Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90–95% of SNP genotypes could be obtained from 25 to 100 pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user’s control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.     

Next generation sequencing of SNPs using the HID-Ion AmpliSeq™ Identity Panel on the Ion Torrent PGM™ platform

The HID-Ion AmpliSeq™ Identity Panel (the HID Identity Panel) is designed to detect 124-plex single nucleotide polymorphisms (SNPs) with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 90 individual identification SNPs (IISNPs) on autosomal chromosomes and 34 lineage informative SNPs (LISNPs) on Y chromosome. In this study, we evaluated performance for the HID Identity Panel to provide a reference for NGS-SNP application, focusing on locus strand balance, locus coverage balance, heterozygote balance, and background signals. Besides, several experiments were carried out to find out improvements and limitations of this panel, including studies of species specificity, repeatability and concordance, sensitivity, mixtures, case-type samples and degraded samples, population genetics and pedigrees following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. In addition, Southern and Northern Chinese Han were investigated to assess applicability of this panel. Results showed this panel led to cross-reactivity with primates to some extent but rarely with non-primate animals. Repeatable and concordant genotypes could be obtained in triplicate with one exception at rs7520386. Full profiles could be obtained from 100 pg input DNA, but the optimal input DNA would be 1 ng–200 pg with 21 initial PCR cycles. A sample with ≥20% minor contributor could be considered as a mixture by the number of homozygotes, and full profiles belonging to minor contributors could be detected between 9:1 and 1:9 mixtures with known reference profiles. Also, this assay could be used for case-type samples and degraded samples. For autosomal SNPs (A-SNPs), F_ST across all 90 loci was not significantly different between Southern and Northern Chinese Han or between male and female samples. All A-SNP loci were independent in Chinese Han population. Except for 18 loci with H_e <0.4, most of the A-SNPs in the HID Identity Panel presented high polymorphisms. Forensic parameters were calculated as >99.999% for combined discrimination power (CDP), 0.999999724 for combined power of exclusion (CPE), 1.390 × 10¹¹ for combined likelihood ratio (CLR) of trios, and 2.361 × 10⁶ for CLR of motherless duos. For Y-SNPs, a total of 8 haplotypes were observed with the value of 0.684 for haplotype diversity. As a whole, the HID Identity Panel is a well-performed, robust, reliable and high informative NGS-SNP assay and it can fully meet requirements for individual identification and paternity testing in forensic science.     

Evaluation of the Early Access STR Kit v1 on the Ion Torrent PGM™ platform

The Early Access STR Kit v1 is designed to detect 25-plex loci with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 16 of 20 expanded Combined DNA Index System (CODIS) core loci (CSF1PO, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D13S317, D16S539, D19S433, D21S11, TH01, TPOX and vWA), 8 non-CODIS core loci (D1S1677, D2S1776, D4S2408, D5S2500.AC008791, D6S1043, D6S474, D9S2157 and D14S1434) and Amelogenin. In this study, we compared the Early Access STR Kit v1 with the Ion Torrent™ HID STR 10-plex to find out its improvements and explored an appropriate analytical threshold to enhance the performance. In addition, seven experiments were conducted to evaluate the Early Access STR Kit v1 such as studies of repeatability, concordance, sensitivity, mixtures, degraded samples, case-type samples and pedigrees. Other than a little discordance (0.95%) with CE-STR results observed at D21S11, NGS-STR results correctly reflected the sample being tested. Repeatable results were obtained from both initial PCRs and emPCRs aside from a few variations of allele coverage. Full profiles could be obtained from 100 pg input DNA and >48.84% profiles from 10 pg input DNA. Mixtures were easily detected at 9:1 and 1:9 ratios. This system could be adapted to case-type samples and degraded samples. As a whole, the Early Access STR Kit v1 is a robust, reliable and reproducible assay for NGS-STR typing and a potential tool for human identification.     

Characterization of microRNA expression profiles in blood and saliva using the Ion Personal Genome Machine® System (Ion PGM™ System)

MicroRNA (miRNA) expression profiling is gaining interest in the forensic community because the intrinsically short fragment and tissue-specific expression pattern enable miRNAs as a useful biomarker for body fluid identification. Measuring the quantity of miRNAs in forensically relevant body fluids is an important step to screen specific miRNAs for body fluid identification. The recent introduction of massively parallel sequencing (MPS) has the potential for screening miRNA biomarkers at the genome-wide level, which allows both the detection of expression pattern and miRNA sequences. In this study, we employed the Ion Personal Genome Machine^® System (Ion PGM™ System, Thermo Fisher) to characterize the distribution and expression of 2588 human mature miRNAs (miRBase v21) in 5 blood samples and 5 saliva samples. An average of 1,885,000 and 1,356,000 sequence reads were generated in blood and saliva respectively. Based on miRDong, a Perl-based tool developed for semi-automated miRNA distribution designations, and manually ascertained, 6 and 19 miRNAs were identified respectively as potentially blood and saliva-specific biomarkers. Herein, this study describes a complete and reliable miRNA workflow solution based on Ion PGM™ System, starting from efficient RNA extraction, followed by small RNA library construction and sequencing. With this workflow solution and miRDong analysis it will be possible to measure miRNA expression pattern at the genome-wide level in other forensically relevant body fluids.     

Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software—An automatic NGS-STR workflow from DNA to profile for forensic science

The Ion Torrent ™ Genexus ™ Sequencer (Genexus) is a highly integrated instrument that can automate library construction, templating, and sequencing in a single-instrument run. By programing the ForeNGS Analysis Software (FNAS), we bridged the gap between sequencing and genotyping without manual intervention. FNAS can automatically transfer sequencing output files from Genexus, analyze the repeat and flanking regions aligned to the GRCh38 assembly, name the alleles according to the ISFG guidelines, and generate user-friendly interactive profiles. Genexus and FNAS can accomplish the fully automatic DNA-to-Profile workflow in forensics. Based on our experiences, the optimal assay parameters on Genexus were validated as follows: 24 cycles of target amplification for library construction; 40 μL of library and 400 bp of template size for templating; 852 flows of dNTPs by order of Ion samba HID2 for sequencing; and 750,000 reads per sample at minimum for 16 samples multiplexed on a lane. By developmental validations of the Precision ID Globalfiler ™ NGS STR Panel v2, Genexus presented competitive performance at the optimal assay parameters qualified to detect commonly used forensic STR markers. It could produce repeatable and reproducible results, and human profiles could be easily separated from nonhuman profiles. Additionally, Genexus was sensitive enough to detect samples with 100 pg of input DNA, and it was suitable for various types of case samples, especially for low copy number samples and degraded samples. Moreover, minor contributors could be detected between the 4:1 and 1:4 mixtures with an analysis threshold of 50 × . The Genexus workflow is a robust and labor-effective solution enabling forensic scientists to obtain NGS-STR profiles within a single day and with only the need to prepare DNA extracts, then set up Genexus, and finally interpret profiles on FNAS.     

Phylogenetic and forensic studies of the Southeast Florida Hispanic population using the next-generation forensic PowerPlex® Y23 STR marker system

The purpose of this study is to examine the robustness and sensitivity of the newly available Y-STR multiplex kit, the PowerPlex® Y23 System, by comparing our data at the 23-loci level to the routinely used 17 loci provided by the AmpFlSTR® Yfiler® PCR Amplification kit. For the first time, allelic and genotypic frequencies for the 23 Y-STR loci included in the PowerPlex® Y23 System are provided for the Southeast Florida Hispanic (SFH) population. In addition, we have characterized the SFH population in terms of intra-population and inter-population parameters. We also compared these indices of forensic and population genetics interest in the SFH population to comparable data of previously published populations to assess their phylogenetic relationships. Our 23-loci data was shown to provide more discriminatory values as compared to the data when using only 17 loci. Also, the R_ST distance values demonstrate the superior capacity of the PowerPlex® Y23 system to discriminate among populations.     

Developmental validation of STRmix™ NGS,a probabilistic genotyping tool for the interpretation of autosomal STRs from forensic profiles generated using NGS

We describe the developmental validation of the probabilistic genotyping software – STRmix™ NGS – developed for the interpretation of forensic DNA profiles containing autosomal STRs generated using next generation sequencing (NGS) also known as massively parallel sequencing (MPS) technologies. Developmental validation was carried out in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Guidelines for the Validation of Probabilistic Genotyping Systems and the International Society for Forensic Genetics (ISFG) recommendations and included sensitivity and specificity testing, accuracy, precision, and the interpretation of case-types samples. The results of developmental validation demonstrate the appropriateness of the software for the interpretation of profiles developed using NGS technology.     

Evaluation of forensic genetic parameters of 12 STR loci in the Korean population using the InvestigatorⓇ HDplex kit

We genotyped and calculated the forensic parameters of 10 non-CODIS loci and 2 CODIS loci of 990 Korean individuals using the Investigator^Ⓡ HDplex kit. No significant deviations from Hardy–Weinberg equilibrium (after Bonferroni correction for multiple testing) or genetic linkage disequilibrium were observed. The calculated matching probability and power of discrimination ranged from 0.0080 to 0.2014, and 0.7986 to 0.9920, respectively. We conclude that the markers of the kit are highly informative corroborative tools for forensic DNA analysis.

     

An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA

Damage to the DNA molecule can occur through exposure to environmental conditions such as ultraviolet light, heat and humidity. Forensic samples are particularly prone to such damage due to their prolonged exposure after deposition at crime scenes or mass disasters. Current methods for typing such samples rely heavily on the intact DNA template, and can be adversely affected by damage that is present. Proposed solutions center around increased access to the smaller remaining fragments and/or increased sensitivity. However, all rely on the polymerase chain reaction to copy the starting material; the required polymerase can be impeded by certain types of damage such as dimer-formation after ultraviolet light exposure, resulting in stochastic effects that can complicate interpretation. In vitro repair of such damage offers the ability to generate high quality profiles using traditional methods without changes to the current amplification reagents or conditions. Typically, repair reactions required large quantities of starting material and a separate repair reaction. Forensic samples, however, usually consist of small quantities, and quality control measures necessitate laboratory procedures that minimize sample manipulation. Here, an optimized protocol for forensic application of the PreCR? Repair Mix to current typing methods is demonstrated for samples damaged by ultraviolet light exposure.     

Developmental validation of the Microreader™ RM-Y ID System: a new rapidly mutating Y-STR 17-plex system for forensic application

International Journal of Legal Medicine - Y-chromosomal short tandem repeats (Y-STRs) are widely applied to evolutionary, genealogical, and kinship analyses of male linages in forensic studies, but...     

Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software

Massively parallel sequencing (MPS) technologies enable the simultaneous analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). MPS also enables the detection of alleles of the minor contributors in imbalanced DNA mixtures. In this study, 59 STRs (amelogenin, 27 autosomal STRs, 7 X-STRs, and 24 Y-STRs) and 94 identity-informative SNPs of 119 unrelated Taiwanese (50 men, 69 women) were sequenced using a commercial MPS kit. Forty-eight nondegraded and 44 highly degraded two-person artificial DNA mixtures with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, and 1:99) were analyzed to examine the performance of this system for detecting the alleles of the minor contributors in DNA mixtures. Likelihood ratios based on continuous model were calculated using the EuroForMix for DNA mixture interpretation. The STR and SNP genotypes of these 119 Taiwanese were obtained. Several sequence variants of STRs were observed. Using EuroForMix software based on the sequence data of autosomal STRs and autosomal SNPs, 97.9% (47/48) and 97.7% (42/43) of minor donors were accurately inferred among the successfully analyzed nondegraded and degraded DNA mixtures, respectively. In conclusion, combined with EuroForMix software, this commercial kit is effective for assignment of the minor contributors in nondegraded and degraded DNA mixtures.

     

Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.     

Performance of two 17 locus forensic identification STR kits-Applied Biosystems's AmpFℓSTR® NGMSElect™ and Promega's PowerPlex® ESI17 kits

We compared the performance of two recently released 17 loci STR multiplexes for human identification: Applied Biosystems's AmpF?STR^® NGMSElect? and Promega's PowerPlex^® ESI17. The comparative parameters were chosen by their relevance in forensic identification and particularly in crime cases. The comparative analyses encompass: amplification ability, heterozygote balance, allelic drop-out, drop-in, stutter analysis and inter-locus balance.Four DNA profiles were analysed in various concentrations in a serial dilution experiment. The amounts of DNA in the PCR ranged from 3 pg to 420 pg and were analysed in triplicate using 28, 29 and 30 PCR cycles. In order to compare the kits, aliquots from each sample were analysed with both kits under identical conditions. Furthermore, DNA profiles from 200 reference profiles were analysed using both kits.The results from the statistical analyses did not indicate any substantial differences of practical relevance between the kits for forensic case work. For all parameters included in this comparative study, the two kits showed no departure from previously observed patterns relative to e.g. the amounts of DNA or amplicon lengths. Based on our analyses, both kits are considered applicable for forensic crime case work.     

Dosimetry of the microSelectron-HDR Ir-192 source using PRESAGE™ and optical CT

Optical CT, using a solid polyurethane (PRESAGE™) radiochromic dosimeter, has been used to evaluate dose distributions produced by the microSelectron-HDR Ir-192 source. The anisotropy functions obtained through optical CT are in good agreement with Monte Carlo and previously published results especially at polar angle above 20°. The results indicated an evident potential for using solid polymer dosimetry as an accurate method for 3-D dosimetry, although refinements to the existing methods are necessary before the technique can be used clinically.     

Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens

Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1 ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system.     

Massively parallel sequencing for the forensic scientist – sequencing archived amplified products of AmpFlSTR Identifiler and PowerPlex Y multiplex kits to capture additional information

In the last few years the cost and ease of massively parallel sequencing (MPS) has reduced dramatically to the point that it can now be considered as a tool for use in forensic case work. An important consideration for the implementation of any new forensic technology is the ability to remain compatible with previous technology. During this study we sequenced the amplicons of two commercial forensic short tandem repeat (STR) multiplexes AmpFlSTR Identifiler and PowerPlex Y using the Illumina MiSeq and Ion PGM Sequencer (Life Technologies) and characterised the sequence data from a forensic perspective. Using the MPS data from both platforms we determined the STR genotypes of forensic samples and found previously undocumented sequence variation in seven STR alleles. By characterising features of the DNA sequence profiles, such as stutter and locus imbalance we identified areas for future development that will be needed prior to casework implementation. The rapid development of this technology has meant many in the forensic community have been ‘left behind’. We also provide an explanation, for forensic scientists, of what is happening at the different stages of the MPS workflow, from library preparation through to bioinformatics, and how this may affect the results.     

Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct systems

Short tandem repeat (STR) analysis remains the primary forensic tool for DNA identification. Because of the success of forensic DNA typing and the use of database searches to develop investigative leads, there is an increased demand for populating forensic DNA databases. Reference samples tend to be of high quantity and quality and are somewhat standardized in format. Being more predictable in quality than unknown forensic casework samples, reference samples lend themselves to alternate methods of analysis such as direct amplification. Two commercially available direct amplification kits for processing reference samples were evaluated. The kits are PowerPlex^® 18D (Promega Corp., Madison, Wisconsin) and Identifiler^® Direct (Life Technologies, Carlsbad, CA). Both kits offer the core CODIS loci plus amelogenin, and the loci D2S1338, D19S433. The PP18D kit offers two additional loci, Penta E and Penta D. To determine the robustness and reliability of the PP18D and ID Direct amplification systems, buccal cell samples (deposited on FTA paper using the EasiCollect? device (Florham Park, NJ)) from 400 anonymous donors were analyzed under conditions to achieve a high rate of successful typing. First-pass success rates were 96.25%, 96.25%, and 95% for PP18D with a 5 s injection, ID Direct with a 10 s injection, and ID Direct with a 5 s injection, respectively. Profiles that could not be typed were not a result of the kits’ performance but were a result of the inherent variation in the amount of DNA obtained with the collection device and buccal cells. Low signal profiles can be re-analyzed by either re-injecting for a longer time or by re-amplification with an additional PCR cycle. Overloaded profiles can be re-analyzed by re-injecting for a shorter time or by re-amplification with one less cycle. All called typing results, when interpretable, were consistent under the prescribed conditions, different injection times, and 26-28 PCR cycles for both chemistries. Peak height ratios for both kits were well balanced with peaks ranging in height >2000 RFUs to those with one or more peaks with heights <100 RFUs. A change in the ILS morphology sloping downward to the right relative to a normal ILS profile for PP18D and ID Direct was an indication of a poor injection. Re-injection effectively overcame the effect manifested by a sloping ILS phenomenon. A subset of samples were subjected to direct amplification using the reagents in Identifiler^® Plus kit and successful typing results were obtained for the majority of samples. However, the profiles displayed increased amounts of non-adenylated products. The results of this study demonstrate that PP18D and ID Direct are both robust kits for direct amplification. The interpretation guidelines used for this study can form a basis for internal validation studies by databasing laboratories.