Assessing the GeneRead SNP panel for analysis of low-template and PCR-inhibitory samples

Investigation into the sequence structure of 23 Y chromosomal STR loci using massively parallel sequencing

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs. These data clearly demonstrate that smaller PCR amplicons provide an attractive alternative to mitochondrial DNA for forensic analysis of degraded DNA.

Assessing heteroplasmic variant drift in the mtDNA control region of human hairs using an MPS approach

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.

Many forensic laboratories have conducted sequence‐based population studies to support the integration of massively parallel sequencing (MPS). However, the data remain limited concerning African populations. MPS enhances allelic representation compared to CE methods. It is hypothesized that this increase will be more pronounced for African populations due to their greater genetic diversity. A systematic review and meta‐analysis were conducted to compile data from population genetic studies using the ForenSeq™ DNA Signature Prep kit, frequently employed in forensic MPS population studies. The aim of the review was to gain insight into global forensic sequence‐based population data, focusing on African and underrepresented populations. The search spanned three databases, resulting in 582 records, where 40 articles met inclusion criteria for the systematic review and 20 qualified for the meta‐analysis. The meta‐analysis aimed to quantify the increase in genetic variation in autosomal short tandem repeat (A‐STR) markers using allele counts and random match probability (RMP). Most population studies were conducted in high‐income countries (65%, 26/40), with none from Africa. Only 14 out of 40 studies included concordance data, with 13 of these reporting rates above 99%. The meta‐analysis covered 35 population groups and found that of the 27 A‐STR markers evaluated, mean allele counts increased by 53.08% from length‐to‐sequence‐based analyses. African ancestry groups showed the highest increase in allele counts and the biggest reduction in RMP. Despite substantial genetic diversity in African populations, their representation in MPS studies is minimal. Addressing this gap is crucial to justify further research in African countries.

Here, we present the results from a population study that evaluated the performance of massively parallel sequencing (MPS) of short tandem repeats (STRs) with a particular focus on DNA intelligence databasing purposes. To meet this objective, 247 randomly selected reference samples, earlier being processed with conventional capillary electrophoretic (CE) STR sizing from the Austrian National DNA Database, were reanalyzed with the PowerSeq 46Y kit (Promega). This sample set provides MPS-based population data valid for the Austrian population to increase the body of sequence-based STR variation. The study addressed forensically relevant parameters, such as concordance and backward compatibility to extant amplicon-based genotypes, sequence-based stutter ratios, and relative marker performance. Of the 22 autosomal STR loci included in the PowerSeq 46GY panel, 99.98% of the allele calls were concordant between MPS and CE. Moreover, 25 new sequence variants from 15 markers were found in the Austrian dataset that are yet undescribed in the STRSeq online catalogue and were submitted for inclusion. Despite the high degree of concordance between MPS and CE derived genotypes, our results demonstrate the need for a harmonized allele nomenclature system that is equally applicable to both technologies, but at the same time can take advantage of the increased information content of MPS. This appears to be particularly important with regard to database applications in order to prevent false exclusions due to varying allele naming based on different analysis platforms and ensures backward compatibility.

Massively parallel sequencing of 10 autosomal STRs in Chinese using the ion torrent personal genome machine (PGM)

To explore the cause of abortion and strategy of prenatal diagnosis for pregnant women with high risk for chromosomal abnormalities by using copy number variation sequencing (CNV-seq) and short tandem repeats (STR) analysis. A total of 36 samples were collected, including amniotic fluid, abortion tissue, whole blood, chorionic villi and umbilical cord blood. CNV-seq and STR analysis were carried out to detect microdeletions, microduplications, chromosomal aneuploidies, mosaicisms and triploidies. Among all samples, 1 was detected with 4p15.1p16.3 and 14q11.1q22.1 duplication, 1 was detected with 19p13.3 deletion, 8 were detected with chromosomal aneuploidies, 4 were detected with mosaicisms, two were detected with triploidies. No definite pathogenic CNVs were detected in 20 samples, which yielded a positive detection rate of 44.44%. As a high-throughput detection method, CNV-seq has the advantages of rapidity, simplicity and high accuracy. It may suit prenatal diagnosis and analysis of abortion factors in combination with STR analysis.

The conditions under which contact DNA analysis yields useful recovery of materials is not well known. Using simulated models of grabbing and struggling, the recovery of male contact DNA transferred from male “assailants” to female “victims” was investigated. The upper and lower arm areas of female subjects (sites for the modeled assaults) were swabbed for male DNA. Extracted DNA was quantified using the Quantifiler® Duo DNA Quantification kit and amplified using the AmpFlSTR® Identifiler® and the AmpFlSTR® YfilerTM PCR Amplification kits. The qPCR quantification results detected male “assailant” DNA from the female “victims”. However, no viable male profiles were obtained when the DNA was subjected to short tandem repeat (STR) analysis with the Identifiler® and Yfiler™ kits. The grab and struggle scenarios yielded up to 20 pg/μL and 40 pg/μL of male contact DNA from female subjects, respectively, DNA amounts which are below the detection threshold of most commercial human STR identification kits. Although contact DNA levels are highly variable, this study showed that the likelihood of DNA deposition on skin from assailant to victim was substantially greater when a struggle ensues. The results of the present study are limited by small sample size, but imply that further research for improving recovery and amplification techniques of contact DNA should be sought to increase the probative value of this evidence in sexual assault investigations.

Massively parallel sequencing (MPS) offers substantial improvements over current forensic DNA typing methodologies such as increased resolution, scalability, and throughput. The Ion PGM™ is a promising MPS platform for analysis of forensic biological evidence. The system employs a sequencing-by-synthesis chemistry on a semiconductor chip that measures a pH change due to the release of hydrogen ions as nucleotides are incorporated into the growing DNA strands. However, implementation of MPS into forensic laboratories requires a robust chemistry. Ion Torrent's Hi-Q™ Sequencing Chemistry was evaluated to determine if it could improve on the quality of the generated sequence data in association with selected genetic marker targets. The whole mitochondrial genome and the HID-Ion STR 10-plex panel were sequenced on the Ion PGM™ system with the Ion PGM™ Sequencing 400 Kit and the Ion PGM™ Hi-Q™ Sequencing Kit. Concordance, coverage, strand balance, noise, and deletion ratios were assessed in evaluating the performance of the Ion PGM™ Hi-Q™ Sequencing Kit. The results indicate that reliable, accurate data are generated and that sequencing through homopolymeric regions can be improved with the use of Ion Torrent's Hi-Q™ Sequencing Chemistry. Overall, the quality of the generated sequencing data supports the potential for use of the Ion PGM™ in forensic genetic laboratories.

Forensic DNA elimination databases are valuable tools for identifying potential contamination risks during forensic investigations. This study provides a comparative analysis of the design, implementation, and effectiveness of forensic DNA elimination databases across seven European countries: Sweden, Germany, Czechia, Poland, the Netherlands, the UK, and Finland. Data were collected through structured inquiries sent to ENFSI member states’ forensic DNA laboratories, focusing on key aspects such as legal frameworks, database sizes, and contamination cases identified through the elimination database. The results reveal significant differences in the establishment and management of these databases, reflecting diverse legal and operational contexts. The findings underscore the need to ensure that all European countries implement their elimination databases to standardize forensic procedures and improve the reliability of DNA evidence. Furthermore, the study highlights the importance of introducing harmonized frameworks for forensic DNA elimination databases to enhance transparency, accessibility, and efficiency in forensic DNA practices across Europe.

Currently, the most commonly used method for human identification and kinship analysis in forensic genetics is the detection of length polymorphism in short tandem repeats (STRs) using polymerase chain reaction (PCR) and capillary electrophoresis (CE). However, numerous studies have shown that considerable sequence variations exist in the repeat and flanking regions of the STR loci, which cannot be identified by CE detection. Comparatively, massively parallel sequencing (MPS) technology can capture these sequence differences, thereby enhancing the identification capability of certain STRs. In this study, we used the ForenSeq™ DNA Signature Prep Kit to sequence 58 STRs and 94 individual identification SNPs (iiSNPs) in a sample of 220 unrelated individuals from the Eastern Chinese Han population. Our aim is to obtain MPS-based STR and SNP data, providing further evidence for the study of population genetics and forensic applications. The results showed that the MPS method, utilizing sequence information, identified a total of 486 alleles on autosomal STRs (A-STRs), 97 alleles on X-chromosome STRs (X-STRs), and 218 alleles on Y-chromosome STRs (Y-STRs). Compared with length polymorphism, we observed an increase of 260 alleles (157, 31, and 72 alleles on A-STRs, X-STRs, and Y-STRs, respectively) across 36 STRs. The most substantial increments were observed in DYF387S1 and DYS389II, with increases of 287.5% and 250%, respectively. The most increment in the number of alleles was found at DYF387S1 and DYS389II (287.5% and 250%, respectively). The length-based (LB) and sequence-based (SB) combined random match probability (RMP) of 27 A-STRs were 6.05E-31 and 1.53E-34, respectively. Furthermore, other forensic parameters such as total discrimination power (TDP), cumulative probability of exclusion of trios (CPEtrio), and duos (CPEduo) were significantly improved when using the SB data, and informative data were obtained for the 94 iiSNPs. Collectively, these findings highlight the advantages of MPS technology in forensic genetics, and the Eastern Chinese Han genetic data generated in this study could be used as a valuable reference for future research in this field.

Chapter 9 - Fundamentals of DNA Separation and Detection

Concurrent genotyping of mitochondrial DNA and nuclear DNA in rootless hair shafts and blood samples for enhanced analysis