Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System.

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the 'separating' and 'merging' strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Sequence-based mutation patterns at 41 Y chromosomal STRs in 2 548 father-son pairs.

A total of 2 548 unrelated healthy father-son pairs from a Northern Han Chinese population were genotyped at 41 Y chromosomal short tandem repeat (Y-STRs) including DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS444, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS522, DYS549, DYS533, DYS557, DYS570, DYS576, DYS593, DYS596, DYS627, DYS635, DYS643, DYS645, Y-GATA-H4, DYF387S1a/b, DYF404S1a/b, DYS385a/b, and DYS527a/b. In 2 548 father samples, 2 387 unique haplotypes were detected with the haplotype diversity and discrimination capacity values of 0.999 956 608 and 0.96 741 007. The average gene diversity (GD) value was 0.6934 with a range from 0.1051 at DYS645 to 0.9657 at DYS385a/b. When comparing alleles at 24 overlapped Y-STRs between the ForenSeq™ deoxyribonucleic acid (DNA) Signature Prep Kit on the MiSeq FGx® Forensic Genomics System and the Goldeneye® DNA ID Y Plus Kit on the Applied Biosystems™ 3730 DNA Analyzer from 308 father samples in mutational pairs, 258 alleles were detected by massively parallel sequencing (MPS) typing including 156 length-based alleles that could be obtained by capillary electrophoresis (CE) typing, 95 repeat region (RR) variant alleles and seven flanking region variant alleles. Hereof, we found 16 novel RR variant alleles and firstly identified two SNPs (rs2016239814 at DYS19 and rs2089968964 at DYS448) and one 4-bp deletion (rs2053269960 at DYS439) that had been validated by the Database of Short Genetic Variation. Sanger sequencing or MPS was employed to confirm 356 mutations from 104 468 allele transfers generated from CE, where 96.63% resulted in one-step mutations, 2.25% in two-step, and 1.12% in multi-step, and the overall ratio of repeat gains versus losses was balanced (173 gains vs. 183 losses). In 308 father-son pairs, 268 pairs occurred mutations at a single locus, 33 pairs at two loci, six pairs at three loci, and one pair at four loci. The average Y-STR mutation rate at 41 Y-STRs was ⁓3.4 × 10-3 (95% confidence intervals: 3.1 × 10-3-3.8 × 10-3). The mutation rates at DYS576 and DYS627 were higher than 1 × 10-2 in Northern Han Chinese, whilst the mutation rates at DYF387S1a/b, DYF404S1a/b, DYS449, DYS518, and DYS570 were lower than initially defined. In this study, the classical molecular factors (the longer STR region, the more complex motif and the order father) were confirmed to drive Y-STR mutation rates increased, but the length of repeat unit did not conform to the convention. Lastly, the interactive graphical and installable StatsY was developed to facilitate forensic scientists to automatically calculate allele and haplotype frequencies, forensic parameters, and mutation rates at Y-STRs. Key points: 308 of 2 548 father-son pairs from Northern Han Chinese occurred at least one mutation(s) across 41 Y-STRs.Sanger sequencing or MPS was employed to confirm those mutations generated from CE.The longer STR region, the more complex motif and the order father drove Y-STR mutation rates increased.StatsY was developed to calculate allele and haplotype frequencies, forensic parameters and mutation rates at Y-STRs.

Splicing forgery localization via noise fingerprint incorporated with CFA configuration.

Noise is the inherent intrinsic fingerprint in digital images and is often used for forgery localization. Most noise-based methods assume that the noise is similar over the whole image and can be considered as white Gaussian noise. However, the noise is different in various regions, which degrade the performance of these noise-based methods. To reduce the impact of impractical assumptions, in this paper, we propose an effective noise fingerprint incorporated with CFA configuration for splicing forgery localization. The noise of interpolated pixels is expected to be suppressed after interpolation, and the relationship between the noise levels of adjacent acquired and interpolated pixels is only related to the interpolation algorithm, which is constant in the original image. We utilize a dual tree wavelet based denoising algorithm to extract the noise from the green channel and compute the standard deviation of the noise for acquired and interpolated pixels, respectively. The noise level of acquired and interpolated pixels are then obtained by the geometric mean of the noise standard deviations. Finally, the ratio of noise levels between acquired and interpolated pixels can be a fingerprint to locate tampered regions. Experiments conducted on publicly available databases demonstrate that the proposed approach outperforms previous methods for detecting splice tampering. Moreover, the proposed method is robust to Gaussian filtering and JPEG compression attacks.

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.

StatsX v2.0: the interactive graphical software for population statistics on X-STR.

StatsX (Statistics for X-STR) v2.0 is developed to facilitate the workflow for analysis of population data on X-chromosomal short tandem repeat (X-STR) marker. The interactive graphical and installable software can estimate allele/haplotype frequencies from female, male, and pooled samples and calculate forensic parameters by importing a single file. In addition, it is able to filter out the invalid raw data and export a series of pre-formatted Arlequin input files that are directly used for testing the significance of the Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), and gender differentiation. As expected, all results of frequencies and forensic parameters can be saved as EXCEL workbooks and also plotted as bar or line graphs. Further, full concordance was obtained by recalculating frequencies and forensic parameters with other established software from a published Han Chinese population.

Exposing splicing forgery based on color temperature estimation.

Splicing is one of the most common tampering techniques for image manipulation in many forensic cases. Normally color shift in images due to color temperature of illumination can be seen as intrinsic features relative to imaging process. In splicing forgeries, copied area and pasted target image come from different imaging process, and are likely to have different color shift. In this paper, a novel automated authentication method is presented to expose splicing manipulation and locate manipulated areas by discriminating the inconsistencies of color shift in an image. In order to minimize human interaction on detection of splicing forgeries as well as localization of manipulated areas, a forensic image is divided into blocks with grid-based strategy. After calculation on color temperature of each blocks with white-point algorithm, reference color temperature is obtained with a random restricted algorithm. Then color temperature distance between each block and reference area is calculated sequentially. At last, by comparing color temperature distance with an optimized threshold determined by OSTU algorithm. This method enables us to judge if splicing has occurred and furthermore localize manipulated area simultaneously. Experiments show that the proposed method can speed up the quantitative detection of possible splicing manipulation and localize manipulated area automatically.