Comparison of three massively parallel sequencing platforms for single nucleotide polymorphism (SNP) genotyping in forensic genetics.

Three MPS platforms are being used in forensic genetic analysis, i.e., MiSeq FGx, Ion S5 XL, and MGISEQ-2000. However, few studies compared their performance. In this study, we sequenced 83 common SNPs of 71 samples using the ForenSeq™ DNA Signature Prep Kit on MiSeq FGx, the Precision ID Identity Panel on Ion S5 XL, and the MGIEasy Signature Identification Library Prep Kit on MGISEQ-2000 and then the performance was compared. Results showed that the MiSeq FGx had the highest sequence quality but the lowest sequencing depth and allele balance. Discordant genotypes were observed at six SNPs, which may be caused by variants at primer binding regions, indel errors, or misalignments. Besides, two kinds of background noises, allele-specific miscalled reads (ASMR) and allele-nonspecific miscalled reads (ANMR), were characterized. MGISEQ-2000 showed the highest level of ASMR while Ion S5 XL had the highest level of ANMR. Site- and genotype-dependent miscalled patterns were observed at several SNPs on Ion S5 XL and MGISEQ-2000, but few on MiSeq FGx. In conclusion, the three MPS platforms perform differently with respect to sequencing quality, sequencing depth, allele balance, concordance, and background noise. These findings may be useful for data comparison, mixture deconvolution, and heteroplasmy analysis in forensic genetics.

Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures.

BACKGROUND: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods. RESULTS: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach. CONCLUSIONS: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Massively parallel sequencing of human skeletal remains in Vietnam using the precision ID mtDNA control region panel on the Ion S5™ system.

Mitochondrial DNA (mtDNA) analysis using Sanger sequencing has been a routine practice for the identification of human skeletal remains. However, this process is usually challenging since DNA from the remains is highly degraded and at low concentration. Recently, the advent and implementation of massively parallel sequencing (MPS) have been offered the ability to improve mtDNA sequence data for forensic analysis. To assess the utility of the Ion S5™ system - an MPS platform for mtDNA analysis in challenging samples, we sequenced the mitochondrial control region of 52 age-old skeletal remains. Using the Precision ID mtDNA Control Region Panel, 50 full and two partial control region haplotypes at relatively high mean coverage of 2494 × were achieved for variant calling. Further variant analysis at 10% threshold for point heteroplasmy showed high degradation degree in terms of DNA damage in our bone samples. A higher point heteroplasmy threshold of 20% was required to diminish most of background noise caused by the damage. The results from this study indicated the potential application of the Ion S5™ system in sequencing degraded samples in Vietnam and provided valuable data sources for forensic analyses in the future.

Characterization of sequence variation at 30 autosomal STRs in Chinese Han and Tibetan populations.

Massively parallel sequencing (MPS) technologies have the ability to reveal sequence variations within STR alleles as well as their nominal allele lengths, which have traditionally been detected by CE instruments. Recently, Thermo Fisher Scientific has updated the MPS-STR panel, named the Precision ID GlobalFiler next-generation sequencing (NGS) STR Panel version 2, with primers redesigned to add two pentanucleotide tandem repeat loci and profile interpretation supported by the Converge software. Using the Ion Chef System, the Ion S5XL System, and the Converge software, genetic variations were characterized within STR repeat and flanking regions of 30 autosomal STR markers in 115 unrelated individuals from two Chinese population groups (58 Tibetans and 57 Hans). Nineteen STRs demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. In total, 390 alleles were identified by their sequences compared with 258 alleles that were identified by length. Of these 92 sequence variants found within the STR repeat regions, 40 variants were located in STR flanking regions. Additionally, the agreement of the results with CE data was evaluated, as was the ability of this new MPS panel to analyze case-type (11 samples) and artificially degraded samples (seven samples in triplicate). The results generated from this study illustrate that extensive sequence variation exists in commonly used STR markers in the selected population samples and indicate that this NGS STR panel has the potential to be used as an effective tool for human forensics.

Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success.

Skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.

Implementing a biogeographic ancestry inference service for forensic casework.

The Centre of Forensic Sciences has validated the Precision ID Ancestry Panel on the Ion S5™ Massively Parallel Sequencing instrument for use in forensic casework. The focus of this paper is the development of reporting guidelines for implementation of the biogeographic ancestry inference service based on the Admixture Prediction results produced using the Torrent Suite™ Software (Thermo Fisher Scientific). The Admixture Prediction algorithm estimates the genetic ancestry of a sample using seven root populations (Europe, East Asia, Oceania, America, Africa, South Asia, and Southwest Asia). For individuals that declared a single ancestry, there was a high correlation between the declared ancestry and the ancestry predicted by the algorithm. However, some individuals with declared ancestries of Southern Europe, Southwest Asia, South Asia and Horn of Africa had Admixture Predictions that were composed of two or more root populations at 20% or greater. For individuals with known admixed ancestry, the major component of their declaration was included in their results in all but one case. Based on these results, reporting guidelines were developed and subsequently evaluated using the Admixture Predictions of additional samples. This paper discusses the development and evaluation of these reporting guidelines, along with an implementation plan for forensic casework.

Massively parallel sequencing-enabled mixture analysis of mitochondrial DNA samples.

The mitochondrial genome has a number of characteristics that provide useful information to forensic investigations. Massively parallel sequencing (MPS) technologies offer improvements to the quantitative analysis of the mitochondrial genome, specifically the interpretation of mixed mitochondrial samples. Two-person mixtures with nuclear DNA ratios of 1:1, 5:1, 10:1, and 20:1 of individuals from different and similar phylogenetic backgrounds and three-person mixtures with nuclear DNA ratios of 1:1:1 and 5:1:1 were prepared using the Precision ID mtDNA Whole Genome Panel and Ion Chef, and sequenced on the Ion PGM or Ion S5 sequencer (Thermo Fisher Scientific, Waltham, MA, USA). These data were used to evaluate whether and to what degree MPS mixtures could be deconvolved. Analysis was effective in identifying the major contributor in each instance, while SNPs from the minor contributor's haplotype only were identified in the 1:1, 5:1, and 10:1 two-person mixtures. While the major contributor was identified from the 5:1:1 mixture, analysis of the three-person mixtures was more complex, and the mixed haplotypes could not be completely parsed. These results indicate that mixed mitochondrial DNA samples may be interpreted with the use of MPS technologies.

Recommendations for the successful identification of altered human remains using standard and emerging technologies: Results of a systematic approach.

Successful DNA-based identification of altered human remains relies on the condition of the corpses and varies between tissue types. Therefore, the aim of this prospective multicenter study was to generate evidence-based recommendations for the successful identification of altered remains. For this, 19 commonly used soft and hard tissues from 102 altered human bodies were investigated. The corpses' condition was categorized into three anatomical regions using a practical scoring system. Besides other data, DNA yields, degradation indices, and short tandem repeat (STR) profile completeness were determined in 949 tissue samples. Additionally, varying degrees of alteration and tissue-specific differences were evaluated using the Next Generation Sequencing (NGS) platform MiSeq FGx™. Selected challenging samples were sequenced in parallel with the Ion S5™ platform to assess platform-specific performances in the prediction of the deceased's phenotype and the biogeographic ancestry. Differences between tissue types and DNA extraction methods were found, revealing, for example, the lowest degradation for vertebral disc samples from corpses with initiating, advanced and high degrees of decomposition. With respect to STR profile completeness, blood samples outperformed all other tissues including even profoundly degraded corpses. NGS results revealed higher profile completeness compared to standard capillary electrophoresis (CE) genotyping. Per sample, material and degradation degree, a probability for its genotyping success, including the "extended" European Standard Set (eESS) loci, was provided for the forensic community. Based on the observations, recommendations for the alteration-specific optimal tissue types were made to improve the first-attempt identification success of altered human remains for forensic casework.

Evaluation of a custom GeneRead™ massively parallel sequencing assay with 210 ancestry informative SNPs using the Ion S5™ and MiSeq platforms.

A custom GeneRead DNAseq SNP panel with 210 markers was evaluated using the Ion S5 and MiSeq sequencing platforms. Sensitivity, PCR cycle number, and the use of half volume of reagents for target enrichment and library preparation were tested. Furthermore, genotype concordance between results obtained with the different sequencing platforms and with known profiles generated using other sequencing assays was analysed. The GeneRead DNASeq SNP assay gave reproducible results with an input of 200 pg DNA on both platforms. A total of 204 loci were successfully sequenced. Three loci failed completely in the PCR amplification, and three additional loci displayed frequent locus drop-outs due to low read depth or high heterozygote imbalance. Overall, the read depth across the loci was more well-balanced with the MiSeq, while the heterozygote balance was less variable with the Ion S5. Noise levels were low on both platforms (median< 0.2 %). Two simple criteria for genotyping were applied: A minimum threshold of 45 reads and an acceptable heterozygote balance range of 0.3-3.0. Complete concordance between platforms was observed except for three genotypes in one of the poorly performing loci, rs1470637. This locus had relatively low read depths on both platforms, skewed heterozygote balance, and frequent locus drop-outs. There was also full genotype concordance between the results from the GeneRead assay and known profiles generated with the QIAseq and Ion AmpliSeq assays. The few discordant results were either due to locus drop-outs in the poorly performing loci or allele drop-outs in the QIAseq assay. Profiles with a minimum of 179 SNPs were obtained from four challenging case work samples (blood swabs, bone, or blood from a corpse). Overall, the GeneRead DNASeq assay showed considerable potential and could provide a reliable method for SNP genotyping in cases involving identification of individuals, prediction of phenotypic traits, and ancestry inference.

Evaluation of a hybridization capture-based hereditary cancer panel for the ion semiconductor-based next-generation sequencing system.

BACKGROUND: There is an unmet need to use the ThermoFisher Ion sequencing platform with a hybridization capture-based next-generation sequencing(NGS) panel. However, the analytical performance of combining an ion semiconductor-based sequencing system and a hybridization capture-based NGS panel has not been thoroughly evaluated. Therefore, we compared the analytic performance of Illumina's NextSeq and Ion S5 XL using a hybridization capture-based target enrichment method. METHODS: We included 31 clinical samples and NA12878 reference material for comparing two different NGS instruments using a hybridization capture-based panel. The hybridization-based capture hereditary cancer predisposition (HCP) panel was designed for Ion S5 XL Sequencer and Illumina platforms, respectively. RESULTS: We obtained comparable data involving sequencing run metrics and analytical performance of two different NGS systems using a hybridization-based capture panel designed for each NGS platform. The sensitivity, specificity, and accuracy of single-nucleotide variant and indel calling were 97.06%, 100%, and 100% for the Ion S5™ XL system and 98.53%, 100%, and 100% for NextSeq 550Dx platform, respectively. CONCLUSIONS: This study demonstrated that a hybrid capture panel kit can be successfully implemented using the ThermoFisher Scientific Ion S5 XL instrument and offers the opportunity to select a variety of hybridization-based capture panels from various manufacturers.

Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age.

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille's overall increased gain of DNA when enough tissue is available and Dabney's improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Building a custom large-scale panel of novel microhaplotypes for forensic identification using MiSeq and Ion S5 massively parallel sequencing systems.

A large number of new microhaplotype loci were identified in the human genome by applying a directed search with selection criteria emphasizing short haplotype length (<120 nucleotides) and maximum levels of polymorphism in the composite SNPs. From these searches, 107 autosomal microhaplotypes and 11 X chromosome microhaplotypes were selected, with well-spaced autosomal positions to ensure their independence in relationship tests. The 118 microhaplotypes were assembled into a single multiplex assay for the analysis of forensic DNA with massively parallel sequencing (MPS). A single AmpliSeq-adapted primer set was made for Illumina MiSeq and Thermo Fisher Ion S5 MPS platforms and the performance of the assay was comprehensively evaluated in both systems. Five microhaplotypes showed critical sequencing failures in both MPS platforms and were removed, while a further 13 required manual checks and the application of sequence quality thresholds in one or both systems to ensure the successful analysis of low-level DNA in these loci. The targeting of short microhaplotype spans during marker selection, with an average length of 51 nucleotides in the 118 loci, led to a high level of sensitivity for the panel when sequencing the very degraded DNA typically encountered in forensic casework and the identification of missing persons.

Forensic evaluation of the Asia Pacific ancestry-informative MAPlex assay.

DNA intelligence, and particularly the inference of biogeographical ancestry (BGA) is increasing in interest, and relevance within the forensic genetics community. The majority of current MPS-based forensic ancestry-informative assays focus on the differentiation of major global populations. The recently published MAPlex (Multiplex for the Asia Pacific) panel contains 144 SNPs and 20 microhaplotypes and aims to improve the differentiation of populations in the Asia Pacific region. This study reports the first forensic evaluation of the MAPlex panel using AmpliSeq technology and Ion S5 sequencing. This study reports on the overall performance of MAPlex including the assay's sequence coverage distribution and stability, baseline noise and description of problematic SNPs. Dilution series, artificially degraded and mixed DNA samples were also analysed to evaluate the sensitivity of the panel with challenging or compromised forensic samples. As the first panel to combine biallelic SNPs, multiple-allele SNPs and microhaplotypes, the MAPlex assay demonstrated an enhanced capacity for mixture detection, not easily performed with common binary SNPs. This performance evaluation indicates that MAPlex is a robust, stable and highly sensitive assay that is applicable to forensic casework for the prediction of BGA.

Response of Bacterial and Fungal Soil Communities to Chinese Fir (Cunninghamia lanceolate) Long-Term Monoculture Plantations.

Successive rotation and monoculture, as common silvicultural practices, are extensively applied worldwide, particularly in subtropical Chinese fir (Cunninghamia lanceolata) plantations in southern China. Although regeneration failure and productivity decline are frequently observed in continuous monoculture plantations, the potential mechanisms are still unclear. In this study, high-throughput sequencing was used to compare the diversity and composition of bacterial and fungal communities among different generations of Chinese fir plantation (first rotation, FRP; second rotation, SRP; third rotation, TRP) and natural forest (NF) in December and June. Our results showed significant declines in richness and diversity of bacterial and fungal communities in TRP compared with FRP and SRP, but no significant difference between FRP and SRP. The fungal phyla with high relative abundance were Basidiomycota (12.9-76.9%) and Ascomycota (14.3-52.8%), while the bacterial phyla with high relative abundance were Acidobacteria (39.1-57.7%) and Proteobacteria (21.2-39.5%) in all treatments at both sampling months. On average, the relative abundance of Basidiomycota in TRP increased by 53.4%, while that of Ascomycota decreased by 37.1% compared with FRP and SRP. Moreover, soil NH4 +-N, pH, and DOC appear to be the key factors in shaping the fungal communities, while soil NH4 +-N, DOCN, and AP primarily drive the changes in bacterial communities. Collectively, our findings highlighted the alteration of soil bacterial and fungal communities induced by changes in soil nutrient environment in different generations of continuously cultivated Chinese fir plantation.

Population genetic data of 74 microhaplotypes in four major U.S. population groups.

Microhaplotypes (microhaps or MHs) are novel forensically relevant genetic markers that demand large and appropriate allele frequency datasets for their implementation in casework. In this study we report on the allele frequency data of 74 microhap loci (230 SNPs) included in a newly developed 74-plex assay. The panel was tested on the Ion S5 system on a total of 347 samples from four main U.S. population groups of African, European, East Asian and Southwest Hispanic descent. Overall, frequencies of individual alleles at each locus varied considerably among the different population groups. An increase in the average value of gene diversity was also observed as the number of SNPs per locus increased. Most microhap markers showed no significant deviation from Hardy-Weinberg ratios within any of the individual population samples displaying an average power of discrimination between 0.74 and 0.81 and an average probability of exclusion between 0.32 and 0.39. Moreover, the four population groups had no clear genetic affinities with the exception of U.S. European and U.S. Southwest Hispanic populations, which showed the lowest FST value. STRUCTURE and principal component analyses (PCA) analysis resulted in effective clustering of the four populations with the U.S. European and Southwest Hispanic showing some overlap. These results support the potential use of this sequence-based 74plex-microhaplotype assay for ancestry inference in addition to previously reported human identification and mixture deconvolution capabilities.

Development and validation of the VISAGE AmpliSeq basic tool to predict appearance and ancestry from DNA.

Forensic DNA phenotyping is gaining interest as the number of applications increases within the forensic genetics community. The possibility of providing investigative leads in addition to conventional DNA profiling for human identification provides new insights into otherwise "cold" police investigations. The ability of reporting on the bio-geographical ancestry (BGA), appearance characteristics and age based on DNA obtained from a crime scene sample of an unknown donor makes the exploration of such markers and the development of new methods meaningful for criminal investigations. The VISible Attributes through GEnomics (VISAGE) Consortium aims to disseminate and broaden the use of predictive markers and develop fully optimized and validated prototypes for forensic casework implementation. Here, the first VISAGE appearance and ancestry tool development, performance and validation is reported. A total of 153 SNPs (96.84 % assay conversion rate) were successfully incorporated into a single multiplex reaction using the AmpliSeq™ design pipeline, and applied for massively parallel sequencing with the Ion S5 platform. A collaborative effort involving six VISAGE laboratory partners was devised to perform all validation tests. An extensive validation plan was carefully organized to explore the assay's overall performance with optimum and low-input samples, as well as with challenging and casework mock samples. In addition, forensic validation studies such as concordance and mixture tests recurring to the Coriell sample set with known genotypes were performed. Finally, inhibitor tolerance and specificity were also evaluated. Results showed a robust, highly sensitive assay with good overall concordance between laboratories.

Developmental validation of a custom panel including 165 Y-SNPs for Chinese Y-chromosomal haplogroups dissection using the ion S5 XL system.

Massively parallel sequencing (MPS) technology has the capability to genotype hundreds to thousands of SNPs from multiple samples, with high coverage in a single experimental run. MPS, which enables high-resolution Y haplogrouping from limited DNA resources, has attracted much interest in Y-chromosomal applications, particularly human forensics and anthropological studies. Previously, we selected and designed a 72 Y-SNPs MPS panel aiming at the Chinese population for providing the basic structure of the haplogroup tree, and then additional Y-SNPs were screened out for further subdivision into different populations by using pyrosequencing technology. In the present study, we designed a customized MPS panel that includes 165 Y-SNPs based on our previous studies and evaluated the sequencing performance, sensitivity and the ability of the MPS-SNP panel to analyze case-type samples using the Ion S5 XL System. In the concordant study, only four Y-SNPs presented missing genotypes due to low coverage reads, and five SNPs were prone to being designated heterozygotes, whereas the designation of the other SNPs were fully consistent with the results obtained from other Y-SNP typing tools. The analyst-interpreted Y-SNP profiles demonstrated that the targeted Y-SNPs, except for the nine poorly performing Y-SNPs, performed well and could acquire achievable high-resolution. Moreover, this MPS panel could be adapted to case-type samples, and single source complete profiles could be stably obtained using 1 ng of input DNA. In summary, this MPS Y-SNP panel offers a straightforward sample-to-haplogroup workflow that would be beneficial for paternal lineage classification and forensic pedigree searches, and the present results provide support for the application of MPS technology in SNP analysis.

Utility of the Ion S5™ and MiSeq FGx™ sequencing platforms to characterize challenging human remains.

Often in missing persons' and mass disaster cases, the samples remaining for analysis are hard tissues such as bones, teeth, nails, and hair. These remains may have been exposed to harsh environmental conditions, which pose challenges for downstream genotyping. Short tandem repeat analysis (STR) via capillary electrophoresis (CE) is still the gold standard for DNA typing; however, a newer technology known as massively parallel sequencing (MPS) could improve upon our current techniques by typing different and more markers in a single analysis, and consequently improving the power of discrimination. In this study, bone and tooth samples exposed to a variety of DNA insults (cremation, embalming, decomposition, thermal degradation, and fire) were assessed and sequenced using the Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System, and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™ system, as well as the GlobalFiler™ PCR Amplification Kit on the 3500™ Genetic Analyzer. The results demonstrated that using traditional CE-based genotyping performed as expected, producing a partial or full DNA profile for all samples, and that both sequencing chemistries and platforms were able to recover sufficient STR and SNP information from a majority of the same challenging samples. Run metrics including profile completeness and mean read depth produced good results with each system, considering the degree of damage of some samples. Most sample insults (except decomposed) produced similar numbers of alleles for both MPS systems. Comparable markers produced full concordance between the two platforms.

Pilot study for forensic evaluations of the Precision ID GlobalFiler™ NGS STR Panel v2 with the Ion S5™ system.

With the continuous development of massively parallel sequencing (MPS), increasing numbers of laboratories have utilized this method for forensic genomic analyses. When sequencing common short tandem repeats (STRs), MPS does have many advantages over the length-based genotyping method that uses traditional capillary electrophoresis (CE) technology. The Precision ID GlobalFiler™ NGS STR Panel v2 was recently released to simultaneously target 31 autosomal STRs (20 expanded Combined DNA Index System (CODIS) core loci and 11 non-CODIS loci) and 4 gender determination loci (Amelogenin, DYS391, SRY and Y-indel (rs2032678)) with the Ion S5™ System. In the current study, we performed a preliminary validation for this novel MPS-STR panel that included the following analyses: repeatability, concordance, stutter and balance, sensitivity, case-type sample testing, stability, mixture and a population investigation. Complete and reliable profiles were obtained using 125 pg of positive control DNA. The commonly encountered types of case samples and artificial mixtures with ratios of 1:1, 1:3 and 3:1 were also fully genotyped. Additional allele sequence variations were detected in samples from 50 unrelated individuals, and subsequently, an increased power of discrimination and power of exclusion were achieved. However, the average depth of coverage (DoC) of the Penta D locus was detected to be dramatically lower than those of other loci, which caused an interlocus imbalance; this could be one of the reasons for the intralocus imbalance of this locus and the 0.18% inconsistent results in the concordance study. Although certain flaws were observed, the informative metrics, including the DoC, sequence coverage ratios (SCRs) and heterozygote balance (Hb), of the novel MPS multiplex in our study were sufficient for reliable sequencing results that were 99.61% in concordance with the capillary electrophoresis (CE) results. In general, the Precision ID GlobalFiler™ NGS STR Panel v2 was demonstrated to be sensitive, reliable and robust and could be a powerful tool for human identification and kinship analyses. Additionally, we look forward to its updated version.

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems.

Next-generation sequencing (NGS) has been used to genotype forensic short tandem repeat (STR) markers for individual identification and kinship analysis. STR data from several NGS platforms have been published, but forensic application trials using the Ion S5™ XL system have not been reported. In this work, we report sensitivity, reproducibility, mixture, simulated degradation, and casework sample data on the Ion Chef™ and S5™ XL systems using an early access 25-plex panel. Sensitivity experiments showed that over 97% of the alleles were detectable with down to 62 pg input of genomic DNA. In mixture studies, alleles from minor contributors were correctly assigned at 1:9 and 9:1 ratios. NGS successfully gave 12 full genotype results from 13 challenging casework samples, compared with five full results using the CE platform. In conclusion, the Ion Chef™ and the Ion S5™ XL systems provided an alternative and promising approach for forensic STR genotyping.