Enhanced sample preparation and data interpretation strategies using massively parallel sequencing for human identification in missing persons' and DVI casework

When the remains of victims from mass disasters, military conflicts, or missing persons’ cases are recovered, identification is the most important objective. The recovered unidentified remains may be intact, fragmented, comingled, highly decomposed, or skeletonized. The DNA within these tissues is often degraded, damaged, and/or contains inhibitory agents depending on the environment in which the remains were discovered. This project explores the use of traditional genotyping and newer DNA sequencing technologies for the identification of challenging human remains commonly recovered from mass disasters and missing persons’ cases. The results of this study will provide the forensic community with additional information on the comparative performance of massively parallel sequencing (MPS) chemistries and platforms with compromised samples, particularly highly inhibited samples. This study was comprised of four projects. First, two CE-based STR megaplex kits (GlobalFiler® PCR Amplification and Investigator® 24plex QS kits) were evaluated for their tolerance to PCR inhibitors (humic acid, melanin, hematin, collagen, calcium) and overall sensitivity of detection for high and low quantity (1 ng and 0.1 ng) DNA samples. The results suggested that the GlobalFiler® kit was more sensitive down to 7.8 pg of DNA while the Investigator® kit was more tolerant to all PCR inhibitors at both DNA concentrations. The GlobalFiler® kit produced more alleles, higher peak heights, and higher peak height ratios when determining sensitivity. Conversely, the Investigator® kit produced more alleles and balanced profiles for every inhibitor and inhibitor concentration than the GlobalFiler® kit. Second, two MPS chemistries and platforms (Ion AmpliSeq™ kit on the Ion PGM and the ForenSeq™ kit on the MiSeq FGx™) were evaluated side-by-side using the same inhibited DNA samples. The AmpliSeq™ and ForenSeq™ kits were found to be tolerant and susceptible to different common PCR inhibitors. The AmpliSeq™ chemistry demonstrated tolerance to collagen and calcium; however, it was highly susceptible to humic acid and hematin. Conversely, the ForenSeq™ kit showed extreme tolerance to hematin and calcium inhibitors but was greatly affected by melanin. The third study focused on determining the effectiveness of common DNA extraction methods to remove inhibitors from forensically relevant samples and their downstream compatibility with two MPS chemistries. Three substrates (blood, hair, and bone) were spiked with high concentrations of four inhibitors (humic acid, melanin, hematin, and calcium) and extracted using five DNA extraction methods (DNA IQ™, QIAamp® DNA Investigator, PrepFiler®, and two total demineralization protocols (bone only)). The results showed that all extraction methods were able to efficiently remove all PCR inhibitors with no sign of inhibition and provide sufficiently pure DNA extracts for sequencing. Although the amount of DNA recovered using the different extraction methods differed, the sequencing data indicated that none of the extraction methods negatively influenced the downstream sequencing performance on either MPS system. The fourth and final study reports the comparative performance of two MPS systems when sequencing challenging human skeletal remains. Thermally degraded, embalmed, cremated, burned, and decomposed bones and teeth (N = 24) were extracted using a total demineralization protocol and processed with two MPS chemistries and platforms in addition to traditional CE-based STR typing. The results demonstrated that CE-based STR profiling was still a valuable approach by providing at least a partial DNA profile for every sample, whereas MPS did fail to produce a profile in some instances. However, these MPS chemistries are still not fully optimized to tolerate such difficult samples and further optimization is warranted. Conversely, MPS has the capability to analyze more markers and multiple marker systems (STRs, SNPs, etc.) simultaneously. Therefore, even though some CE samples produced more complete profiles, the additional markers within MPS multiplexes may result in higher powers of discrimination for identification, and thereby provide results to assist with solving missing persons’, forensic, and DVI cases

Utility of the Ion S5™ and MiSeq FGx™ sequencing platforms to characterize challenging human remains

Often in missing persons’ and mass disaster cases the samples remaining for analysis are hard tissues such as bones, teeth, nails, and hair. These remains may have been exposed to harsh environmental conditions, which pose challenges for downstream genotyping. Short tandem repeat analysis (STR) via capillary electrophoresis (CE) is still the gold standard for DNA typing; however, a newer technology known as massively parallel sequencing (MPS) could improve upon our current techniques by typing different and more markers in a single analysis, and consequently improving the power of discrimination. In this study, bone and tooth samples exposed to a variety of DNA insults (cremation, embalming, decomposition, thermal degradation, and fire) were assessed and sequenced using the Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System, and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™ system, as well as the GlobalFiler™ PCR Amplification Kit on the 3500™ Genetic Analyzer. The results demonstrated that using traditional CE-based genotyping performed as expected, producing a partial or full DNA profile for all samples, and that both sequencing chemistries and platforms were able to recover sufficient STR and SNP information from a majority of the same challenging samples. Run metrics including profile completeness and mean read depth produced good results with each system, considering the degree of damage of some samples. Most sample insults (except decomposed) produced similar numbers of alleles for both MPS systems. Comparable markers produced full concordance between the two platforms

Comparative sensitivity and inhibitor tolerance of GlobalFiler® PCR Amplification and Investigator® 24plex QS kits for challenging samples

In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit

Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors

Human remains can be severely affected by the environment, and the DNA may be damaged, degraded, and/or inhibited. In this study, a DNA sample (at 1 ng DNA target input in triplicate) was spiked with five concentrations of five inhibitors (humic acid, melanin, hematin, collagen, and calcium) and sequenced with both the HID-Ion AmpliSeq (TM) Library Kit and ID panel on the Ion PGM (TM) System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx (TM). The objective of this study was to compare the baseline tolerance of the two sequencing chemistries and platforms to common inhibitors encountered in human remains recovered from missing person cases. The two chemistries generally were comparable but not always susceptible to the same inhibitors or at the same capacity. The HID-Ion AmpliSeq (TM) Library Kit and ID panel and the ForenSeq DNA Signature Prep Kit both were susceptible to humic acid, melanin, and collagen; however, the ForenSeq kit showed greater inhibition to melanin and collagen than the AmpliSeq (TM) kit. In contrast, the ForenSeq kit was resistant to the effects of hematin and calcium, whereas the AmpliSeq (TM) kit was highly inhibited by hematin. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) showed the same trend among inhibitors when using the ForenSeq kit. Generally, locus read depth, heterozygote allele balance, and the numbers of alleles typed were inversely correlated with increasing inhibitor concentration. The larger STR loci were affected more so by the presence of inhibitors compared to smaller STR amplicons and SNP loci. Additionally, it does not appear that sequence noise is affected by the inhibitors. The noise percentage, however, does increase as the inhibitor concentration increases, due to the decrease in locus read depth and not likely because of chemistry effects

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount