Advancing human genotyping: The Infinium HTS iSelect Custom microarray panel (Rita) development study.

Single Nucleotide Polymorphisms (SNPs), as the most prevalent type of variation in the human genome, play a pivotal role in influencing human traits. They are extensively utilized in diverse fields such as population genetics, forensic science, and genetic medicine. This study focuses on the 'Rita' BeadChip, a custom SNP microarray panel developed using Illumina Infinium HTS technology. Designed for high-throughput genotyping, the panel facilitates the analysis of over 4000 markers efficiently and cost-effectively. After careful clustering performed on a set of 1000 samples, an evaluation of the Rita panel was undertaken, assessing its sensitivity, repeatability, reproducibility, precision, accuracy, and resistance to contamination. The panel's performance was evaluated in various scenarios, including sex estimation and parental relationship assessment, using GenomeStudio data analysis software. Findings show that over 95 % of the custom BeadChip assay markers were successful, with better performance of transitions over other mutations, and a considerably lower success rate for Y chromosome loci. An exceptional call rate exceeding 99 % was demonstrated for control samples, even with DNA input as low as 0.781 ng. Call rates above 80 % were still obtained with DNA quantities under 0.1 ng, indicating high sensitivity and suitability for forensic applications where DNA quantity is often limited. Repeatability, reproducibility, and precision studies revealed consistency of the panel's performance across different batches and operators, with no significant deviations in call rates or genotyping results. Accuracy assessments, involving comparison with multiple available genetic databases, including the 1000 Genome Project and HapMap, denoted over 99 % concordance, establishing the Rita panel's reliability in genotyping. The contamination study revealed insights into background noise and allowed the definition of thresholds for sample quality evaluation. Multiple metrics for differentiating between negative controls and true samples were highlighted, increasing the reliability of the obtained results. The sex estimation tool in GenomeStudio proved highly effective, correctly assigning sex in all samples with autosomal loci call rates above 97 %. The parental relationship assessment of family trios highlighted the utility of GenomeStudio in identifying genotyping errors or potential Mendelian inconsistencies, promoting the application of arrays such as Rita in kinship testing. Overall, this evaluation confirms the Rita microarray as a robust, high-throughput genotyping tool, underscoring its potential in genetic research and forensic applications. With its custom content and adaptable design, it not only meets current genotyping demands but also opens avenues for further research and application expansion in the field of genetic analysis.

Unveiling genetic diversity and forensic utility of SureID® human DNA identification kits: a comprehensive analysis of 44 autosomal STR loci in English and Irish populations.

Background: Human identification and kinship testing in forensic science rely on Short Tandem Repeat (STR) multiplex kits, typically containing loci recommended by standard sets. However, complementary kits with additional STR loci can be valuable in complex cases. Allele frequency databases specific to the population are essential for accurate forensic analysis.Aim: This study aimed to generate allele frequencies and population genetic data for 44 autosomal STR loci from SureID® PanGlobal and 27comp kits in English and Irish populations for forensic casework, human identification, and kinship testing.Subjects and methods: Buccal swab samples from 645 White Caucasians (365 English, 280 Irish) were collected. DNA was extracted and amplified using the mentioned kits. Quality control, statistical analysis, and genetic distance calculations were performed.Results: Both kits demonstrated robustness with no significant deviations from Hardy-Weinberg Equilibrium (HWE). Variant alleles and minor discordances between kits were observed. Syntenic STR pairs were identified but showed no significant linkage. A close genetic relationship was found between English and Irish populations, allowing for combined databases.Conclusions: The SureID® PanGlobal and 27comp kits showed high discriminatory power and reliability in the English and Irish populations. Care is needed when handling variant alleles, discordances, and syntenic loci. Combining data from both populations is feasible for a comprehensive database. Further studies are required to explore their effectiveness in diverse populations.

Exploring the gut microbiota: lifestyle choices, disease associations, and personal genomics.

The gut microbiota is a rich and dynamic ecosystem that actively interacts with the human body, playing a significant role in the state of health and disease of the host. Diet, exercise, mental health, and other factors have exhibited the ability to influence the gut bacterial composition, leading to changes that can prevent and improve, or favor and worsen, both intestinal and extra-intestinal conditions. Altered gut microbial states, or 'dysbiosis', associated with conditions and diseases are often characterized by shifts in bacterial abundance and diversity, including an impaired Firmicutes to Bacteroidetes ratio. By understanding the effect of lifestyle on the gut microbiota, personalized advice can be generated to suit each individual profile and foster the adoption of lifestyle changes that can both prevent and ameliorate dysbiosis. The delivery of effective and reliable advice, however, depends not only on the available research and current understanding of the topic, but also on the methods used to assess individuals and to discover the associations, which can introduce bias at multiple stages. The aim of this review is to summarize how human gut microbial variability is defined and what lifestyle choices and diseases have shown association with gut bacterial composition. Furthermore, popular methods to investigate the human gut microbiota are outlined, with a focus on the possible bias caused by the lack of use of standardized methods. Finally, an overview of the current state of personalized advice based on gut microbiota testing is presented, underlining its power and limitations.

Validation of reduced volume VeriFiler™ Express PCR Amplification Kit for buccal swab samples extracted using Prep-n-Go™ Buffer.

The efficiency of reduced volume PCR amplification was studied using the VeriFiler™ Express PCR Amplification Kit. Full (25 µL) and reduced (5 µL) volumes were tested in parallel to identify any differences in template DNA sensitivity and other electropherogram parameters. Both volumes produced full DNA profiles down to 0.08 ng/µL DNA concentration at 26 PCR cycles; however, reduced volume produced higher peak heights due to increased signal intensities. Significant difference (p-value ≤ 0.05) in heterozygote peak height ratios was observed between both volumes, where the reduced volume threshold was lowered to 0.6 to accommodate all data points. However, no significant difference (p-value > 0.05) was identified in the stutter ratios between both volumes. The analytical threshold for reduced volume was also determined to be 150 RFU with the presence of template DNA in PCR amplification. When the optimized reduced volume parameters were tested on DNA extracted from buccal swab samples using Prep-n-Go™ Buffer, good quality DNA profiles were produced. Overall, the reduced volume not only showed better results compared to the full volume, but also enable more samples to be processed with a PCR amplification kit, thus reduced the cost.

Population genetics data for 22 autosomal STR loci in European, South Asian and African populations using SureID® 23comp Human DNA Identification Kit.

Allele frequency data for 22 short tandem repeat loci; D18S1364, D1S1656, D13S325, D5S2800, D9S1122, D4S2366, D3S1744, D12S391, D11S2368, D21S2055, D20S482, D8S1132, D7S3048, D2S441, D19S253, D10S1248, D17S1301, D22-GATA198B05, D16S539, D6S474, D14S1434 and D15S659 from the SureID® 23comp Human DNA Identification Kit have been determined for unrelated individuals in European, South Asian and African populations. Deviations from Hardy-Weinberg equilibrium were observed in loci D1S1656 and D19S253 in European; D18S1364, D6S474 and D14S1434 in South Asian; and D9S1122 and D8S1132 in African populations (p-value <0.05). However, after Bonferroni correction no significant deviations were observed (p-value <0.002). The most discriminating loci were D1S1656 and D12S391 for European (PD=0.977), D21S2055 for South Asian (PD=0.980), and D21S2055 and D7S3048 for African (PD=0.972) populations. The match probabilities were 1 in 6.7×1025 for European, 1 in 1.4×1026 for South Asian and 1 in 1.6×1026 for African populations. These findings established the high discriminatory capacity and robustness of the tested STR loci for forensic identification and kinship testing.

Development of a multiplex system to assess DNA persistence in taphonomic studies.

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single-copy nuclear recombination activation gene (RAG-1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature-24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.