Persistence of DNA in the Singapore context.

The relevance and not merely the presence of one's DNA at a crime scene has become the emerging issue in courtrooms all over the world today. By studying the length of time DNA is likely to persist in an environment until detection, a more holistic assessment of DNA evidence in the context of a case can be made. The current study looks at the persistence of DNA from blood, keratinocytes, and several types of mock exhibits under various conditions, in the tropical rainforest climate of Singapore. While DNA on articles left outdoors showed highly variable persistence subject to the presence of rainfall, DNA from items placed indoors at ambient temperature and under controlled temperature and humidity is comparatively stable. The information gathered from this study, while not exhaustive, serves to provide investigators and the courts with a better understanding of the relevance of DNA recovered from crime scenes of different environmental conditions.

A study of DNA transfers onto plastic packets placed in personal bags.

The ability to detect low level DNA brings with it the uncertainty of whether the detected DNA is a result of transfer. To address this uncertainty, a simulation study was conducted in which a mock illicit drug packet was placed into the personal bags of individuals. When the average transit time of the packets was increased from around 2 h to more than 14 h, the percentage of the DNA profiles recovered from the packets which could be attributed to the individuals increased greatly from 5.3% to 48.6%. We found that drug packers who were poor shedders could not be included as contributors to the DNA profiles from the drug packets at all and there was a higher chance that individuals other than themselves could be included as contributors to the DNA profile recovered from drug packets. We also found that it was equally likely that the drug packers who had direct contact with the drug packets and bag owners who did not, could be included as contributors to the DNA profiles recovered from the packets. The results in this study highlight the importance of taking into consideration the transit time of drug packet, the shedder status of the alleged packer and the history of an item, when evaluating DNA evidence in the context of illicit drug activities.

Long-Term Tissue Preservation at Ambient Temperature for Post-Mass Fatality Incident DNA-Based Victim Identification.

In a mass fatality incident (MFI), effective preservation of tissue samples is the cornerstone for downstream DNA-based identification of victims. This is commonly achieved through freezing of tissue samples excised from bodies/fragmented remains which may be buried or stored in refrigerated containers. This may, however, not be possible depending on the nature of the MFI; in particular, during armed conflict/war where extended periods of electrical outages would be expected. The present study compared the effectiveness of long-term tissue preservation at ambient temperatures using two commercial products (non-iodized kitchen salt and a 40% alcoholic beverage) against a chemical preservative (Allprotect™ Tissue Reagent (Qiagen, Germantown, MD, USA)) and freezing at -20 °C. Bovine muscle tissue, used as a proxy for human tissue, was treated with the four preservation methods and sampled at six different time-points over a 24-month period. All four methods were able to preserve the bovine tissue, generally yielding STR-PCR (Short Tandem Repeat-Polymerase Chain Reaction) amplicons > 200 bp in size even at the end of 24 months. Gel electrophoresis, however, indicated that salt was more effective in preserving DNA integrity with high-molecular-weight DNA clearly visible as compared to the low-molecular-weight DNA smears observed in the other methods. This study also proposes a simple process for the rapid and low-cost preservation of tissue samples for long-term storage at ambient temperatures in support of post-incident victim identification efforts.

Shedder status-An analysis over time and assessment of various contributing factors.

The shedder status of a person is an important consideration when evaluating probabilities of DNA transfer during activity-level assessments. As an extension of our previously published study, the shedder statuses of 38 individuals were reassessed 1 year later. The study found that shedder status may change over time for some individuals and was associated with one's gender, number of items touched, and mobile phone usage. In 29% of touch events, no DNA allele was detected and in 99% of touch events, the amount of DNA deposited was <2 ng. The study also found that in 0.6% of touch events, the participant could be excluded as a contributor of the observed DNA profile, with another person being included. Additionally, our investigations suggest that the current three-category system for shedder status classification may require further refinement to better represent the individuals' shedder status in a population.

Uncertainty in estimating the number of contributors from simulated DNA mixture profiles, with and without allele dropout, from Chinese, Malay, Indian, and Caucasian ethnic populations.

Determining the number of contributors (NOC) accurately in a forensic DNA mixture profile can be challenging. To address this issue, there have been various studies that examined the uncertainty in estimating the NOC in a DNA mixture profile. However, the focus of these studies lies primarily on dominant populations residing within Europe and North America. Thus, there is limited representation of Asian populations in these studies. Further, the effects of allele dropout on the NOC estimation has not been explored. As such, this study assesses the uncertainty of NOC in simulated DNA mixture profiles of Chinese, Malay, and Indian populations, which are the predominant ethnic populations in Asia. The Caucasian ethnic population was also included to provide a basis of comparison with other similar studies. Our results showed that without considering allele dropout, the NOC from DNA mixture profiles derived from up to four contributors of the same ethnic population could be estimated with confidence in the Chinese, Malay, Indian and Caucasian populations. The same results can be observed on DNA mixture profiles originating from a combination of differing ethnic populations. The inclusion of an overall 30% allele dropout rate increased the probability (risk) of underestimating the NOC in a DNA mixture profile; even a 3-person DNA mixture profile has a > 99% risk of underestimating the NOC as two or fewer contributors. However, such risks could be mitigated when the highly polymorphic SE33 locus was included in the dataset. Lastly there was a negligible level of risk in misinterpreting the NOC in a mixture profile as deriving from a single source profile. In summary, our studies showcased novel results representative of the Chinese, Malay, and Indian ethnic populations when examining the uncertainty in NOC estimation in a DNA mixture profile. Our results would be useful in the estimation of NOC in a DNA mixture profile in the Asian context.

Artificial neural network, predictor variables and sensitivity threshold for DNA methylation-based age prediction using blood samples.

Regression models are often used to predict age of an individual based on methylation patterns. Artificial neural network (ANN) however was recently shown to be more accurate for age prediction. Additionally, the impact of ethnicity and sex on our previous regression model have not been studied. Furthermore, there is currently no age prediction study investigating the lower limit of input DNA at the bisulfite treatment stage prior to pyrosequencing. Herein, we evaluated both regression and ANN models, and the impact of ethnicity and sex on age prediction for 333 local blood samples using three loci on the pyrosequencing platform. Subsequently, we trained a one locus-based ANN model to reduce the amount of DNA used. We demonstrated that the ANN model has a higher accuracy of age prediction than the regression model. Additionally, we showed that ethnicity did not affect age prediction among local Chinese, Malays and Indians. Although the predicted age of males were marginally overestimated, sex did not impact the accuracy of age prediction. Lastly, we present a one locus, dual CpG model using 25 ng of input DNA that is sufficient for forensic age prediction. In conclusion, the two ANN models validated would be useful for age prediction to provide forensic intelligence leads.

Differentiation of Asian population samples using the Illumina ForenSeq kit.

The Kidd set of ancestry informative SNPs are included in Illumina's ForenSeq DNA Signature Kit. We had previously reported on the capability of these SNPs together with some phenotypic SNPs with ancestry informative properties in differentiating individuals from the Chinese, Malay and Indian populations in Singapore. The Singapore population is primarily made up of Chinese, Malays and Indians, with individuals from other Asian countries making up the rest. In this study, we evaluated the ancestry prediction capabilities of the ForenSeq kit in 484 unrelated individuals of self-declared Bangladeshi, Burmese, Filipino, Indonesian and Vietnamese origin. 750 Chinese, Malay and Indian individuals previously reported were included in this study. 48 ancestry SNPs and 12 phenotypic SNPs with ancestry informative properties were selected for analyses. Ancestry modelling in STRUCTURE showed that the eight tested populations could be better classified as five. Principal component analysis also showed that the eight populations clustered in five groups based on general geographic location within Asia; with Chinese clustering with Vietnamese, Malays clustering with Indonesians, Indians clustering with Bangladeshi, and the Burmese and Filipino populations clustering in-between and overlapping with the Chinese and Malay populations. The 60 SNPs analysed could account for only 23 % of the variation between the populations. The lack of distinction between the populations resulted in poor (43 % correct self-classification) cross-validation using Snipper. While this was improved by merging the co-clustering populations into five groups (East, South-East, South Asian, Burmese & Filipino), successful self-classification was still relatively low (69 %). While the 60 tested ancestry informative markers were able to differentiate between individuals of East, South-East and South Asian origin, they are not sufficiently informative to effectively discriminate between Chinese, Malays and Indians, and Bangladeshi, Burmese, Filipino, Indonesian and Filipino populations in the country.

A practical study on direct PCR amplification using the GlobalFiler™ PCR Amplification Kit on human bloodstains collected with microFLOQ™ Direct swabs.

Rapid DNA profiling of casework samples is a powerful tool that can support law enforcement agencies in the quick apprehension of perpetrators before they re-offend or escape the jurisdiction. This present study evaluated the feasibility of direct PCR amplification, using the microFLOQ™ Direct swab, for generating DNA profiles (from bloodstains) within 3 h. The swab tip is coated with nylon fibers pre-treated with cell lysing agent, which allows for the direct PCR amplification of collected samples without DNA extraction and quantification, thereby shortening the time required to obtain a DNA profile. Samples collected were directly amplified using GlobalFiler™ PCR Amplification Kit with and without the presence of a PCR additive. Addition of the PCR additive enhanced the peak heights of DNA profiles by approximately 2 fold. Hence, an additive could improve results obtained in the absence of a DNA purification step, especially since casework samples may contain PCR inhibitors. Subsequently, these swabs, amplified using the GlobalFiler™ PCR Amplification Kit with PCR additive, were evaluated on common substrates encountered in routine casework samples submitted with bloodstains, such as denim jeans, knife blade, tissue paper, leather belt, shirt, and blood swabs. The minimum peak heights observed were generally above the analytical and stochastic thresholds established by the laboratory. Finally, the microFLOQ™ Direct swab workflow was compared to the laboratory's standard workflow of DNA profiling comprising of conventional processing steps such as extraction using the DNA-IQ™ chemistry on Maxwell® 16, followed by quantification, amplification and capillary electrophoresis. The average peak heights of the DNA profiles generated by direct PCR amplification were similar or exceeded those generated using the standard workflow. This study clearly demonstrates that direct PCR amplification using microFLOQ™ Direct swab can be used in a rapid workflow to obtain DNA profiles from casework samples.

Simple DNA extraction of urine samples: Effects of storage temperature and storage time.

Urine samples are commonly analysed in cases with suspected illicit drug consumption. In events of alleged sample mishandling, urine sample source identification may be necessary. A simple DNA extraction procedure suitable for STR typing of urine samples was established on the Promega Maxwell® 16 paramagnetic silica bead platform. A small sample volume of 1.7mL was used. Samples were stored at room temperature, 4°C and -20°C for 100days to investigate the influence of storage temperature and time on extracted DNA quantity and success rate of STR typing. Samples stored at room temperature exhibited a faster decline in DNA yield with time and lower typing success rates as compared to those at 4°C and -20°C. This trend can likely be attributed to DNA degradation. In conclusion, this study presents a quick and effective DNA extraction protocol from a small urine volume stored for up to 100days at 4°C and -20°C.

Sequence polymorphism of the mitochondrial DNA hypervariable regions I and II in 205 Singapore Malays.

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were analyzed in 205 unrelated ethnic Malays residing in Singapore as an initial effort to generate a database for forensic identification purposes. Sequence polymorphism was detected using PCR and direct sequencing analysis. A total of 152 haplotypes was found containing 152 polymorphisms. Out of the 152 haplotypes, 115 were observed only once and 37 types were seen in multiple individuals. The most common haplotype (16223T, 16295T, 16362C, 73G, 146C, 199C, 263G, and 315.1C) was shared by 7 (3.41%) individuals, two haplotypes were shared by 4 individuals, seven haplotypes were shared by 3 individuals, and 27 haplotypes by 2 individuals. Haplotype diversity and random match probability were estimated to be 0.9961% and 0.87%, respectively.

Ancestry prediction in Singapore population samples using the Illumina ForenSeq kit.

The ability to predict bio-geographic ancestry can be valuable to generate investigative leads towards solving crimes. Ancestry informative marker (AIM) sets include large numbers of SNPs to predict an ancestral population. Massively parallel sequencing has enabled forensic laboratories to genotype a large number of such markers in a single assay. Illumina's ForenSeq DNA Signature Kit includes the ancestry informative SNPs reported by Kidd et al. In this study, the ancestry prediction capabilities of the ForenSeq kit through sequencing on the MiSeq FGx were evaluated in 1030 unrelated Singapore population samples of Chinese, Malay and Indian origin. A total of 59 ancestry SNPs and phenotypic SNPs with AIM properties were selected. The bio-geographic ancestry of the 1030 samples, as predicted by Illumina's ForenSeq Universal Analysis Software (UAS), was determined. 712 of the genotyped samples were used as a training sample set for the generation of an ancestry prediction model using STRUCTURE and Snipper. The performance of the prediction model was tested by both methods with the remaining 318 samples. Ancestry prediction in UAS was able to correctly classify the Singapore Chinese as part of the East Asian cluster, while Indians clustered with Ad-mixed Americans and Malays clustered in-between these two reference populations. Principal component analyses showed that the 59 SNPs were only able to account for 26% of the variation between the Singapore sub-populations. Their discriminatory potential was also found to be lower (GST=0.085) than that reported in ALFRED (FST=0.357). The Snipper algorithm was able to correctly predict bio-geographic ancestry in 91% of Chinese and Indian, and 88% of Malay individuals, while the success rates for the STRUCTURE algorithm were 94% in Chinese, 80% in Malay, and 91% in Indian individuals. Both these algorithms were able to provide admixture proportions when present. Ancestry prediction accuracy (in terms of likelihood ratio) was generally high in the absence of admixture. Misclassification occurred in admixed individuals, who were likely offspring of inter-ethnic marriages, and hence whose self-reported bio-geographic ancestries were dependent on that of their fathers, and in individuals of minority sub-populations with inter-ethnic beliefs. The ancestry prediction capabilities of the 59 SNPs on the ForenSeq kit were reasonably effective in differentiating the Singapore Chinese, Malay and Indian sub-populations, and will be of use for investigative purposes. However, there is potential for more accurate prediction through the evaluation of other AIM sets.

A preliminary evaluation study of new generation multiplex STR kits comprising of the CODIS core loci and the European Standard Set loci.

The GlobalFiler™ (Life Technologies), Investigator® 24plex QS (Qiagen), and PowerPlex® Fusion 6C (Promega) kits are the latest generation 6-dye fluorescent chemistry STR-PCR amplification kits. These kits allow for the simultaneous amplification of the CODIS core loci and the European Standard Set loci, as well as a few Y-STR loci in addition to the standard sex-determining marker Amelogenin. The present study was designed to be a preliminary evaluation of the three STR-PCR kits in terms of sensitivity, profile recovery from degraded DNA samples, tolerance to PCR inhibitors, and detection of minor components in DNA mixtures. The results showed that the three STR-PCR kits had relatively similar performance with each kit faring better for the different aspects studied. The PowerPlex® Fusion 6C and the Investigator® 24plex QS kits were shown to tolerate inhibitors better, while the GlobalFiler™ kit appeared to have a higher mean percentage recovery of alleles from low template DNA samples and for minor components in DNA mixtures.

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Population study of 11 Y-chromosomal STR loci in Singapore Chinese.

In this study of 212 unrelated Singapore Chinese males, allelic frequencies and gene diversities of 11 Y-chromosome specific STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS438 and DYS439) were established. A total of 184 haplotypes were observed in the 212 individuals studied, of which 165 (89.67%) were unique. The most common haplotype was observed in five (2.35%) individuals. The overall haplotype diversity for the 11 Y-STR loci was 99.81%, and the discrimination capacity was 86.79%.

Optimization of spermatozoa detection using immunofluorescent staining and laser micro-dissection.

The present study evaluated the use of an immunofluorescence-based assay for the microscopic detection of human spermatozoa, following which the fluorescence-labelled spermatozoa could be excised with a laser micro-dissection system. The Sperm Hy-Liter™ PI kit was able to detect spermatozoa from as little as 20nL of semen. No interference or non-specificity were observed when the kit was used on semen mixed with various body fluids such as blood and urine, as well as when semen was spiked onto different types of fabric. Good results could also be obtained with rectal samples which contain auto-fluorescent fecal materials through the use of dual FITC/PI filters. We also developed a method for concurrent testing of two protein biomarkers of semen (semenogelin and prostate-specific antigen) and detection of spermatozoa. This approach would maximize the evidential value from a single piece of sexual assault exhibit. The results also showed that staining by Sperm Hy-Liter™ PI does not interfere with DNA recovery, facilitating the generation of clear male DNA profiles from dissected spermatozoa, thereby making profile interpretation less complex. In summary, Sperm Hy-Liter™ PI staining was demonstrated to be sensitive, robust and specific.

Evaluation of the RapidHIT™ 200 System: A comparative study of its performance with Maxwell(®) DNA IQ™/Identifiler(®) Plus/ABI 3500xL workflow.

RapidHIT(™) System is a rapid DNA instrument that is capable of processing forensic samples from extraction through to capillary electrophoresis and profile generation within two hours. Evaluation of the RapidHIT(™) 200 System was conducted to examine several key performance indicators of the instrument, including reproducibility, contamination, sensitivity, versatility and the possibility of sample re-extraction. Results indicated that the RapidHIT(™) 200 System was capable of generating high quality DNA profiles which were comparable to those from the standard protocol comprising of Maxwell(®) 16 DNA IQ(™) System, Identifiler(®) Plus and ABI 3500xL. No contamination was detected during the studies. Results also showed that the instrument was able to generate DNA profiles from samples containing lower amounts of DNA (0.5 µl of blood) albeit with more allele and locus dropouts when compared to the standard protocol. The ability to process blood swabs, blood-stained FTA punches, semen swabs, buccal swabs, product of conception (POC), bone marrow, fingernail clippings and cigarette butts at a good success rate indicated the robustness and versatility of the RapidHIT(™) 200 System. Furthermore, additional alleles could be recovered via re-analysis of the failed samples using the standard protocol. In summary, our results showed that the RapidHIT(™) 200 System was able to process casework samples for the purpose of providing rapid intelligence through DNA database searches and reference matching. Confirmative DNA results can be obtained through either concurrent processing of duplicate samples via standard protocol or re-extraction of samples retrieved from the RapidHIT(™) sample cartridge.

Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a "do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.