Collection and direct amplification methods using the GlobalFiler™ kit for DNA recovered from common pipe bomb substrates

When analyzing DNA from exploded pipe bombs, quantities are often in trace amounts, making DNA typing extremely difficult. Amplifying minute amounts of DNA can cause stochastic effects resulting in partial or uninterpretable profiles. Therefore, the initial DNA collection from “touch” evidence must be optimized to maximize the amount of DNA available for analysis. This proof-of-concept study evaluated two different swab types with two direct amplification strategies to identify the most effective method for recovering DNA from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wire spiked with epithelial cells were swabbed with cotton or microFLOQ® Direct Swabs and amplified directly or via a pre-treatment prior to STR amplification. Not only was the microFLOQ® Direct Swab protocol the quickest method with the least risk of contamination, but in combination with direct amplification, the microFLOQ® Direct Swabs also generated the most complete STR profiles

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

Development and evaluation of a rapid PCR method for the PowerPlex (R) S5 system for forensic DNA profiling

Forensic DNA profiling is a multi-step process taking approximately 10 h to complete. A reduction in the amount of time required for the amplification step would allow for faster human identification and increase laboratory throughput. The goal of this work was to optimize and evaluate a rapid PCR method for the PowerPlex (R) S5 system for forensic DNA profiling. By pairing fast chemistries with a fast thermal cycler, we were able to reduce the amplification time by 70% (1 h). Sensitivity and heterozygous peak height ratios were comparable between the fast and standard protocols. However, there was a notable decrease (5%) in peak height ratio at the D18S51 locus with the fast cycling method. An increase in average mean stutter for combined loci of 2.6% was observed in profiles amplified using the fast protocol compared to the standard system. Our results suggest that with further optimization and validation the fast protocol can be used to replace the standard amplification conditions. (C) 2014 Elsevier Ireland Ltd. All rights reserved

Development and validation of a rapid PCR method for the PowerPlex® 16 HS system for forensic DNA identification

Currently, the amplification step of most forensic DNA profiling systems takes 3–4\ua0h to complete. A decrease in the amplification time would allow for increased laboratory throughput, which may help reduce backlog when is due to limited cycling capacity. By using the SpeedSTAR™ HS Polymerase (Takara Bio, Otsu, Japan) in combination with the Veriti® (Applied Biosystems, Foster City, CA, USA) rapid thermal cycler, the amplification time for the PowerPlex® 16 HS (Promega, Madison, WI, USA) kit was reduced by 66\ua0% (1\ua0h). The sensitivity of this fast method was comparable to the standard system (0.13\ua0ng). Although this rapid protocol showed an increase in average stutter ratios (2.8\ua0%) and a decrease in average peak height ratios (7\ua0%) across all loci when compared with standard conditions, it was able to consistently generate reliable DNA profiles. The results of this study indicate that the rapid protocol could be implemented in forensic laboratories with an optimal range of 0.25–2\ua0ng of input DNA using appropriate analytical interpretation guidelines

The effects of extra PCR cycles when amplifying skeletal samples with the GlobalFiler® PCR Amplification Kit

When samples with low amounts of DNA are amplified using short tandem repeats (STRs), stochastic effects such as allele and locus dropout or drop-in, allele imbalance, and increased stutter often occur making data interpretation more difficult. The most common approach to improving STR results from low template samples is to increase the number of PCR cycles. Although more alleles may be recovered, stochastic effects may be exaggerated resulting in more complicated STR profiles. This work reports the effect of additional PCR cycles (29 vs. 30, 31, and 32) on STR success from environmentally challenged bone and tooth samples using the GlobalFiler DNA Amplification Kit (Thermo Fisher Scientific). In addition, we compared the efficiency of two DNA extraction kits for skeletal samples: QIAamp DNA Investigator (QIAGEN) and PrepFiler BTA™ Forensic DNA Extraction (Thermo Fisher Scientific) kits. Results showed that more DNA was recovered from samples using the PrepFiler BTA™ kit; but regardless of the extraction method, the number of alleles detected and the peak heights both increased with an increase in PCR cycle number. Although more alleles were reported in almost all samples, the most notable improvement was observed in samples with the DNA template < 120 pg. A general increase in the number of PCR artifacts was detected in STR profiles generated using 30–32 cycles. Overall, this study provides supporting evidence that STR profile completeness and quality may be improved when low template skeletal samples are amplified with extra PCR cycles (up to 32 cycles) using the GlobalFiler DNA Amplification Kit

An alternate method for extracting DNA from environmentally challenged teeth for improved DNA analysis

A grinding-free method to extract DNA from teeth via a direct minimal-invasive retrograde approach to the pulp cavity and dentine was compared to a standard grinding/pulverisation method. This alternate method uses endodontic dental files to access the root canals and pulp cavity for tissue and dentine harvest via the apical end of the roots and avoids mechanical damage to the crown and root morphology. In contrast, other methods require pulverisation of the whole root or tooth, transection or destruction of the occlusal surface to gain access to the DNA in the root canals and pulp chamber. This study compared two methods for preparing dentine powder from the roots of environmentally challenged teeth for forensic DNA analysis.We found that although the filing method was more laborious, and produced less dentine powder, the amount of amplifiable DNA per milligram of powder was substantially higher with the filing method compared to grinding the entire root. In addition, the number of short tandem repeat (STR) alleles detected and the peak height ratios of the STR profiles were notably higher. Although several other methods of extracting DNA-rich tissue from the pulp chamber of teeth have previously been reported, the method presented in this study is minimally invasive, thereby allowing the preservation of tooth and crown morphology. (C) 2015 Elsevier Ireland Ltd. All rights reserved

In-field collection and preservation of decomposing human tissues to facilitate rapid purification and STR typing

Short tandem repeats (STR) are currently the gold standard in human identification for forensic casework purposes, and successful STR typing is dependent on sufficient quantity and quality DNA. In the aftermath of a mass disaster and some forensic cases, human remains are recovered for identification in various stages of decomposition, and ideally these remains are transported to a refrigerated facility in order to halt the decomposition process and preserve the integrity of DNA within the tissue. However, in situations where refrigeration is not available (e.g., after a mass disaster or in rural forensic casework), remains continue to be exposed to environmental insults after collection, causing further DNA damage and degradation. Therefore, successful STR typing is dependent on the time of collection and preservation of the DNA sample. This study aims to test two simple in-field collection and preservation methods for decomposing human tissues that are subsequently stored at room temperature for up to six months either in a tissue preservative solution (modified TENT buffer) or on an FTA Elute Card. In addition, these collection and preservation methods were tested for their ability to facilitate more direct and faster processing of DNA from preserved tissues or DNA leached into the surrounding TENT preservative solution for STR typing. Pre-PCR methods tested in this study include a quick lysis of FTA Elute Cards, silica-based purification (QIAquick), enzyme-based extractions (PDQeX), and simple dilution of liquid preservative. The traditional DNA analysis pipeline, which includes DNA extraction and quantification, will be compared to an alternate direct PCR method, thereby allowing the elimination of these two time-consuming and costly steps. The results indicate that modified TENT preservative and FTA Elute Cards both preserved DNA from relatively fresh tissue for up to six months at room temperature. However, mostly partial profiles were produced from decomposed tissues (day 6 – day 14 in this study) when stored for up to six months compared to when tissues were processed immediately following collection. Overall, the modified TENT preservative produced higher DNA concentrations and more successful STR results than FTA Elute Cards. In addition, a rapid DNA extraction platform (PDQeX) generated the most successful STR typing results from the decomposed tissues stored in TENT for up to six months at room temperature. The direct PCR method used in this study generated comparable STR results to the traditional DNA analysis approach, warranting further investigation of direct PCR methods for forensic casework type samples

Evaluation of a powder-free DNA extraction method for skeletal Remains

Bones are often recovered in forensic investigations, including missing persons and mass disasters. While traditional DNA extraction methods rely on grinding bone into powder prior to DNA purification, the TBone Ex buffer (DNA Chip Research Inc.) digests bone chips without powdering. In this study, six bones were extracted using the TBone Ex kit in conjunction with the PrepFiler BTA™ DNA extraction kit (Thermo Fisher Scientific) both manually and via an automated platform. Comparable amounts of DNA were recovered from a 50 mg bone chip using the TBone Ex kit and 50 mg of powdered bone with the PrepFiler BTA™ kit. However, automated DNA purification decreased DNA yield (p < 0.05). Nevertheless, short tandem repeat (STR) success was comparable across all methods tested. This study demonstrates that digestion of whole bone fragments is an efficient alternative to powdering bones for DNA extraction without compromising downstream STR profile quality