less than 200 bp, respectively [
42‐
44]. A practical example of the utility of ForenSeq’s smaller amplicon sizes, relative to CE-based methods, on degraded (ancient) remains was reported by Xavier and Parson [
38].
Due to hard structures such as enamel and bone that provide environmental protection, dental tissues are a valuable source of genomic and/or mitochondrial DNA, in cases when other biological fluid or tissue may not be available [
45]. Few studies have reported on the application of MPS technology to nuclear DNA analysis of dental tissues [
38,
45,
46]. Here, we report on evaluation of nuclear DNA utility as obtained from human dental tissues assayed with DFK, InnoQuant HY, and the MiSeq FGx Forensic Genomics System for identification purposes and to generate investigative leads.
Conclusions and future directions
The data described in this study demonstrate the robust performance of a comprehensive sample-to-sequencing workflow capable of achieving informative nuclear DNA results from challenging, low-level, and/or degraded samples. The DFK method provided efficient recovery of the dental pulp and root cement tissues from teeth samples, resulting in two samples per tooth suitable for nuclear DNA extraction (and other types of forensic examination) while preserving the tooth. InnoQuant HY quantification of QuickExtract FFPE Kit extracted teeth DNA and organic extracted blood samples provided a highly sensitive and accurate assessment of nuclear DNA quantity and quality; actionable results were obtained for teeth samples down to 0.01 ng/μL short target and 0.0001 ng/μL long target concentrations with corresponding DI of 76, and for blood samples down to 0.17 ng/μL short target and 0.0004 ng/μL long target concentrations with corresponding DI of 460. The accurate quant-based characterization of DNA quality and quantity enabled data-informed amplification targeting 231 STR and SNP markers with the ForenSeq DNA Signature Prep Kit.
Compared with CE amplification kits, ForenSeq provides a larger multiplex with smaller amplicon sizes (191 STR and SNP makers with maximum amplicon size ≤ 200 bp) to more effectively type degraded DNA samples. Sequencing of 13 teeth samples on the MiSeq FGx instrument generated 24 to 82 total STR allele calls per sample (58 STR loci targeted plus Amelogenin) and simultaneously typed 38 to 94 iSNP loci per sample (94 iSNP loci targeted). The tooth sample that yielded the least amount of data (22% of autosomal STR and 40% of iSNP loci typed) resulted in an autosomal STR RMP of 1 in ~ 860 million and an iSNP RMP of 1 in ~ 350,000. Sequencing of the most severely degraded blood sample (DI of 460 with 2 pg total long target DNA input) generated data for 21% of autosomal STR loci and 74% of iSNP loci, with a corresponding autosomal STR RMP of 1 in ~ 202 million and iSNP RMP of 1 in ~ 2.9 trillion. These results demonstrate highly discriminatory genotype frequency statistics even with diminishing data recovery in challenging samples.
Collectively, application of the DFK method for tissue recovery and DNA extraction, followed by InnoQuant HY quantification and MiSeq FGx System genotyping of dental samples generates a significant amount of usable data for successful sample identification, with the added benefit of potential investigative leads through biogeographical ancestry and phenotype estimation. The demonstrated success of this three-part comprehensive workflow on teeth samples and degraded DNA suggests that the application of these combined methods may also benefit nuclear DNA processing results for aged skeletal remains (i.e., bone samples). The DFK, InnoQuant HY, and MiSeq FGx workflows were employed in a preliminary test of one 45-year PMI jawbone sample resulting in the detection of 48 STR alleles and 59 iSNP loci. Further evaluation to verify the success of this method with bone samples is warranted. Additionally, comparison of different extraction methods (e.g. organic vs QuickExtract FFPE) utilized on the DFK recovered tissues, as well as the addition of a purification/concentration step (e.g., filter concentrators) may further optimize the workflow and improve results for the most challenging scenarios.
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