Establishment of a co-analysis system for personal identification and body fluid identification: a preliminary report

Analysis of genetic markers can provide clues for case investigation. Short tandem repeat (STR) detection and analysis are widely used for both personal identification and parentage testing. However, DNA analysis currently cannot provide sufficient information for body fluid identification. Tissue or cell sources of samples can be identified by detecting body fluid-specific mRNA markers, which have been studied thoroughly. Integrating STR profiling and mRNA expression patterns can provide more information than conventional methods for investigations and the reconstruction of crime scenes; this can be achieved by DNA/RNA co-extraction technology, which is economical, efficient, and suitable for low-template samples. Here, we propose a co-analysis system based on the PowerPlex 16 kit. This system can simultaneously amplify 25 markers, including 15 STRs, one non-STR amelogenin, and nine mRNA markers (three blood-specific, two saliva-specific, two semen-specific, and two housekeeping gene markers). The specificity and sensitivity of the co-analysis system were determined and aged and degraded samples were used to validate the stability of the co-analysis system. Finally, different DNA/RNA ratios and various carriers were evaluated. The results showed that the DNA/RNA co-analysis system correctly identified different types of body fluid stains. The STR profiles obtained using the co-analysis system were identical to those obtained using the PP16 kit, which demonstrates that the mRNA primers used did not affect STR profiling. Complete STR and mRNA profiles could be obtained from 1/8 portions of buccal swabs, 1/16 portions of swabs of blood and semen samples, 0.1 cm² of blood samples, 0.25 cm² of semen samples, and 1.0 cm² saliva samples. Additionally, our findings indicate that complete STR and mRNA profiles can be obtained with this system from blood and semen samples when the DNA/RNA ratio is 1:1/32. This study suggests that the co-analysis system could be used for simultaneous personal identification and body fluid identification.

     

Evaluating the forensic application of 19 target microRNAs as biomarkers in body fluid and tissue identification

RNA‐based body fluid and tissue identification has evolved as a promising and reliable new technique to classify type and source of biological evidence in crime cases. In particular, mRNA‐based approaches are currently on the rise to replace conventional protein‐based methods and are increasingly implemented into forensic casework. However, degradation of these nucleic acid molecules can cause issues on laboratory scale and need to be considered for a credible investigation. For this reason, the analysis of miRNAs using qPCR has been proposed to be a sensitive and specific approach to identify the origin of a biological trace taking advantage of their small size and resistance to degradation. Despite the straightforward workflow of this method, suitable endogenous controls are inevitable when performing real-time PCR to ensure accurate normalization of gene expression data in order to allow a meaningful interpretation. In this regard, we have validated reference genes for a set of forensically relevant body fluids and tissues (blood, saliva, semen, vaginal secretions, menstrual blood and skin) and tested 15 target genes aiming to identify abovementioned sample types. Our data showed that preselected endogenous controls (miR26b, miR92 and miR484) and miR144, initially selected as potential marker for the detection of menstrual blood, were the most stable expressed genes among our set of samples. Normalizing qPCR data with these four validated references revealed that only five miRNA markers are necessary to differentiate between the six different cell types selected in this study. Nevertheless, our observations in the present study indicate that miRNA analysis methods may not provide straightforward data interpretation strategies required for an implementation in forensic casework.     

RNA/DNA co-analysis from human skin and contact traces – results of a sixth collaborative EDNAP exercise

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.     

Evaluation of a co-extraction kit for mRNA,miRNA and DNA methylation-based body fluid identification

Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.     

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material.In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol^® Reagent, Invitrogen; NucleoSpin^® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy^® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling.We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of ‘RNA integrity number’ (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates.By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin^® miRNA Kit excelling at DNA co-extraction and RNA results, respectively.Overall, there was no ‘best’ method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.     

STR sequence analysis for characterizing normal, variant, and null alleles

DNA sequence variation is known to exist in and around the repeat region of short tandem repeat (STR) loci used in human identity testing. While the vast majority of STR alleles measured in forensic DNA laboratories worldwide type as "normal" alleles compared with STR kit allelic ladders, a number of variant alleles have been reported. In addition, a sequence difference at a polymerase chain reaction (PCR) primer binding site in the DNA template can cause allele drop-out (i.e., a "null" or "silent" allele) with one set of primers and not with another. Our group at the National Institute of Standards and Technology (NIST) has been sequencing variant and null alleles supplied by forensic labs and cataloging this information on the NIST STRBase website for the past decade. The PCR primer sequences and strategy used for our STR allele sequencing work involving 23 autosomal STRs and 17 Y-chromosome STRs are described along with the results from 111 variant and 17 null alleles.     

A capillary electrophoresis method for identifying forensically relevant body fluids using miRNAs

Body fluid identification (BFID) can provide crucial information during the course of an investigation. In recent years, microRNAs (miRNAs) have shown considerable body fluid specificity, are able to be co-extracted with DNA, and their small size (18–25 nucleotides) make them ideal for analyzing highly degraded forensic samples. In this study, we designed a preliminary 8-marker system for BFID including an endogenous reference gene (let-7g) to differentiate between venous blood (miR-451a and miR-142-3p), menstrual blood (miR-141-3p and miR-412-3p), semen (miR-891a and miR-10b), and saliva (miR-205) using a capillary electrophoresis approach. This panel uses a linear primer system in order to incorporate additional miRNA markers by forming a multiplex system. The miRNA system was able to distinguish between venous blood, menstrual blood, semen, and saliva using a rudimentary data interpretation strategy. All STR amplifications from co-extracted DNA yielded complete profiles from human identification purposes.     

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48 × 10⁻²¹, higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA.In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.     

Best of both: A simultaneous analysis of mRNA and miRNA markers for body fluid identification

The body fluid identification of traces found at crime scenes is crucial in relation of the circumstance of crime. For this reason, the body fluid identification (BFI) by molecular biological methods has been increasingly investigated in recent decades. Especially the use of messenger RNA (mRNA) has been established and validated by various studies. mRNAs can resist degradation for several decades under dry and dark environmental conditions, but degradation increases greatly e.g., in humid environments and UV radiation. In contrast, the shorter and protein-protected micro RNAs (miRNAs) are less susceptible to degradation, but not all potential markers are tissue-specific. The aim of this study was to develop a simultaneous mRNA/miRNA multiplex assay to take advantage of both types of RNA. The final assay was tested for various body fluids, dilutions, and mixtures. To demonstrate the advantage of a combined mRNA/miRNA assay, older and mostly degraded samples were examined and compared to an established mRNA assay. Initial results from degraded samples show that tissue-specific miRNAs expected could be detected for 93% of the degraded samples compared to mRNA markers with 25% of the mRNA assay. The result is a simultaneous mRNA/miRNA multiplex assay on capillary electrophoresis (CE) for the first time.     

RNA/DNA co-analysis from human saliva and semen stains – Results of a third collaborative EDNAP exercise

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10–0.01 μl saliva, 5–0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.     

A proof-of-principle study on implementing polymerase chain displacement reaction (PCDR) to improve forensic low-template DNA analysis

Polymerase chain reaction (PCR) plays an important role in forensic DNA analysis. However, the amplification of low-template DNA (LTDNA) samples usually encounters unsatisfactory results for the limited efficiency of PCR, which would interfere with the subsequent profile interpretation. Polymerase chain displacement reaction (PCDR) is a highly-efficient technique characterized by combining PCR and strand displacement reaction into a single PCDR cycle. This study explored the feasibility of PCDR for improving forensic LTDNA analysis. STR markers commonly used in forensic genetics were subjected to PCDR amplification and capillary electrophoresis detection. The results of singleplex reactions indicated that PCDR surpassed original PCR in efficiency for STR amplification. The average peak height of alleles in PCDR profiles was linearly correlated to the number of outer primers adopted for initiating the strand displacement process. Further, we assessed the multiplexing potential of PCDR by incorporating 17 STRs included in the expanded CODIS core loci and Amelogenin gene into a multiplex PCDR system. For pristine DNA templates ranged from 200 pg to 12.5 pg, the multiplex PCDR system consistently exhibited higher allele peak height as well as less allele dropout compared to the multiplex PCR references. Meanwhile, a significant reduction of stutter ratio was extensively observed in PCDR profiles. We also tested mock casework samples to verify the practical ability of multiplex PCDR for LTDNA detection. With DNA input varying from 48.1 pg to 6.6 pg, the multiplex PCDR system consistently obtained more allelic information than multiplex PCR methods. Our data collectively suggested that it is feasible to apply PCDR in forensic LTDNA analysis.     

Identification of organ tissue types and skin from forensic samples by microRNA expression analysis

The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers.Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models.     

The art of traditional native PAGE: The APLP 48-ID assay for human identification

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing. Forty-seven SNPs were selected from all 22 autosomes, showing high diversity in European, Nigerian, Han Chinese, and Japanese population in the HapMap data. PCR primers were designed to generate amplicons 40–100 bp in length to increase the robustness of the PCR.The APLP 48-ID assay consisted of four independent PCR reactions followed by electrophoretic run on four lanes in a polyacrylamide gel. Complete profiles were obtained when more than 1.2 ng of DNA was used. We applied this assay for genotyping of 236 Japanese individuals. The random matching probability was 3.3E-20, and the power of exclusion was greater than 0.9999999. This method is a rapid, robust, and cost-effective approach for human identification and paternity testing.     

Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification

Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturer's repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann–Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may be useful for restoring STR profiles from damaged DNA, but further work is required to develop a generalized approach.     

Characterizing the amplification of STR markers in multiplex polymerase chain displacement reaction using massively parallel sequencing

Polymerase chain displacement reaction (PCDR) showed advantages in forensic low-template DNA analysis with improved amplification efficiency, higher allele detection capacity, and lower stutter artifact than PCR. However, characteristics of STR markers after PCDR amplification remain unclarified for the limited resolving power of capillary electrophoresis (CE). This issue can be addressed by massively parallel sequencing (MPS) technology with higher throughput and discriminability. Here, we developed a multiplex PCDR system including 24 STRs and amelogenin. In addition, a PCR reference was established for comparison. After amplification, products were subjected to PCR-free library construction and sequenced on the Illumina NovaSeq system. We implemented a sequence-matching pipeline to separate different amplicon types of PCDR products from the combination of primers. In the sensitivity test, the PCDR multiplex obtained full STR profiles with as low as 125 pg 2800M control DNA. Based on that, single-source DNA samples were tested. First, highly concordant genotypes were observed among the PCDR multiplex, the PCR reference, and CE-based STR kits. Next, read counts of different PCDR amplicon types were investigated, showing a relative abundance of 78:12:12:1 for the shortest amplicon S, the two medium amplicons M1 and M2, and the longest amplicon L. We also analyzed the stutter artifacts for distinct amplicon types, and the results revealed the reduction of N − 1 and N − 2 contraction stutters, and the increase of N + 1 and N + 2 elongation stutters in PCDR samples. Moreover, we confirmed the feasibility of PCDR for amplifying degraded DNA samples and unbalanced DNA mixtures. Compared to the previous proof of principle study, our work took a further step to characterize the complete profile of STR markers in the PCDR context. Our results suggested that the PCDR-MPS workflow is an effective approach for forensic STR analysis. Corresponding findings in this study may help the development of PCDR-based assays and probabilistic methods in future studies.