Individual Identification with Short Tandem Repeat Analysis and Collection of Secondary Information Using Microbiome 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.

Global non-random abundance of short tandem repeats in rodents and primates.

Background: While of predominant abundance across vertebrate genomes and significant biological implications, the relevance of short tandem repeat (STR) abundance to speciation remains largely elusive and attributed to random coincidence for the most part. In a model study, here we collected whole-genome abundance of mono-, di-, and trinucleotide STRs in nine species, encompassing rodents and primates, including rat, mouse, olive baboon, gelada, macaque, gorilla, chimpanzee, bonobo, and human. The obtained unnormalized and normalized data were used to analyze hierarchical clustering of the STR abundances in the selected species. Results: We found massive differential abundances between the rodent and primate orders. In addition, while numerous STRs had random abundance across the nine selected species, the global abundance conformed to three consistent <clusters>, as follows: <rat, mouse>, <gelada, macaque, olive baboon>, <gorilla, chimpanzee, bonobo, human>, which coincided with the phylogenetic distances of the selected species (p< 4E-05). Exceptionally, in the trinucleotide STR compartment, human was significantly distant from all other species. Conclusion: We propose that the global abundance of STRs is non-random in rodents and primates, and probably had a determining impact on the speciation of the two orders. We also propose the STRs and STR lengths which specifically coincided with the phylogeny of the selected species.

Development of a multiplex PCR short tandem repeat typing scheme for Candida krusei

Candida krusei is a human pathogenic yeast that can cause candidemia with the lowest 90-day survival rate in comparison to other Candida species. Infections occur frequently in immunocompromised patients and several C. krusei outbreaks in health care facilities have been described. Here, we developed a short tandem repeat (STR) typing scheme for C. krusei to allow for fast and cost-effective genotyping of an outbreak and compared identified relatedness of ten isolates to SNP calling from whole-genome sequencing (WGS). From a selection of 14 novel STR markers, six were used to develop two multiplex PCRs. Additionally, three previously reported markers were selected for a third multiplex PCR. In total, 119 C. krusei isolates were typed using these nine markers and 79 different genotypes were found. STR typing correlated well with WGS SNP typing, as isolates with the same STR genotype varied by 8 and 19 SNPs, while isolates that differed in all STR markers varied at least tens of thousands of SNPs. The STR typing assay was found to be specific for C. krusei , stable in 100 subcloned generations, and comparable to SNP calling by WGS. In summary, this newly developed C. krusei STR typing scheme is a fast, reliable, easy-to-interpret and cost-effective method compared to other typing methods. Moreover, the two newly developed multiplexes showed the same discriminatory power as all nine markers combined, indicating that multiplexes M3-1 and M9 are sufficient to type C. krusei .

First experiences with the Spectrum Compact CE System

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.

Rapidly Mutating Y Chromosomal STRs in Deep-Rooted Endogamous Pedigrees

Y-chromosome short tandem repeat polymorphisms (Y-STRs) are important in many areas of human genetics. Y chromosomal STRs being normally utilized in the field of forensic exhibit low haplotype diversity in endogamous populations and fail to discriminate among male relatives from same pedigree. Rapidly mutating Y-STRs (RM Y-STRs) have been paid much attention in last decade. These 13 RM Y STRs have high mutation rates (>10-2) and have considerably higher haplotype diversity and discrimination capacity than conventionally used Y-STRs showing remarkable power, when it comes to differentiation in paternal lineages in endogamous populations. Previously, we have analyzed 2–4 generation, 99 pedigrees covering 1568 pairs of men covering 1–6 meioses from all over Pakistan and 216 male relatives from 18 deep rooted endogamous Sindhi pedigrees covering 1-7 meioses. Here we are presenting 861 pairs of men from 63 endogamous pedigrees covering 1-6 meioses from Punjabi population of Punjab, Pakistan. Mutations were frequently observed at DYF399 and DYF403 while no mutation was observed at DYS526a/b. The rate of differentiation ranged from 29.70% (first meiosis) to 80.95% (fifth meiosis) while overall (1 to 6 meiosis) differentiation was 59.46%. Combining previously published data with newly generated data, an overall differentiation rate was 38.79% based on 5176 pairs of men related by 1–20 meioses, while Y-filer differentiation was 9.24% based on 3864 pairs. Using father-son pair data from the present and previous studies, we also provide updated RM Y-STR mutation rates.

Utilization of Short Tandem Repeat Analysis to Resolve Specimen Mislabeling in the Anatomic Pathology Laboratory

Introduction/Objective A mislabeled specimen is an example of preanalytical error that can have significant consequences on patient care. These errors can be difficult to detect and resolve. One method to confirm genetic identity is short tandem repeat (STR) analysis, which is utilized in forensic investigations, paternity studies, and post- hematopoietic stem cell transplantation monitoring. Herein we present application of STR analysis to resolve a suspected specimen mislabeling prior to receipt in our anatomic pathology laboratory. Methods/Case Report DNA was extracted from paraffin embedded tissues. Chimerism testing was performed by STR analysis using the Globalfiler (ThermoFisher Scientific) and analyzed by Chimermarker (Softgenetics) automated chimerism software. Results (if a Case Study enter NA) Colon biopsies were received for a single patient (#1) with two requisition forms. Each specimen (A-F) was labeled with the patient’s name, with specimens A-D noted on first page of requisition and specimens E-F on the second requisition page. After the case was signed out, the lab was contacted looking for biopsy results on another patient (#2) who was seen on the same day as patient #1. Review of all the patients seen in the endoscopy suite on the given date raised suspicion that specimens E-F from the second page of the requisition actually pertained to patient #2. STR analysis performed on specimens confirmed that specimens E-F were genetically distinct from those labelled A-D. Tissue from a subsequent biopsy on patient #2 was analyzed by STR testing, which was identical to STR results performed on specimens E-F. Conclusion Here we utilized STR testing to resolve a suspected mislabeled specimen, allowing the appropriate diagnosis to be attributed to the correct patient. This is a unique application of a common method, which could be implemented in anatomic pathology laboratories to resolve cases of specimen mix-ups.