Integrated Sample Cleanup and Microchip Capillary Array Electrophoresis for High-Performance Forensic STR Profiling

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 analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.

Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis

Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use of genomic DNA has increased with the development of DNA sequencing technology, thereby enabling additional analysis during criminal investigations when additional legal evidence is required. In this study, we used next-generation sequencing to facilitate the generation of complementary data in order to analyze human evidence obtained through short tandem repeat (STR) analysis. We examined the applicability and potential of analyzing microbial genome communities. Microbiological supplementation information was confirmed for two of four failed STR samples. Additionally, the accuracy of the gargle sample was confirmed to be as high as 100% and was highly likely to be classified as a body fluid sample. Our experimental method confirmed that anthropological and microbiological evidence can be obtained by performing two experiments with one extraction. We discuss the advantages and disadvantages of using these techniques, explore prospects in the forensic field, and highlight suggestions for future research.

A high-risk gestational trophoblastic neoplasia derived from a complete hydatidiform mole with coexisting fetus identified by short tandem repeats analysis: A case report

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.

Sibship determination for any two persons whose parents have died is one of the most fundamental issues of personal identification, second only to those of a parent-child relationship. By automated fluorescence analysis of a PCR-amplified short tandem repeat (STR) system in conjunction with capillary electrophoresis, a panel of up to 15 polymorphic, autosomal, unlinked STR loci was used to investigate sibship index (SI) values in a cohort of 126 true sibling pairs. These SI values were then compared with those of 126 random pairs. The 15-loci STR panel provides a cumulative power of exclusion of 0. 9999997. Of the 126 random pairs, 124 (98.4%) had cumulative sibship indices (CSIs) of <1.0, and none had a CSI of >3.0 (median, 0.0101; range, 0.0000003-2.5376). In contrast, 107 (85%) of the 126 sibling pairs had a CSI of >100 (median, 5,579.9853; range, 0.0747-9,406,829, 249.8461). However, five pairs (4%) of the sibling group had a CSI of <3.0. True sibship was confirmed for this particular group by additional paternity testing and mitochondrial DNA sequence analysis. Among a total of 1890 observations (15 loci x 126 pairs), two alleles per locus were shared 760 times (40.21%) (mean, 6.03 loci; range, 1-10) in the sibling group, but only 192 times (10.16%) in the random group (mean, 1.52 loci; range, 1-5) (p<0.001). No alleles were shared 696 times (36.83%) in the unrelated pairs, as compared to 176 times (9.31%) in the sibling group (p<0.001). A polarized distribution was not noted in the sharing of single alleles in either the random or the sibling group: 1002 observations (53.02%) and 954 observations (50.48%), respectively. Highly polymorphic STR analysis can be discriminative in most sibship determinations, and the sharing of two alleles per locus is most informative in indicating sibship. Complementary mitochondrial DNA sequence analysis is mandatory in a few cases to exclude or establish true sibship when CSIs are equivocal and neither parent is available.

Forensic DNA analysis has revolutionized criminal investigations by providing valuable insights into identifying perpetrators, exonerating the innocent and unravelling mysteries of the past. Various molecular biology techniques, such as Short Tandem Repeat (STR) analysis, Single Nucleotide Polymorphism (SNP) profiling, mitochondrial DNA (mtDNA) sequencing, epigenetics and DNA methylation analysis, have played crucial roles in forensic investigations. While these techniques offer exceptional sensitivity and specificity, they also come with unique challenges. This review explores the current approaches of forensic DNA analysis, highlighting each technique. STR analysis, regarded as the gold standard in forensic DNA profiling, provides high discrimination power. However, it is limited in its ability to analyze degraded or mixed samples. On the other hand, SNP profiling offers advantages in analyzing highly degraded DNA samples but lacks the discriminatory power of STRs. Meanwhile, mtDNA analysis, particularly useful in cases involving compromised nuclear DNA, presents challenges due to its maternal inheritance pattern and lower discriminatory power. Furthermore, explore the exciting realm of epigenetics and the analysis of DNA methylation in forensic investigations. Epigenetic markers offer insights into gene expression patterns influenced by environmental factors, potentially aiding in the determination of tissue origin and chronological age estimation. DNA methylation analysis holds promise in forensic applications, providing additional layers of information for identity verification and tissue differentiation. Despite these advancements, several challenges persist in forensic DNA analysis, including the interpretation of complex DNA mixtures, standardization of methodologies, ethical considerations, and privacy concerns associated with the use of genetic information. Moreover, the integration of multi-omics data and machine learning approaches presents both opportunities and challenges in enhancing the accuracy and reliability of forensic DNA analysis. Looking ahead, future directions in molecular biology research for forensic DNA analysis involve the development of novel techniques with increased sensitivity, scalability, and robustness. By addressing these challenges and embracing emerging technologies, the field of forensic DNA analysis is poised for further advancements, offering enhanced capabilities in criminal justice and humanitarian efforts.

The content of the 13th Mutation Detection meeting (Leiden, April 2015) is summarized in this report. Topics discussed at the meeting included current challenges of clinical NGS, advances in bioinformatics, data quality control, single cell analysis and RNA sequencing, among others. Social, ethical and regulatory challenges of genomic data handling and data sharing were the focus of an expert panel debate. The 14th International Symposium on Variants in the Genome will take place in Santiago de Compostela, June 5-8, 2017. http://isv.variome.org.

Systematic analysis of short tandem repeats in 38,095 exomes provides an additional diagnostic yield

Usher syndrome encompasses a group of genetically and clinically heterogeneous autosomal recessive disorders with hearing deficiencies and retinitis pigmentosa. The mechanisms underlying the Usher syndrome are highly variable. In the present study, a Chinese family with Usher syndrome was recruited. Whole exome sequencing (WES), Sanger sequencing, homozygosity mapping, short tandem repeat (STR) analysis and segregation analysis were performed. Functional domains of the pathogenic variant for USH2A were analysed. We identified a homozygous frameshift variant c.99_100insT (p.Arg34Serfs*41) in the USH2A gene in the proband that showed discordant segregation in the father. Further homozygosity mapping and STR analysis identified an unusual homozygous variant of proband that originated from maternal uniparental disomy (UPD). The p.Arg34Serfs*41 variant produced a predicted truncated protein that removes all functional domains of USH2A. The variant was not included in the 1000 Human Genomes Project database, ExAC database, HGMD or gnomAD database, but was included in the ClinVar databases as pathogenic. Although USH2A is an autosomal recessive disease, the effects of UPD should be informed in genetic counselling since the recurrence risk of an affected child is greatly reduced when the disease is due to the UPD mechanism. To test potential patients, WES, combined with STR analysis and homozygosity mapping, provides an accurate and useful strategy for genetic diagnosis. In summary, our discoveries can help further the understanding of the molecular pathogenesis of Usher syndrome type IIA to advance the prevention, diagnosis and therapy for this disorder.

Initially categorized 46,XY embryo transfer ending with 45,X products of conception—a case report and a review of discordant result management

To conduct high-throughput mutational analysis in 6 commonly used head and neck cancer cell lines. Comprehensive mutation analysis of primary head and neck squamous cell carcinoma (HNSCC) tumors has recently been reported, and mutations in the NOTCH receptors, TP53 and CDKN2A, were key findings. Established cell lines are valuable tools to study cancer in vitro. Similar high-throughput mutational analysis of head and neck cancer cell lines is necessary to confirm their mutational profile. DNA was extracted from American Type Culture Collection (ATCC) cell lines Cal27, Detroit562, FaDu, SCC4, SCC15, and SCC25. Cell line identity was confirmed by short tandem repeat (STR) analysis, and human papillomavirus (HPV) infection status was assessed by real-time polymerase chain reaction. A total of 535 cancer-associated genes were sequenced through a limited exome capture on the Illumina HiSeq system. London Regional Cancer Program. The identity of the 6 cell lines was confirmed by STR analysis, and all lines tested negative for HPV infection. We achieved an average of 129-fold coverage with paired-end 100 base-pair reads. Sequencing revealed an average of 38 damaging mutations in each cell line (range, 30-45). The TP53 mutations, predicted to confer loss of function, were noted in all cell lines, and damaging CDKN2A mutations were found in all lines except SCC15. High-throughput sequencing of head and neck cancer cell lines revealed similar mutations to those observed in primary tumors. Thus, these lines reflect the tumor biology of HNSCC and can serve as valuable models to study HNSCC in vitro.

DNA technologies such as restriction fragment length polymorphism (RFLP)-based genotyping and polymerase chain reaction (PCR)-based genotyping, were first applied to forensic samples in the mid 1980s. This chapter discusses some of the more recent developments in forensic DNA analysis such as next-generation sequencing, also known as massively parallel sequencing. The clonal nature of this technology allows the deconvolution of mixtures, a common and challenging category of crime scene evidence. The strategy discussed here is the construction of a shotgun library, targeted capture by hybridization with a probe panel, and sequencing on the IIlumina MiSeq platform, a protocol suited to the small DNA fragments found in many forensic samples. Sequencing the entire mitochondrial DNA genome, a haploid lineage marker allows deconvolution of mixtures by counting individual clonal sequence reads and assigning them to one of the contributors, aided by a phylogenetic-based software Mixemt. The chapter also discusses the use of massively parallel sequencing using a probe capture strategy for the analysis of short tandem repeats (STRs) using the web-based software toaSTR. In addition, this chapter reviews the implementation of Rapid DNA technology, instruments capable of automating the DNA extraction, amplification of the short tandem repeat markers, and analysis by capillary electrophoresis.

Candida (Candidozyma) auris has distinct genetic clades. Clade distinction is relevant for infection control and epidemiological purposes. State-of-the-art typing methodologies for clade distinction are based on genomic approaches, such as short tandem repeat (STR) analysis and whole-genome sequencing (WGS). However, they are time-consuming and expensive. Fourier transform infrared spectroscopy (FT-IR) is an alternative tool for strain typing based on their unique biochemical spectral profiles. To apply FT-IR to differentiate C. auris clades and evaluate epidemiological relationships based on biochemical data among isolates from a multicenter C. auris outbreak in the state of Pernambuco, northeastern Brazil. Sixty-nine C. auris strains from clades I, II, III, and IV were analysed. Fifty-nine were clade IV strains obtained from three outbreaks that took place in Pernambuco state, northeastern Brazil. An adjusted FT-IR spectroscopy protocol was applied to obtain carbohydrates and lipid fingerprints. Short Tandem Repeat (STR) analysis was used in order to validate the spectroscopy approach. The adjusted preparation protocol for FT-IR analysis improved the spectral quality by 31.42% compared to standard protocol. FT-IR allowed us to discriminate C. auris clades I to IV. Moreover, important similarities were observed in C. auris clade IV strains obtained from two of the three hospitals, based on polysaccharides (1300-800 cm-1) plus lipids (3000-2800 cm-1 and 1500-1400 cm-1) spectra. STR confirmed the similarity results obtained by FT-IR, clustering the strains from two different hospitals. The IR Biotyper is fast, easy-to-use, and a promising alternative for moderate-to-high-complexity laboratories to differentiate C. auris clades. Furthermore, this technique has the potential for isolate-level source tracking, which could be valuable for monitoring transmission routes in clinical settings.