[Show abstract][Hide abstract] ABSTRACT: STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively-affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6–200 ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62 pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250 pg of input DNA, and 62 pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.
[Show abstract][Hide abstract] ABSTRACT: Background
Massively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGMTM) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeqTM (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM.
24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%.
In this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.
[Show abstract][Hide abstract] ABSTRACT: The majority of STR loci are not ideal for the analysis of forensic samples with
degraded and/or low template DNA. One alternative to overcome these limitations is
the use of bi-allelic markers, which have low mutation rates and shorter amplicons.
Human identification (HID) InDel marker panels have been described in several
countries, including Brazil. The commercial kit available is, however, mostly suitable for
Europeans, with lower discrimination power for other population groups. Recently a
combination of 49 InDel markers used in four different ethnic groups in the United
States has been shown to be more informative than another panel from Portugal,
already tested in a Rio de Janeiro sample. However, these 49 InDels have yet to be
applied to other admixed or isolated populations. We assessed the efficiency of this
panel in two urban admixed populations (Rio de Janeiro, Brazil; Tripoli, Libya), and one
isolated Native Brazilian community. All markers are in Hardy-Weinberg equilibrium
(HWE) after the Bonferroni correction, and no Linkage disequilibrium was detected.
Assuming loci independence and no substructure effect, cumulative RMP were 2.7x10-
18, 1.5x10-20, and 4.5x10-20 for Native Brazilian, Rio de Janeiro, and Tripoli
populations, respectively. The overall Fst value was 0.05512. Rio de Janeiro and
Tripoli showed similar admixture levels, however for Native Brazilians one parental
cluster represented over 60% of the total parental population. We conclude that this
panel is suitable for HID on these urban populations, but is less efficient for the isolated
International Journal of Legal Medicine 01/2015; · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The TruSeq™ Forensic Amplicon library preparation protocol, originally designed to attach sequencing adapters to chromatin-bound DNA for chromatin immunoprecipitation sequencing (TruSeq™ ChIP-Seq), was used here to attach adapters directly to amplicons containing markers of forensic interest. In this study, the TruSeq™ Forensic Amplicon library preparation protocol was used to detect 160 single nucleotide polymorphisms (SNPs), including human identification SNPs (iSNPs), ancestry, and phenotypic SNPs (apSNPs) in 12 reference samples. Results were compared with those generated by a second laboratory using the same technique, as well as to those generated by whole genome sequencing (WGS). The genotype calls made using the TruSeq™ Forensic Amplicon library preparation protocol were highly concordant. The protocol described herein represents an effective and relatively sensitive means of preparing amplified nuclear DNA for massively parallel sequencing (MPS).
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 11/2014; · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples.
[Show abstract][Hide abstract] ABSTRACT: STRait Razor (the STR Allele Identification Tool – Razor) was developed as a bioinformatic software tool to detect short tandem repeat (STR) alleles in massively parallel sequencing (MPS) raw data. The method of detection used by STRait Razor allows it to make reliable allele calls for all STR types in a manner that is similar to that of capillary electrophoresis. STRait Razor v2.0 incorporates several new features and improvements upon the original software, such as a larger default locus configuration file that increases the number of detectable loci (now including X-chromosome STRs and Amelogenin), an enhanced custom locus list generator, a novel output sorting method that highlights unique sequences for intra-repeat variation detection, and a genotyping tool that emulates traditional electropherogram data. Users also now have the option to choose whether the program detects autosomal, X-chromosome, Y-chromosome, or all STRs. Concordance testing was performed, and allele calls produced by STRait Razor v2.0 were completely consistent with those made by the original software.
[Show abstract][Hide abstract] ABSTRACT: Allele frequencies for 15 autosomal STR loci (N = 290) and haplotype data for 17 Y-STR loci (N = 157) were determined for an admixed population from Belize. There were no detectable departures from Hardy-Weinberg equilibrium expectations at any autosomal STR loci except for the D8S1179 locus (p = 0.002). The combined power of discrimination (PD) and combined power of exclusion (PE) were greater than 0.99999999 and 0.99999951, respectively. In addition, a total of 144 distinct Y-STR haplotypes were observed with 133 Y-STR haplotypes observed only once. The most common Y-STR haplotype was observed three times for two separate haplotypes. The various analyses of these forensically relevant STR loci showed that these markers are informative in the Belize population for forensic and parentage testing applications.
International Journal of Legal Medicine 09/2014; · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type sequencing. Consequently sequencing of a relatively-large target such as the mitochondrial genome (mtGenome) is laborious and time consuming. Thus, sequencing typically focuses on the control region due to its high concentration of variation. Massively parallel sequencing (MPS) has become more accessible in recent years allowing for high-throughput processing of large target areas. In this study, Nextera® XT DNA Sample Preparation Kit and the Illumina MiSeq™ were utilized to generate quality whole genome mitochondrial haplotypes from 283 individuals in a both cost-effective and rapid manner. Results showed that haplotypes can be generated at a high depth of coverage with limited strand bias. The distribution of variants across the mitochondrial genome was described and demonstrated greater variation within the coding region than the non-coding region. Haplotype and haplogroup diversity were described with respect to whole mtGenome and HVI/HVII. An overall increase in haplotype or genetic diversity and random match probability, as well as better haplogroup assignment demonstrates that MPS of the mtGenome using the Illumina MiSeq system is a viable and reliable methodology.
[Show abstract][Hide abstract] ABSTRACT: Vuorio A, Laukkala T, Navathe P, Budowle B, Eyre A, Sajantila A. Aircraft-assisted pilot suicides: lessons to be learned. Aviat Space Environ Med 2014; 85:841–6. Aircraft assisted suicides were studied in the United States, United Kingdom, Germany, and Finland during 1956-2012 by means of literature search and accident case analysis. According to our study the frequency varied slightly between the studies. Overall, the new estimate of aircraft assisted suicides in the United States in a 20-yr period (1993-2012) is 0.33% (95% CI 0.21-0.49) (24/7244). In the detailed accident case analysis, it was found that in five out of the eight cases from the United States, someone knew of prior suicidal ideation before the aircraft assisted fatality. The caveats of standard medico-legal autopsy and accident investigation methods in investigation of suspected aircraft assisted suicides are discussed. It is suggested that a psychological autopsy should be performed in all such cases. Also the social context and possibilities of the prevention of aviation-related suicides were analyzed. In addition, some recent aircraft assisted suicides carried out using commercial aircraft during scheduled services and causing many casualties are discussed.
Aviation Space and Environmental Medicine 08/2014; 85(8). · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.
Investigative genetics. 07/2014; 5:9.
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[Show abstract][Hide abstract] ABSTRACT: Short tandem repeat (STR) typing is used routinely for associating or excluding individuals with biological evidence left at a crime scene. Improvements have been made to reduce the turnaround time and labor involved with profile generation, but there is still some lag time between sample collection and interpretation of results. The RapidHIT(®) (IntegenX; Pleasanton, CA, USA) system is an automated instrument that is configured to perform DNA extraction, bead-based DNA normalization, amplification, electrophoresis of PCR amplicons, and data analysis of five reference swabs simultaneously. The RapidHIT system provided reliable STR profiles from reference buccal swabs in approximately 90min with nominal "hands-on" sample loading time with no evidence of contamination between samples. The overall success rate of typing buccal swabs was comparable to standard typing systems. In the event of a failed run due to instrument failure, the swab can be removed from the cartridge and reanalyzed in the RapidHIT system or with standard STR genotyping workflows.
[Show abstract][Hide abstract] ABSTRACT: Success of DNA typing is related to the amount of target material recovered from an evidentiary item. Generally, the more DNA that is recovered, the better the chance is of obtaining a typing result that will be robust and reliable. One method of collecting stain materials is by swabbing. Recovery of DNA from a number of commercially available swabs is not an efficient process. The X-Swab™ (Diomics Corporation, La Jolla, CA) is a unique bio-specimen collection material with highly absorptive properties and can be dissolved during certain extraction conditions. Therefore, more DNA may be collected from a substrate and be released from the swab matrix than other swabs. The ability to recover DNA from X-Swab material and success in STR typing were compared with the Copan 4N6FLOQSwab™ (Brescia, Italy), a device which utilizes a proprietary flocked-swab technology to maximize DNA collection and elution efficiency. Both types of swabs were impregnated with known amounts of DNA and body fluids and allowed to air dry. In addition, blood was placed onto glass slides, allowed to dry and collected using both types of swabs. DNA recovery was assessed by DNA quantitation and by STR typing. Results suggested that X-Swab material yielded greater DNA recovery, particularly of low quantity samples (defined as diluted neat samples), compared with the 4N6FLOQSwab. Results also indicated that X-Swab material itself enhances yield of PCR products.
[Show abstract][Hide abstract] ABSTRACT: DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow(®) (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex(®) ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8ng±1 to 900ng±159 of DNA compared with the organic method ranging from 0.5ng±0.9 to 855ng±156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial genome (mtGenome) contains genetic information amenable to numerous applications such as medical research, population and evolutionary studies, and human identity testing. However, inconsistent nomenclature assignment makes haplotype comparison difficult and can lead to false exclusion of potentially useful profiles. Massively Parallel Sequencing (MPS) is a platform for sequencing large datasets and potentially whole populations with relative ease. However, the data generated are not easily parsed and interpreted. With this in mind, mitoSAVE has been developed to enable fast conversion of Variant Call Format (VCF) files. mitoSAVE is an Excel-based workbook that converts data within the VCF into mtDNA haplotypes using phylogenetically-established nomenclature as well as rule-based alignments consistent with current forensic standards. mitoSAVE is formatted for human mitochondrial genome; however, it can easily be adapted to support other reasonably small genomes.
[Show abstract][Hide abstract] ABSTRACT: One parameter that impacts the robustness and reliability of forensic DNA analyses is the amount of template DNA used in the polymerase chain reaction (PCR). With short tandem repeat (STR) typing, low copy number (LCN) DNA samples can present exaggerated stochastic effects during the PCR that result in heterozygote peak height imbalance, allele drop out, and increased stutter. Despite these effects, there has been little progress toward decreasing the formation of stutter products and heterozygote peak imbalance effects during PCR. In an attempt to develop a more robust system that is less refractory to stochastic effects, the PCR additives, betaine, DMSO, PEG, and PCRboost®, were investigated on low-quantity DNA samples. The effects of the additives were assessed by evaluating STR typing results. Of the four additives, the only positive effects were observed with betaine treatment. Betaine, at a final concentration of 1.25 mol/L, was found to improve the robustness of the amplification, specifically by decreasing stutter in a dual locus system. In contrast, the addition of 1.25 mol/L betaine to commercial STR amplification kits did not affect stutter ratios. However, the addition of betaine did lead to increased yield of PCR products in all commercial kits tested. The results support that betaine can improve amplification efficiency of LCN DNA samples.
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 05/2014; · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results.
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 05/2014; 128(6). · 2.69 Impact Factor