ForenSeq DNA Signature Prep Kit Research Articles

Performance and characterization of 94 identity-informative SNPs in Northern Han Chinese using ForenSeq ™ DNA signature prep kit

Target and flanking region (FR) variation at 94 identity-informative SNPs (iSNPs) are investigated in 635 Northern Han Chinese using the ForenSeq DNA Signature Prep Kit on the MiSeq FGx Forensic Genomics System. The dataset presents the following performance characteristics (average values): ≥60% bases with a quality score of 20 or higher (%≥ Q20); >700 × of depth of coverage (DoC) from both Sample Details Reports and Flanking Region Reports; >80% of effective reads; ≥60% of allele coverage ratio (ACR); and ≥70% of inter-locus balance, while some stable low-performance characteristics are also observed: low DoC at rs1736442, rs1031825, rs7041158, rs338882, rs2920816, rs1493232, rs719366, and rs2342747; high noise at rs891700; and imbalanced ACR at rs6955448 and rs338882. The average amplicon length is 69 bp, suitable for detecting degraded samples. Bioinformatic concordance achieves 99.99% between the ForenSeq Universal Analysis Software (UAS) and the Integrative Genomic Viewer (IGV) inspection. Discordance results from flanking region deletions of rs10776839, rs8078417, rs2831700, and rs1454361. Due to FR variants within amplicons detected by massively parallel sequencing (MPS), the increases in the number of unique alleles, effective alleles (Ae), and observed heterozygosity (Hobs) are 46.81%, 4.51%, and 3.29%, respectively. Twelve FR variants are first reported to dbSNP, such as rs1252699848, rs1665500714, rs1771121532, rs2097285015, rs1851671415, rs2045669877, rs2046758811, rs2044248635, rs1251308240, rs1968822112, rs1981638299, and rs1341756746. All 94 iSNPs from target and amplicon data are in Hardy-Weinberg equilibrium (HWE) and independent within autosomes. As expected, forensic parameters from the amplicon data increase significantly on the combined power of discrimination (CPD = 1 – 3.9876 × 10−38) and the combined power of exclusion (CPE = 1 – 6.6690 × 10−8). Additionally, the power of the system effectiveness (CPD = 1 − 6.7054 × 10−72 and CPE = 1 − 4.4719 × 10−20) with sequence-based 27 autosomal STRs and 94 iSNP amplicons in combination is substantially improved compared to one type of marker alone. In conclusion, we have established a traditional length-based and current sequence-based reference database with 58 STRs and 94 iSNPs in the Northern Han Chinese population. We hope these data can serve as a solid reference and foundation for forensic practice.

Analysis of homozygous allele mismatches in paternity tests with massively parallel sequencing.

In paternity testing, short tandem repeats (STRs) allele mismatches are often detected. Nowadays, polymerase chain reaction- and capillary electrophoresis (CE)-based STR genotyping is the most commonly used method to distinguish alleles based on their length. However, it could not detect alleles of the same size with sequence differences. Massively parallel sequencing (MPS) can determine not only allele sizes but also sequences, which could explain the causes of allele mismatches. Additionally, more types of genetic markers can be detected in a single assay, which increases the discriminatory power and facilitates the analysis of paternity tests. In this study, we analyzed 11 cases with homozygous allele mismatches from routine DNA trio paternity tests using the CE platform. Samples were sequenced using the ForenSeq DNA Signature Prep Kit and the MiSeq FGx Sequencing System. The results show that of the eight father-child mismatch cases and three mother-child mismatch cases, five cases with D5S818 and D8S1179 and one case at D13S317 were classified as non-amplification. The other three cases and two cases could be defined as mutations. This study suggests that MPS-based STR genotyping can provide additional information that allows more accurate interpretation of allelic mismatches in paternity testing.

Forensic identity SNPs: Characterisation of flanking region variation using massively parallel sequencing.

Single nucleotide polymorphisms (SNPs) can be analysed for identity or kinship applications in forensic genetics to either provide an adjunct to traditional STR typing or as a stand-alone approach. The advent of massively parallel sequencing technology (MPS) has provided a useful opportunity to more easily deploy SNP typing in a forensic context, given the ability to simultaneously amplify a large number of markers. Furthermore, MPS also provides valuable sequence data for the targeted regions, which enables the detection of any additional variation seen in the flanking regions of amplicons. In this study we genotyped 977 samples across five UK-relevant population groups (White British, East Asian, South Asian, North-East African and West African) for 94 identity-informative SNP markers using the ForenSeq DNA Signature Prep Kit. Examination of flanking region variation allowed for the identification of 158 additional alleles across all populations studied. Here we present allele frequencies for all 94 identity-informative SNPs, both including and excluding the flanking region sequence of these markers. We also present information on the configuration of these SNPs in the ForenSeq DNA Signature Prep Kit, including performance metrics for the markers and investigation of bioinformatic and chemistry-based discordances. Overall, the inclusion of flanking region variation in the analysing workflow for these markers reduced the average combined match probability 2175 times across all populations, with a maximum reduction of 675,000-fold in the West African population. The gain due to flanking region-based discrimination increased the heterozygosity of some loci above that of some of the least useful forensic STR loci; thus demonstrating the benefit of enhanced analysis of currently targeted SNP markers for forensic applications.

High-resolution genotyping of 58 STRs in 635 Northern Han Chinese with MiSeq FGx ® Forensic Genomics System

Sequence polymorphisms were characterized at 27 autosomal STRs (A-STRs), 7 X chromosomal STRs (X-STRs), and 24 Y chromosomal STRs (Y-STRs) in 635 Northern Han Chinese with the ForenSeq DNA Signature Prep Kit on the MiSeq FGx Forensic Genomics System. Since repeat region (RR) and flanking region (FR) variation can be detected by massively parallel sequencing (MPS), the increase in the number of unique alleles and the average of gene diversity was 78.18% and 3.51% between sequence and length, respectively. A total of 74 novel RR variants were identified at 33 STRs compared with STRSeq and previous studies, and 13 FR variants (rs1770275883, rs2053373277, rs2082557941, rs1925525766, rs1926380862, rs1569322793, rs2051848492, rs2051848696, rs2016239814, rs2053269960, rs2044518192, rs2044536444, and rs2089968964) were first submitted to dbSNP. Also, 99.94% of alleles were concordant between the ForenSeq DNA Signature Prep Kit and commercial CE kits. Discordance resulted from the low performance at D22S1045 and occasionally at DYS392, flanking region deletions at D7S820 and DXS10074, and the strict alignment algorithm at DXS7132. Null alleles at DYS505 and DYS448 and multialleles at DYS387S1a/b, DYS385a/b, DYS448, DYS505, DXS7132, and HPRTB were validated with other MPS and CE kits. Thus, a high-resolution sequence-based (SB) and length-based (LB) allele frequencies dataset from Northern Han Chinese has been established already. As expected, forensic parameters increased significantly on combined power of discrimination (PD) and combined power of exclusion (PE) at A-STRs, mildly on combined PD and combined mean exclusion chance (MEC) at X-STRs, and barely on discrimination capacity (DC) at Y-STRs. Additionally, MiSeq FGx quality metrics and MPS performance were evaluated in this study, which presented the high-quality of the dataset at 20 consecutive runs, such as ≥ 60% bases with a quality score of 20 or higher (%≥ Q20), > 60% of effective reads, > 2000 × of depth of coverage (DoC), ≥ 60% of allele coverage ratio (ACR) or heterozygote balance, ≥ 70% of inter-locus balance, and ≤ 0.4 of the absolute value of observed minus expected heterozygosity (|Hexp – Hobs|). In conclusion, MiSeq FGx can help us generate a high-resolution and high-quality dataset for human identification and population genetic studies.

Variability of 27 Autosomal STR Loci for the Population of the Republic of Belarus Based on the Mass Parallel Sequencing Data

Variability of 27 autosomal STR loci of the ForenSeq DNA Signature Prep Kit (Illumina) commercial panel was studied using the technology of mass parallel sequencing (MPS) in 733 unrelated individuals representing the population of the Republic of Belarus as well as a population base of MPS allele frequencies for expert probabilistic calculations in human identification and paternity establishment was evaluated. The agreement between genotypes obtained by MPS and capillary electrophoresis (CE) was 99.96%. The number of MPS alleles increased more than two times for eight loci (D12S391, D21S11, D2S1338, vWA, D3S1358, D8S1179, D13S317, D9S1122). Thirteen alleles detected were not included in the STRSeq BioProject catalog of the international online database STRbase 2.0. The random match probability of 27-locus MPS profiles decreased from 1.43 × 10–31 to 2.89 × 10–35, and the combined paternity index increased from 2.08 × 1010 to 3.25 × 1012 compared to CE data.

Population data for 94 identity SNPs in four U.S. population groups

The U.S. National Institute of Standards and Technology (NIST) sequenced 1036 human DNA samples from four United States population groups (African American, Asian, Hispanic, and Caucasian) using the ForenSeq DNA Signature Prep Kit with Primer Mix B (DPMB) on a MiSeq FGx instrument. In addition to STR markers, DPMB includes amplification primers for single nucleotide polymorphisms (SNPs) used for individual identification (iiSNPs, n = 94), ancestry inference (aiSNPs, n = 56), and phenotype prediction (piSNPs, n = 22). Resulting sequencing coverage information was interpreted for the 94 iiSNP markers. Here we present performance characteristics of the ForenSeq DNA Signature Prep Kit in the population studied.

Advancing FDSTools by integrating STRNaming 1.1

The introduction of massively parallel sequencing in forensic analysis has been facilitated with typing kits, analysis software and allele naming tools such as the ForenSeq DNA Signature Prep (DSP) kit, FDSTools and STRNaming respectively. Here we describe how FDSTools 2.0 with integrated and refined STRNaming nomenclature was validated for implementation under ISO 17025 accreditation for the ForenSeq DSP kit. Newly-added options result in efficient automatic allele calling for the majority of markers while specific settings are applied for ‘novel’ sequence variants to avoid the calling of remaining variable noise observed in samples sequenced with the ForenSeq DSP kit that seem to arise in the PCR. Genome-wide built-in reference data allows for greatly simplified configuration of allele naming for human targets.

Evaluation of the VISAGE basic tool for appearance and ancestry inference using ForenSeq® chemistry on the MiSeq FGx® system

The possibility of providing investigative leads when conventional DNA identification methods fail to solve a case can be of extreme relevance to law enforcement. Therefore, the forensic genetics community has focused research towards the broadened use of DNA, particularly for prediction of appearance traits, bio-geographical ancestry and age. The VISible Attributes through GEnomics (VISAGE) Consortium expanded the use of DNA phenotyping by developing new molecular and statistical tools for appearance, age and ancestry prediction. The VISAGE basic tool for appearance (EVC) and ancestry (BGA) prediction was initially developed using Ampliseq chemistry, but here is being evaluated using ForenSeq chemistry. The VISAGE basic tool offers a total of 41 EVC and 115 BGA SNPs and thus provides more predictions, i.e., skin color, than achieved with the ForenSeq DNA Signature Prep kit that is based on 24 EVC and 56 BGA SNPs. Five VISAGE laboratories participated in collaborative experiments to provide foreground for developmental validation of the assay. Assessment of assay performance and quality metrics, reproducibility, sensitivity, inhibitor tolerance and species specificity are described. Furthermore, the assay was tested using challenging samples such as mock casework samples and artificially degraded DNA. Two different analysis strategies were applied for this study and output on genotype calls and read depth was compared. Overall, inter-laboratory, inter-method and concordance with publicly available data were analysed and compared. Finally, the results showed a reliable and robust tool, which can be easily applied for laboratories already using a MiSeq FGx with ForenSeq reagents.

A multi-dimensional evaluation of the ‘NIST 1032’ sample set across four forensic Y-STR multiplexes

This manuscript reports Y-chromosomal short tandem repeat (Y-STR) haplotypes for 1032 male U.S. population samples across 30 Y-STR loci characterized by three capillary electrophoresis (CE) length-based kits (PowerPlex Y23 System, Yfiler Plus PCR Amplification Kit, and Investigator Argus Y-28 QS Kit) and one sequence-based kit (ForenSeq DNA Signature Prep Kit): DYF387S1, DYS19, DYS385 a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS505, DYS518, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS627, DYS635, DYS643, and Y-GATA-H4. The length-based Y-STR haplotypes include six loci that are not reported in the sequence-based kit (DYS393, DYS449, DYS456, DYS458, DYS518, and DYS627), whereas three loci included in the sequence-based kit are not present in length-based kits (DYS505, DYS522, and DYS612). For the latter, a custom multiplex was used to generate CE length-based data, allowing 1032 samples to be evaluated for concordance across the 30 Y-STR loci included in these four commercial Y-STR typing kits. Discordances between typing methods were analyzed further to assess underlying causes such as primer binding site mutations and flanking region insertions/deletions. Allele-level frequency and statistical information is provided for sequenced loci, excluding the multi-copy loci DYF387S1 and DYS385 a/b, for which locus-specific haplotype-level frequencies are provided instead. The resulting data reveals the degree of information gained through sequencing: 88% of sequenced Y-STR loci contain additional sequence-based alleles compared to length-based data, with the DYS389II locus containing the most additional alleles (51) observed by sequencing. Despite these allelic increases, only minimal improvement was observed in haplotype resolution by sequence, with all four commercial kits providing a similar ability to differentiate length-based haplotypes in this sample set. Finally, a subset of 369 male samples were compared to their corresponding additionally sequenced father samples, revealing the sequence basis for the 50 length-based changes observed, and no additional sequence-based mutations. GenBank accession numbers are reported for each unique sequence, and associated records are available in the STRSeq Y-Chromosomal STR Loci National Center for Biotechnology Information (NCBI) BioProject, accession PRJNA380347. Haplotype data is updated in the Y-STR Haplotype Reference Database (YHRD) for the ‘NIST 1032’ data set to now achieve the level of maximal haplotype of YHRD. All supplementary files including revisions to previously published Y-STR data are available in the NIST Public Data Repository: U.S. population data for human identification markers, DOI 10.18434/t4/1500024.

Inference of recent admixture using genotype data

The inference of biogeographic ancestry (BGA) has become a focus of forensic genetics. Misinference of BGA can have profound unwanted consequences for investigations and society. We show that recent admixture can lead to misclassification and erroneous inference of ancestry proportions, using state of the art analysis tools with (i) simulations, (ii) 1000 genomes project data, and (iii) two individuals analyzed using the ForenSeq DNA Signature Prep Kit. Subsequently, we extend existing tools for estimation of individual ancestry (IA) by allowing for different IA in both parents, leading to estimates of parental individual ancestry (PIA), and a statistical test for recent admixture. Estimation of PIA outperforms IA in most scenarios of recent admixture. Furthermore, additional information about parental ancestry can be acquired with PIA that may guide casework.

Pairwise kinship testing with microhaplotypes: Can advancements be made in kinship inference with these markers?

Kinship testing based on genetic relatedness is one of the major tasks in forensic genetics. Although short tandem repeats (STRs) are the “gold standard” biomarkers for relationship testing, microhaplotypes (MHs) have also emerged as viable options for kinship elucidation. In this work, the kinship testing efficiency of 54 highly polymorphic MHs was studied in two extended families consisting of parent-offspring, full siblings, grandparent-grandchildren, uncle/aunt-nephew/nieces, and first cousins. In addition, ten-thousand pairs of different degrees of relationships were simulated using various datasets including 54 MHs, 27 STRs plus 94 single nucleotide polymorphisms (SNPs) that were included in the ForenSeq DNA Signature Prep Kit (ForenSeq), 54 MHs plus loci in ForenSeq, and different subsets of 417-published MHs. The panels’ system effectiveness in the kinship analysis were accessed by likelihood ratio distributions. The results showed that 54 MHs could be used in first-degree relationship testing with high reliability. The effectiveness of 54 MHs was slightly lower than ForenSeq but only by a narrow margin. Both 54 MHs and ForenSeq were not sufficient for distant relationship testing, and approximately 200 microhaplotypes with an average expected heterozygosity (He) = 0.79 were enough to determine second-degree relationships, but a panel of 417 MHs with an average He = 0.72 was not sufficient to first cousins testing according to the simulation analysis. In conclusion, 54 MHs could be used to serve as supplement markers for kinship testing; and well-established STR markers plus well-performing microhaplotype markers may become collective tools in forensic applications, though an enlarged pool of forensic markers is needed for distant relationship testing.

Forensic nanopore sequencing of STRs and SNPs using Verogen's ForenSeq DNA Signature Prep Kit and MinION.

The MinION nanopore sequencing device (Oxford Nanopore Technologies, Oxford, UK) is the smallest commercially available sequencer and can be used outside of conventional laboratories. The use of the MinION for forensic applications, however, is hindered by the high error rate of nanopore sequencing. One approach to solving this problem is to identify forensic genetic markers that can consistently be typed correctly based on nanopore sequencing. In this pilot study, we explored the use of nanopore sequencing for single nucleotide polymorphism (SNP) and short tandem repeat (STR) profiling using Verogen’s (San Diego, CA, USA) ForenSeq DNA Signature Prep Kit. Thirty single-contributor samples and DNA standard material 2800 M were genotyped using the Illumina (San Diego, CA, USA) MiSeq FGx and MinION (with R9.4.1 flow cells) devices. With an optimized cutoff for allelic imbalance, all 94 identity-informative SNP loci could be genotyped reliably using the MinION device, with an overall accuracy of 99.958% (1 error among 2926 genotypes). STR typing was notably error prone, and its accuracy was locus dependent. We developed a custom-made bioinformatics workflow, and finally selected 13 autosomal STRs, 14 Y-STRs, and 4 X-STRs showing high consistency between nanopore and Illumina sequencing among the tested samples. These SNP and STR loci could be candidates for panel design for forensic analysis based on nanopore sequencing.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00414-021-02604-0.

Noninvasive Prenatal Paternity Testing with a Combination of Well-Established SNP and STR Markers Using Massively Parallel Sequencing.

Cell-free fetal DNA (cffDNA) from maternal plasma has made it possible to develop noninvasive prenatal paternity testing (NIPPT). However, most studies have focused on customized single nucleotide polymorphism (SNP) typing systems and few have used conventional short tandem repeat (STR) markers. Based on massively parallel sequencing (MPS), this study used a widely-accepted forensic multiplex assay system to evaluate the effect of noninvasive prenatal paternity testing with a combination of well-established SNP and STR markers. Using a ForenSeq DNA Signature Prep Kit, NIPPT was performed in 17 real parentage cases with monovular unborn fetuses at 7 to 24 gestational weeks. Different analytical strategies for the identification of paternally inherited allele (PIA) were developed to deal with SNPs and STRs. Combined paternity index (CPI) for 17 real trios as well as 272 unrelated trios was calculated. With the combination of SNPs and A-STRs, 82.35% (14/17), 88.24% (15/17), 94.12% (16/17), and 94.12% (16/17) of real trios could be accurately determined when the likelihood ratio (LR) threshold for paternity inclusion was set to 10,000, 1000, 100, and 10, respectively. This reveals that simultaneous surveys of SNP and STR markers included in the ForenSeq DNA Signature Prep Kit offer a promising method for NIPPT using MPS technology.

Evaluation of OpenArray™ as a Genotyping Method for Forensic DNA Phenotyping and Human Identification.

A custom plate of OpenArray™ technology was evaluated to test 60 single-nucleotide polymorphisms (SNPs) validated for the prediction of eye color, hair color, and skin pigmentation, and for personal identification. The SNPs were selected from already validated subsets (Hirisplex-s, Precision ID Identity SNP Panel, and ForenSeq DNA Signature Prep Kit). The concordance rate and call rate for every SNP were calculated by analyzing 314 sequenced DNA samples. The sensitivity of the assay was assessed by preparing a dilution series of 10.0, 5.0, 1.0, and 0.5 ng. The OpenArray™ platform obtained an average call rate of 96.9% and a concordance rate near 99.8%. Sensitivity testing performed on serial dilutions demonstrated that a sample with 0.5 ng of total input DNA can be correctly typed. The profiles of the 19 SNPs selected for human identification reached a random match probability (RMP) of, on average, 10−8. An analysis of 21 examples of biological evidence from 8 individuals, that generated single short tandem repeat profiles during the routine workflow, demonstrated the applicability of this technology in real cases. Seventeen samples were correctly typed, revealing a call rate higher than 90%. Accordingly, the phenotype prediction revealed the same accuracy described in the corresponding validation data. Despite the reduced discrimination power of this system compared to STR based kits, the OpenArray™ System can be used to exclude suspects and prioritize samples for downstream analyses, providing well-established information about the prediction of eye color, hair color, and skin pigmentation. More studies will be needed for further validation of this technology and to consider the opportunity to implement this custom array with more SNPs to obtain a lower RMP and to include markers for studies of ancestry and lineage.

Sequence-based U.S. population data for 7 X-STR loci

The National Institute of Standards and Technology (NIST) U.S. population sample set of unrelated individuals was used to determine allele and haplotype frequencies for seven X-chromosome short tandem repeat (STR) loci in four linkage groups. DXS7132, DXS7423, DXS8378, DXS10074, DXS10103, DXS10135, and HPRTB were sequenced using the ForenSeq DNA Signature Prep Kit on a MiSeq FGx instrument from Verogen. Capillary electrophoresis data produced using the Qiagen Investigator Argus X-12 was compared to ForenSeq length-based alleles and found to be 99 % concordant. For three loci (DXS10103, DXS10074, and HPRTB) the length-based allele call is affected by the extent of flanking region included in the reported sequence. Six of the seven loci gained alleles by sequencing compared to length-based determinations. The increase in alleles are found in both the repeat and flanking region sequences. All sequences for which frequencies are reported in this dataset were cataloged as GenBank records in the STRSeq NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject/380127). Frequency information for both the loci and linkage groups is reported, along with results of statistical tests including gene diversity, polymorphism information content, power of discrimination, and linkage disequilibrium. All supplemental files are available at the NIST Public Data Repository – U.S. population data for Human Identification Markers (https://doi.org/10.18434/t4/1500024).

Identification of sequence polymorphisms at 58 STRs and 94 iiSNPs in a Tibetan population using massively parallel sequencing

Massively parallel sequencing (MPS) has rapidly become a promising method for forensic DNA typing, due to its ability to detect a large number of markers and samples simultaneously in a single reaction, and sequence information can be obtained directly. In the present study, two kinds of forensic genetic markers, short tandem repeat (STR) and identity-informative single nucleotide polymorphism (iiSNP) were analyzed simultaneously using ForenSeq DNA Signature Prep Kit, a commercially available kit on MPS platform. A total of 152 DNA markers, including 27 autosomal STR (A-STR) loci, 24 Y chromosomal STR (Y-STR) loci, 7 X chromosomal STR (X-STR) loci and 94 iiSNP loci were genotyped for 107 Tibetan individuals (53 males and 54 females). Compared with length-based STR typing methods, 112 more A-STR alleles, 41 more Y-STR alleles, and 24 more X-STR alleles were observed at 17 A-STRs, 9 Y-STRs, and 5 X-STRs using sequence-based approaches. Thirty-nine novel sequence variations were observed at 20 STR loci. When the flanking regions were also analyzed in addition to target SNPs at the 94 iiSNPs, 38 more alleles were identified. Our study provided an adequate genotype and frequencies data of the two types of genetic markers for forensic practice. Moreover, we also proved that this panel is highly polymorphic and informative in Tibetan population, and should be efficient in forensic kinship testing and personal identification cases.

Sequenced-based French population data from 169 unrelated individuals with Verogen’s ForenSeq DNA signature prep kit

Massively Parallel Sequencing (MPS) applied to forensic genetics allows the simultaneous analysis of hundreds of genetic markers and the access to full amplicon sequences which help to increase available allele diversity. Meanwhile, sequence variation within the repeat regions represents the majority of the allele diversity, flanking regions adjacent to the repeat core provide an additional degree of variation. The forensic genetics community needs access to population data, from relevant parts of the world that contain this new sequence diversity in order to perform statistical calculations. In this study, we report sequence-based Short Tandem Repeat (STR) and identity Single Nucleotide Polymorphism (iSNPs) allele data for 169 French individuals across 58 STRs and 92 SNPs included in the Verogen ForenSeq DNA Signature Prep kit. 42 STRs out of 58 showed an increased number of alleles due to sequence variation in the repeat motif and/or the flanking regions. D9S1122 showed the largest overall gain with an increase of observed heterozygosities of almost 25 %. The combined match probability combining 27 autosomal STRs and 91 identity SNPs was 1.11E-69. Sequence-based allele frequencies included in this publication will help forensic laboratories to increase the power of discrimination for identification, kinship analysis and mixture interpretation.

Characterizing stutter variants in forensic STRs with massively parallel sequencing

Despite improvements in characterizing stutters of short tandem repeats (STRs), the relationships among the amounts of stutter variants and the relationships among motifs are not well understood yet. In the present study, 750 peripheral blood samples from human subjects were included to characterize the stutters of 58 STRs via the ForenSeq DNA Signature Prep Kit on a MiSeq FGx instrument. Alleles and corresponding stutter products were identified with a sequence simplification procedure. After screening, 26,921 alleles were included, that resulted in over 50 million reads, among which 8.69% were stutter products. Among these stutter products, 83.44% were N-1 stutters. Additionally, N-4, N-3, N-2, N0, N + 1, and N + 2 variants accounted for 0.11%, 0.77%, 6.45%, 3.01%, 5.95%, and 0.25% of the stutter products, respectively. For backward stutters, stutter products correlated best with the corresponding one-unit-longer stutter (or parental allele), which may represent a good predictor for backward stutters. For forward stutters, the N + 2 stutter correlated best with the N + 1 stutter, whereas the N + 1 stutter correlated best with the N-1 stutter rather than the expected parental allele, which indicated that the patterns were more complex for forward stutters. Additionally, some interesting findings were obtained for D21S11. For two adjacent contiguous motifs, co-stuttering patterns were observed where one motif tended to increase one repeat unit while the other motif decreased one repeat unit, whereas the inter-motif dependency was not significant for interrupted motifs. In conclusion, with massively parallel sequencing technology and our sequence simplification strategy, sequence variations within alleles and stutter products were identified, which was useful to determine the origin of stutters, identify more stutter variants, and explore the relationships among motifs. These findings may be helpful for allele designation, a deeper understanding of the mechanism of stutter, and improving resolution in forensic mixture analyses.

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

Evaluation of the performance of the beta version of the ForenSeq DNA signature Prep Kit on the MiSeq FGx forensic genomics system

Massively Parallel Sequencing (MPS) are innovative approaches of analysis of genetic markers, supported by the forensic community. The Illumina Beta Version of the ForenSeq DNA Signature Prep Kit is a high-throughput, multiplexing kit allowing simultaneous, high resolution sequencing of 153 identity informative markers (including 27 autosomal STRs, 7 X-chromosomal, 24 Y-chromosomal haplotype markers and 94 SNPs) plus 56 biogeographical ancestry and 22 phenotypic informative SNPs. Implementation of MPS technology in forensic casework laboratories needs comprehensive, internal validation according to SWGDAM guidelines. The aim of this study was to determine with ForenSeq DNA Signature Prep Kit, method’s precision/accuracy, sensitivity as well as possibility of analysis of two persons mixtures. Analysis of challenging casework samples coming from various tissues were performed and compared to results obtained with capillary electrophoresis (CE). In order to evaluate the quality and reliability of the acquired results, informative metrics of STR and SNP markers including depth of coverage (DoC), allele coverage ratio (ACR), and sequence coverage ratios (SCRs) were assessed.