The use of single nucleotide polymorphisms (SNPs) in forensic genetics has been limited to challenged samples with low template and/or degraded DNA. The recent introduction of massively parallel sequencing (MPS) technologies has expanded the potential applications of these markers and increased the discrimination power of well-established loci by considering variation in the flanking regions of target loci. The ForenSeq Signature Preparation Kit contains 165 SNP amplicons for ancestry- (aiSNPs), identity- (iiSNPs), and phenotype-inference (piSNPs). In this study, 714 individuals from four major populations (African American, AFA; East Asian, ASN; US Caucasian, CAU; and Southwest US Hispanic, HIS) previously reported by Churchill a al. [Forensic Sci Int Genet 30 (2017) 81-92; DOI : https://doi.org/10.1016/j.fsigen.2017.06.004] were assessed using STRait Razor v2s to determine the level of diversity in the flanking regions of these amplicons. The results show that nearly 70% of loci showed some level of flanking region variation with 22 iiSNPs and 8 aiSNPs categorized as microhaplotypes in this study. The heterozygosities of these microhaplotypes approached, and in one instance surpassed, those of some core STR loci. Also, the impact of the flanking region on other forensic parameters (e.g., power of exclusion and power of discrimination) was examined. Sixteen of the 94 iiSNPs had an effective allele number greater than 2.00 across the four populations. To assess what effect the flanking region information had on the ancestry inference, genotype probabilities and likelihood ratios were determined. Additionally, concordance with the ForenSeq UAS and Nextera Rapid Capture was evaluated, and patterns of heterozygote imbalance were identified. Pairwise comparison of the iiSNP diplotypes determined the probability of detecting a mixture (i.e., observing >= 3 haplotypes) using these loci alone was 0.9952. The improvement in random match probabilities for the full regions over the target iiSNPs was found to be significant. When combining the iiSNPs with the autosomal STRs, the combined match probabilities ranged from 6.40 x 10(-73) (ASN) to 1.02 x 10(-79) (AFA).

Ancestry informative markers (AIMs) can be used to determine population affiliation of the donors of forensic samples. In order to examine ancestry evaluations of the four major populations in the USA, 23 highly informative AIMs were identified from the International HapMap project. However, the efficacy of these 23 AIMs could not be fully evaluated in silico. In this study, these 23 SNPs were multiplexed to test their actual performance in ancestry evaluations. Genotype data were obtained from 189 individuals collected from four American populations. One SNP (rs12149261) on chromosome 16 was removed from this panel because it was duplicated on chromosome 1. The resultant 22-AIMs panel was able to empirically resolve the four major populations as in the in silico study. Eight individuals were assigned to a different group than indicated on their samples. The assignments of the 22 AIMs for these samples were consistent with AIMs results from the ForenSeq(TM) panel. No departures from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were detected for all 22 SNPs in four US populations (after removing the eight problematic samples). The principal component analysis (PCA) results indicated that 181 individuals from these populations were assigned to the expected groups. These 22 SNPs can contribute to the candidate AIMs pool for potential forensic identification purposes in major US populations.

Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis. The INDEL marker allelic states range in size from 2 to 6 base pairs, enabling small amplicon size. In addition, heterozygote balance may be increased by minimizing preferential amplification of the smaller allele, as is more common with STR markers. Multiplexing a large number of INDELs allows for generating panels with high discrimination power. The Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA) and massively parallel sequencing (MPS) on the Illumina MiSeq were used to sequence 68 well-characterized INDELs in four major US population groups. In addition, the STR Allele Identification Tool: Razor (STRait Razor) was used in a novel way to analyze INDEL sequences and detect adjacent single nucleotide polymorphisms (SNPs) and other polymorphisms. This application enabled the discovery of unique allelic variants, which increased the discrimination power and decreased the single-locus random match probabilities (RMPs) of 22 of these well-characterized INDELs which can be considered as microhaplotypes. These findings suggest that additional microhaplotypes containing human identification (HID) INDELs may exist elsewhere in the genome.

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).

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.

Massively parallel sequencing (MPS) technology is capable of determining the sizes of short tandem repeat (STR) alleles as well as their individual nucleotide sequences. Thus, single nucleotide polymorphisms (SNPs) within the repeat regions of STRs and variations in the pattern of repeat units in a given repeat motif can be used to differentiate alleles of the same length. In this study, MPS was used to sequence 28 forensically-relevant Y-chromosome STRs in a set of 41 DNA samples from the 3 major U.S. population groups (African Americans, Caucasians, and Hispanics). The resulting sequence data, which were analyzed with STRait Razor v2.0, revealed 37 unique allele sequence variants that have not been previously reported. Of these, 19 sequences were variations of documented sequences resulting from the presence of intra-repeat SNPs or alternative repeat unit patterns. Despite a limited sampling, two of the most frequently-observed variants were found only in African American samples. The remaining 18 variants represented allele sequences for which there were no published data with which to compare. These findings illustrate the great potential of MPS with regard to increasing the resolving power of STR typing and emphasize the need for sample population characterization of STR alleles.

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 (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg 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 250pg of input DNA, and 62pg 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.

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.

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.

Forensic DNA typing can provide useful information on human identification, such as in criminal cases and mass disasters. Short tandem repeats (STRs) are the primary genetic markers used because of their high discrimination power and relatively short amplicon size. However, some evidence samples are highly degraded and may not be characterized well with the current battery of STRs. Single nucleotide polymorphisms (SNPs) are an alternate set of markers that may be applied successfully to degraded samples. Most SNP containing amplicons can be designed to be smaller than 150 bp and, potentially, as short as 50-60 bp in length. However, current instrumentation in a typical crime laboratory is not amenable to typing a large battery of SNPs.

Massively parallel sequencing offers the potential to type a large battery of forensically, relevant markers of a large number of individuals simultaneously. This summary describes progress, on development of STR, SNP, and mtDNA marker typing systems and demonstrates that transition to this technology is promising.

The Ion AmpliSeq™ HID single nucleotide polymorphism (SNP) panel, a primer pool of 103 autosomal SNPs and 33 Y-SNPs, was evaluated using the Ion 314™ Chip on the Ion PGM™ Sequencer with four DNA samples. The study focused on the sequencing of DNA at three different initial target quantities, related interpretation issues, and concordance of results with another sequencing platform, i.e., Genome Analyzer IIx. With 10 ng of template DNA, all genotypes at the 136 SNPs were detected. With 1 ng of DNA, all SNPs were detected and one SNP locus in one sample showed extreme heterozygote imbalance on allele coverage. With 100 pg of DNA, an average of 1.6 SNP loci were not detected, and an average of 4.3 SNPs showed heterozygote imbalance. The average sequence coverage was 945–600× at autosomal SNPs and 465–209× at Y-SNPs for 10 ng–100 pg of DNA. The average heterozygote allele coverage ratio was 89.6–61.8 % for 10 ng–100 pg of DNA. At 10 ng of DNA, all genotypes of the 95 SNPs shared between the two different sequencing platforms were concordant except for one SNP, rs1029047. The error was due to the misalignment of a flanking homopolymer. Overall, the data support that genotyping a large battery of SNPs is feasible with massively parallel sequencing. With barcode systems, better allele balance, and specifically designed alignment software, a more comprehensive rapid genotyping and more cost-effective results may be obtained from multiple samples in one analysis than are possible with current typing and capillary electrophoresis systems.

Recent studies have demonstrated the capability of second generation sequencing (SGS) to provide coverage of short tandem repeats (STRs) found within the human genome. However, there are relatively few bioinformatic software packages capable of detecting these markers in the raw sequence data. The extant STR-calling tools are sophisticated, but are not always applicable to the analysis of the STR loci commonly used in forensic analyses. STRait Razor is a newly developed Perl-based software tool that runs on the Linux/Unix operating system and is designed to detect forensically-relevant STR alleles in FASTQ sequence data, based on allelic length. It is capable of analyzing STR loci with repeat motifs ranging from simple to complex without the need for extensive allelic sequence data. STRait Razor is designed to interpret both single-end and paired-end data and relies on intelligent parallel processing to reduce analysis time. Users are presented with a number of customization options, including variable mismatch detection parameters, as well as the ability to easily allow for the detection of alleles at new loci. In its current state, the software detects alleles for 44 autosomal and Y-chromosome STR loci. The study described herein demonstrates that STRait Razor is capable of detecting STR alleles in data generated by multiple library preparation methods and two Illumina(®) sequencing instruments, with 100% concordance. The data also reveal noteworthy concepts related to the effect of different preparation chemistries and sequencing parameters on the bioinformatic detection of STR alleles.

For very challenged biological samples, mitochondrial DNA (mtDNA) analysis can often provide results when the more traditional nuclear DNA markers fail. While reliable, the current method of mtDNA analysis by Sanger sequencing is expensive, labor-intensive, and time-consuming and is limited by its inability to quantify mixed samples. The Abbott PLEX-ID™ instrument, which enables analysis of mtDNA amplicons via electrospray ionization mass spectrometry (ESI-MS), produces comparable accuracy and sensitivity while offering a faster and less expensive alternative to Sanger sequencing. Unlike Sanger sequencing, this system is capable of quantifying DNA species and thus may be exploited for evaluating heteroplasmy and, possibly, mixture deconvolution. Validation studies of the PLEX-ID™ mtDNA assay confirmed that the instrument is highly sensitive and capable of yielding reproducible results. Samples commonly encountered in a forensic setting, as well as population samples, were typed correctly. The PLEX-ID™ mtDNA assay yields reliable results for single-source samples, which are the same sample types currently examined in forensic laboratories via Sanger sequencing, at a level that meets or exceeds that of the current method. While the instrument has the demonstrated capability to quantify mixed samples, the specific assay design for mtDNA analysis can be used only in a limited fashion to analyze mixtures due to the formation of chimeric mtDNA products.

Studies of DNA transfer have focused largely on the transfer of sloughed off epithelial cells from individuals' hands. This research examines primary, secondary, and tertiary transfer events involving DNA originating from saliva, a commonly encountered body fluid. More routine human behaviors were simulated to evaluate transfer, and the effects of drying time, moisture, and surface composition were investigated. The results agree with previous findings which indicate that the presence of moisture, as well as a smooth nonporous surface as the primary substrate, increases the efficiency of transfer. Previous transfer studies have found that the last individual to come into contact with an item is usually the major contributor to the resulting DNA mixture, unless conditions are simulated in which a "good shedder" serves as the primary depositor and a poor shedder serves as the secondary depositor. The results of this study indicate that when saliva is the source of the transferred DNA, the primary depositor is often the major contributor. These findings suggest that shedder status is less relevant with regard to touch DNA samples in a forensic setting and emphasize the need for caution when analyzing such samples.