Zygotic-splitting after in vitro fertilization and prenatal parenthood testing after suspected embryo mix-up - a case report.

After in vitro fertilization with a single embryo, the parents learned about being pregnant with twins in the 10th week with various indications that an embryonic mix-up could have taken place. The affected couple thus expressed the urgent desire for a clarification of parenthood considering an abortion. However, the prenatal test results would not have been available until the 14/15th week of pregnancy. Legally, then, severe physical or mental distress of the pregnant woman must be claimed by physicians to justify an abortion after the twelfth week. However, a lack of genetic relatedness could lead to serious psychological distress for the parents, making a pregnancy termination possible even after the twelfth week, which is discussed in this case study alongside the interdisciplinary team's ethical, legal, and medical considerations.For the invasive relationship testing, cultivated chorionic villi samples (CVS) from both unborn and saliva samples from the putative parents were genetically analyzed using classical short tandem repeats (STR) analysis. The perfect match of both CVS profiles suggested the occurrence of an unusual late twin shaft, for which, fortunately, parenthood could be confirmed. To our knowledge, this is the first report on a prenatal investigation of a suspected embryo mix-up after assisted reproductive technology (ART), in which parenthood should be fixed. We want to draw attention to this unthinkable scenario, which may increase in the future with ART-induced rising multiple pregnancies.

Genetic Structure Analysis of 155 Transboundary and Local Populations of Cattle (Bos taurus, Bos indicus and Bos grunniens) Based on STR Markers.

Every week, 1-2 breeds of farm animals, including local cattle, disappear in the world. As the keepers of rare allelic variants, native breeds potentially expand the range of genetic solutions to possible problems of the future, which means that the study of the genetic structure of these breeds is an urgent task. Providing nomadic herders with valuable resources necessary for life, domestic yaks have also become an important object of study. In order to determine the population genetic characteristics, and clarify the phylogenetic relationships of modern representatives of 155 cattle populations from different regions of the world, we collected a large set of STR data (10,250 individuals), including unique native cattle, 12 yak populations from Russia, Mongolia and Kyrgyzstan, as well as zebu breeds. Estimation of main population genetic parameters, phylogenetic analysis, principal component analysis and Bayesian cluster analysis allowed us to refine genetic structure and provided insights in relationships of native populations, transboundary breeds and populations of domestic yak. Our results can find practical application in conservation programs of endangered breeds, as well as become the basis for future fundamental research.

About the influence of environmental factors on the persistence of DNA - a long-term study.

DNA persistence and DNA transfer are important features in the assessment of a crime scene. The question how long DNA may persist at a certain location is similarly important as the one how the DNA has been transferred to this location. Depending on the source of the DNA as well as the conditions at the crime scene, the answer to this question is quite difficult. In this study, persistence of DNA from epithelial abrasions, blood cells, and saliva cells in indoor and outdoor scenarios has been investigated with regard to exposure time and exposure conditions including sunlight, temperature, and humidity in summer and winter scenarios. Overall, we generated 338 epithelial samples, 572 blood samples, and 572 saliva samples. A complete profile of the cell/DNA donor after exposure could be obtained in 47%, 65%, and 58% of epithelial abrasions, blood samples, and saliva samples, respectively. Regarding blood samples, there were no differences between supporting materials cloth and plastic; however, the percentage of complete profiles was higher for saliva samples on plastic and for epithelial samples on cloth. In indoor scenarios, complete profiles could be recovered from nearly all blood and saliva samples up to 9 months, whereas the amount of epithelial complete profiles already started to decline after 3 months. In outdoor scenarios, we observed a tipping point at an exposure time of 3 months. Blood and saliva samples collected after this period displayed complete profiles in less than 25% of samples. After 12 months, no outdoor sample showed a complete profile. The results of this study facilitate decisions on the relevance of recovered DNA from crime scenes.

"I've never been at the crime scene!" - gloves as carriers for secondary DNA transfer.

Over recent years, DNA profiling techniques have become highly sensitive. Even small amounts of DNA at crime scenes can be analysed leading to new defence strategies. At court, defence lawyers rarely question the existence of a DNA trace (source level) but challenge how the DNA was transferred to the scene (activity level). Nowadays, the most common defence strategy is to claim that somebody else had stolen the defendant's gloves and used them while breaking and entering. In this study we tested this statement. Using gloves made of different material (cloth, leather, rubber) and varying secondary transfer surfaces (wood, metal, glass), we simulated a few of the most likely transfer scenarios that occur during breaking and entering. While we detected the presence of DNA on the outside of 92 of the 98 gloves tested, we observed only one case of secondary transfer in a total of 81 transfer experiments. This data demonstrates that secondary transfer under conditions resembling realistic conditions is a very rare event.

Forensic nanopore sequencing of STRs and SNPs using Verogen's ForenSeq DNA Signature Prep Kit and MinION.

The MinION nanopore sequencing device (Oxford Nanopore Technologies, Oxford, UK) is the smallest commercially available sequencer and can be used outside of conventional laboratories. The use of the MinION for forensic applications, however, is hindered by the high error rate of nanopore sequencing. One approach to solving this problem is to identify forensic genetic markers that can consistently be typed correctly based on nanopore sequencing. In this pilot study, we explored the use of nanopore sequencing for single nucleotide polymorphism (SNP) and short tandem repeat (STR) profiling using Verogen's (San Diego, CA, USA) ForenSeq DNA Signature Prep Kit. Thirty single-contributor samples and DNA standard material 2800 M were genotyped using the Illumina (San Diego, CA, USA) MiSeq FGx and MinION (with R9.4.1 flow cells) devices. With an optimized cutoff for allelic imbalance, all 94 identity-informative SNP loci could be genotyped reliably using the MinION device, with an overall accuracy of 99.958% (1 error among 2926 genotypes). STR typing was notably error prone, and its accuracy was locus dependent. We developed a custom-made bioinformatics workflow, and finally selected 13 autosomal STRs, 14 Y-STRs, and 4 X-STRs showing high consistency between nanopore and Illumina sequencing among the tested samples. These SNP and STR loci could be candidates for panel design for forensic analysis based on nanopore sequencing.

Enrichment of circulating trophoblasts from maternal blood using laminar microscale vortices.

OBJECTIVE: Enrichment of circulating trophoblasts (CTs) from maternal blood at week 11-13 of gestation, using laminar microscale vortices, and evaluation of the performance of the VTX-1 Liquid Biopsy System in terms of CT recovery and purity. METHOD: Eight mililiter of blood was collected from 15 pregnant women and processed with the VTX-1 Liquid Biopsy System. Y-chromosome specific quantitative PCR was performed to estimate the number of enriched male CTs. To evaluate the VTX-1 performance, the target cell recovery was characterized by spiking experiments with a trophoblast cell line. Furthermore, the total quantity of DNA after enrichment was used to calculate the number of retained maternal cells. RESULTS: Successful recovery of male CTs was established in 7 out of 10 first trimester samples from pregnant women carrying a male fetus. The number of CTs, recovered from 8 ml of blood, was estimated between two and six. Spiking experiments resulted in a CT recovery of ±35 % with ±1524 retained maternal blood cells. CONCLUSION: CTs can be enriched from maternal blood with high purity, using laminar microscale vortices, starting from 8 ml of blood.

Effect of sodium hypochlorite decontamination on the DNA recovery from human teeth.

Genetic identification of skeletal human remains is often realized by short tandem repeat (STR) genotyping of nuclear DNA. Dental DNA is preferred to DNA from bone for the better protection of the endogenous DNA. Especially if whole tooth grinding is intended to access the DNA, contaminations with exogenous DNA have to be avoided. The immersion of the tooth in sodium hypochlorite (NaOCl, known as bleach) is one common procedure to clean the outer surface from extraneous DNA and PCR inhibitors. To investigate the impact of bleaching on endogenous DNA and the decontamination success, 71 recently extracted teeth were differently treated with sodium hypochlorite (2.5 or 5.0% NaOCl for 30 or 60 s, 5.0% NaOCl for 10 min, and control group) in the beginning of the extraction process, whereas equally handled afterwards. Quantitative and qualitative evaluation of the extracted DNA was performed. There was a great variation for the DNA concentration of the extracts even within a group of the same NaOCl treatment. Complete DNA profiles from single persons with alleles for the 16 ESS (European Standard Set) STR loci were obtained for all regarded teeth. A statistically significant difference between the DNA yields of the treatment groups was not determined. Moreover, a negative effect of NaOCl (2.5% and 5.0%) on the DNA recovery could not be observed. Significant larger amounts of DNA were extracted from anterior teeth in contrast to posterior teeth.

Cleaning a crime scene 2.0-what to do with the bloody knife after the crime?

The persistence of DNA on washed items as well as the DNA transfer has become a major subject of research in recent years, especially after the detectability of minor DNA traces was heavily increased by sensitive analysis methods. Nowadays, the attribution of a DNA trace to an individual is only rarely questioned, whereas the way of application of this DNA to an item is subject to much discussion and speculation. Additionally, the removal of DNA by cleaning or its possible persistence on an item despite a cleaning process are often important problems in court. The aim of this study was to investigate whether DNA traces (blood, saliva, epithelial cells) on different objects (knives, plates, glasses, and plastic lids) can persist on the surface despite cleaning by different methods like hand-washing or the use of a dishwasher. In total, 120 samples were collected from artificially constructed blood, saliva, and epithelial cell stains on objects with smooth surfaces after washing and analyzed by STR amplification. Samples taken after rinsing or hand-washing resulted mainly in complete DNA profiles (62.5% of samples), while cleaning in the dishwasher rendered almost everything completely DNA-free. Since in the hand-washing experiments a secondary transfer of DNA through the water could not be ruled out, additional transfer experiments were conducted with blood and saliva samples on plates. Here, a carryover of DNA traces could be demonstrated up to the fifth washed item.

Identification of tetragametic human chimerism by routine DNA profiling.

Chimerism in humans is defined as the presence of two genetically different cell lines within the same organism. It is usually an acquired condition that is restricted to certain tissues and can be explained by therapeutic interventions such as blood transfusion or the transplantation of allogenic hematopoietic cells. Implications of such patients for forensic DNA testing have been described in the literature. In some rare cases, true inherited chimerism is observed. This so called tetragametic chimerism occurs via the fertilization of the two ova by two spermatozoa, followed by the fusion of early embryos and the development of an organism with intermingled cell lines. Such examples have been found in mice and other mammalian species including humans. We describe a phenotypically normal woman in whom tetragametic chimerism (46,XX/46,XX) was unexpectedly identified by STR typing during routine DNA profiling. Cytogenetic analysis proved to be a valuable tool for both independent confirmation and direct visualization of the two coexisting cell lines.

Unintentional effects of cleaning a crime scene-when the sponge becomes an accomplice in DNA transfer.

DNA transfer in aqueous solutions as well as the persistence of DNA on washed items has become a major subject of research in recent years and is often a significant problem in court. Despite these approaches, the question about the "mobility" of DNA especially in capital offenses cannot be answered in every case, since a variety of scenarios for DNA transfer are possible. The aim of this study was to investigate whether DNA traces could be distributed by cleaning an object. For this purpose, a large table surface and fabric piece were artificially provided with skin contact traces and body fluids (saliva and blood) in two series of experiments and then wiped off with water or with soap water (218 samples in total). These experiments resulted in a clear "carry over" of DNA traces especially for body fluid samples (100% of blood samples and 75% of saliva samples led to a complete profile). The results could be confirmed in a second experimental set-up with 384 samples using different cleaning agents and more intense cleaning actions. Even small amounts of 5-10 µl body fluid led to complete profiles in around 45% of the samples, while 20 µl led to nearly 65% complete profiles. A strong impact of the amount of traces and the chosen surface could be demonstrated, while the active component of the cleaning agent seemed to be of less influence with the explicit exception of chloric agents which rendered almost everything completely DNA-free. In summary, a distribution of DNA traces by wiping or scrubbing an object could be clearly proven.

Establishment and characterization of a cell line HCS1220 from human liver metastasis of colon cancer.

BACKGROUND: To establish one primary cell line of human liver metastasis of colon cancer. METHODS: HCS1220 cell line was derived from one liver metastasis of colon cancer patient's resected tumor sample. The characterization of the cell line was defined by karyotype analysis, short tandem repeat (STR) analysis and mycoplasma contamination. Subcutaneous injection 1 × 106 cells to four BALB/c nude mice, the viable tumors were developed and diagnosed (H&E staining). The expression of biomarkers CK20 and CDX2 for colon cancer were determined by immunocytochemistry assay. RESULTS: HCS1220 cell line can grow stably and continuously passage. During the grow process, the contact loss in the growth process and superimposed growth, which could be defined as proliferation of malignant tumor. Chromosome analysis revealed the cells derived from human female. The cells were not contaminated by mycoplasma. By immunohistochemistry, the cell line was proven to express the biomarkers of colon cancer CK20 and CDX2, while a-fetoprotein, hep-1 and glypican-3 were stained negative, which demonstrated that the HCS1220 cell line originating from the intestinal tissue. CONCLUSIONS: HCS1220 cell line has the characteristics of primary human liver metastasis of colon cancer. The results of STR have genetically showed that cell line is original, which can provided cell materials for research in vitro and can also help for establishing the mechanism model of liver metastasis of colon cancer and preparing, screening and evaluating anti-tumor drugs.

Impact of several wearers on the persistence of DNA on clothes-a study with experimental scenarios.

The detection of DNA of a certain person on the inside of a piece of clothing involved in a crime scene is usually seen as confirmation that this person is the owner or bearer and therefore participated in this crime. However, besides the possibilities of secondary or even tertiary transfer of DNA, the accused often argues that he lent the garment to another person who by chance did not leave any DNA while committing the crime. Then, forensic genetic scientists have to answer the question how long DNA persists on an item used in daily routine and how long a piece of clothing must be worn to definitively leave detectable DNA behind. In an attempt to answer these questions, several scenarios with two or three individuals wearing the same sweatband for different time periods were set up. DNA left on the sweatbands was isolated, quantified, and then analyzed using the Powerplex® ESX17fast kit. The majority of samples displayed all alleles of both/all three wearers on the outside (67%) as well as on the inside (80%) of the sweatbands. In contrast, a single profile of the first wearer could only be found once among all 204 samples, a single profile of the second wearer in 7% of samples. Wearing the sweatband for only 10 min was enough to result in a complete profile of the second wearer in 79% of samples. So, it is highly unlikely to wear/use a piece of clothing for even a short period of time without leaving own DNA behind.

Comparison of different methods for repairing damaged DNA from buffered and unbuffered formalin-fixed tissues.

Formalin fixation is considered an important process for preservation of human tissue samples for long periods. However, this process not only results in cross-linking complicating isolation of nucleic acid but also introduces polymerase "blocks" during polymerase chain reaction (PCR). At present, many protocols have already been developed aiming at extracting high amounts of amplifiable DNA from formalin-fixed tissues (FFTs). However, there are few methods for repairing formalin-damaged DNA. In this study, we compared the effectiveness of several post-extraction enzymatic repair techniques, including Taq DNA polymerase, DNA polymerase I and T4 DNA ligase, the PreCR™ Repair Mix and Restorase® DNA Polymerase, in restoring STR profiles from formalin-damaged DNA. Our results indicated that formalin-damaged DNA may be repaired partly with Taq DNA polymerase and the Restorase® DNA Polymerase, and lost alleles may be restored and STR peak heights may increase upon repair with them. Moreover, the repair ability of the protocol 2 with Taq DNA polymerase surpasses the Restorase® DNA Polymerase.

Persistence of DNA on clothes after exposure to water for different time periods-a study on bathtub, pond, and river.

DNA traces on clothes of drowned bodies can provide important evidence for police investigations, especially in cases of suspected suicides or homicides. However, it is generally assumed that the water "erodes" a large part of the DNA depending especially on the exposure time. In forensic casework, DNA of suspects could be found frequently on clothes of drowned bodies after hours, sometimes days of exposure to water. This study was conducted to attempt a general statement about the conditions under which sufficient DNA remains can be expected for molecular genetic analysis. For this purpose, different scenarios were designed including DNA from three to five people, different types of waters (tap, pond, bathtub and river) for various time periods, with higher water pressure, different temperature, and soapy water (bathtub). Epithelial cells and blood cells were mounted on cotton cloths, and the DNA left after exposure was analyzed using the Powerplex® ESX17fast kit. In the indoor experiments, complete profiles could be seen even after 10 min rinsing of clothes under the tap and after 1 week in the bathtub. Outdoors, the results differed considerably between summer and winter as well as between pond and river. The longest exposure time still resulting in a complete profile was 2 weeks for a sample with skin cells in the pond during winter. In summer, the time period for erasing the bulk of DNA was 4 hours regarding epithelial samples and more than 1 day for blood samples in pond and river environments. All in all, the results demonstrate that DNA could still be recovered from clothes exposed to water for more than 1 week.

Laundry in a washing machine as a mediator of secondary and tertiary DNA transfer.

The aim of this work was to investigate the possibility of secondary and tertiary DNA transfer during laundry. The modes of transfer tested were mixed and separate laundry of worn and unworn garments in household and public washing machines. In addition, the possibility of a background DNA carry-over from a washing machine's drum was investigated. In the mixed (worn and unworn garments washed together) laundry experiment, 22% of samples from new unworn socks with no traceable DNA prior to experiment produced DNA profiles post-laundry. In the tertiary DNA transfer experiment performed in a public washing machine (unworn garments only), no detectable DNA profiles were observed. Samples collected from the internal drum of 25 washing and drying machines did not produce detectable STR profiles. The implications of these results are discussed in the context of forensic DNA casework analysis. Graphical Abstract ᅟA real-life scenario of secondary DNA transfer between worn and unworn garments during machine washing has been evaluated. Experiments demonstrated this scenario is possible (22% of samples) and may in fact result in high quality DNA profiles. On the contrary, testing washing machine's interior for deposition of biological material between separate washing cycles to serve as a mediator of tertiary DNA transfer resulted in no DNA profiles.

Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study.

OBJECTIVES: The human salivary gland (HSG) cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study radiation therapy, Sjögren's syndrome, pleomorphic adenoma, mucocele, epithelial-to-mesenchymal transition, and epigenetics. However, the American Type Culture Collection (ATCC) has recently released a list of cross-contaminated cell lines that included HSG. Despite this notice, some research laboratories still use HSG as a salivary cell model. Therefore, this study examined the authenticity of HSG sampled from three different laboratories. METHODS: DNA was extracted from HSG and additional salivary cell lines (NS-SV-AC, NS-SV-DC, A253, HSY) and submitted for cell line authentication with short tandem repeat (STR) analysis. RESULTS: All HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases. This confirmed that HSG sampled from three different laboratories and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles. CONCLUSION: Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.

Probabilistic peak detection in CE-LIF for STR DNA typing.

In this work, we present a novel probabilistic peak detection algorithm based on a Bayesian framework for forensic DNA analysis. The proposed method aims at an exhaustive use of raw electropherogram data from a laser-induced fluorescence multi-CE system. As the raw data are informative up to a single data point, the conventional threshold-based approaches discard relevant forensic information early in the data analysis pipeline. Our proposed method assigns a posterior probability reflecting the data point's relevance with respect to peak detection criteria. Peaks of low intensity generated from a truly existing allele can thus constitute evidential value instead of fully discarding them and contemplating a potential allele drop-out. This way of working utilizes the information available within each individual data point and thus avoids making early (binary) decisions on the data analysis that can lead to error propagation. The proposed method was tested and compared to the application of a set threshold as is current practice in forensic STR DNA profiling. The new method was found to yield a significant improvement in the number of alleles identified, regardless of the peak heights and deviation from Gaussian shape.

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.

A preliminary assessment of the ForenSeq™ FGx System: next generation sequencing of an STR and SNP multiplex.

The ForenSeq™ FGx System (Illumina, San Diego, CA) was initially evaluated in concordance with SWGDAM guidelines for internal validation to determine the quality of the system's components: the ForenSeq™ DNA Signature Prep Kit reagents, the MiSeq FGx™ instrument, and the ForenSeq™ Universal Analysis Software, for the analysis of targeted, forensically informative single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). This multiplex consisted of STRs (autosomal, X, and Y) and SNPs (identity, ancestry, and phenotypic) that were run using one preparation process. Overall, the ForenSeq™ FGx System performed as well as the traditional capillary electrophoresis-based method in producing usable profile information, along with additional information that could aid in investigative leads. The MiSeq FGx™ System was validated using DNA samples in studies testing reproducibility, repeatability, concordance, sensitivity, and mock case single donor samples. Overall, genotyping results for STRs and SNPs were concordant with the profiles generated from conventional STR analysis using Identifiler and SNPs typed by 23andMe analysis. Genotypes of the ForenSeq™ aSNPs were used to evaluate biogeographical ancestry estimations using ForenSeq™ Universal Analysis Software, FROG-kb database (KIDD aiSNP 55 panel), and 23andMe. The system was shown to provide reproducible genotypes and reliable results were obtained at levels as low as 50 pg. All mock case samples were concordant with the donor profile. The results support consideration of the ForenSeq™ FGx System as an acceptable alternative to current STR and SNP analysis, pending formal developmental and internal validation studies.

Genetic profiling reveals an alarming rate of cross-contamination among human cell lines used in China.

Cell lines are widely used as in vitro model systems in biologic and medical research. However, much of the research has been invalidated by the unwitting use of false cell lines. A significant proportion of the research involving human cell lines was initiated in China. Paradoxically, the cell lines used in China have never been authenticated. Here, we present a comprehensive survey of cross-contamination in 380 samples from 113 independent sources in China using short tandem repeat profiling methods. High levels of cross-contamination were uncovered (95 of 380, 25%). Notable false cell lines (e.g., KB and WISH) are still actively used under their false identity and tissue attributions. Most strikingly, 85.51% of lines established in China were misidentified (59 of 69) and accounted for over half of the misidentifications (59 of 95, 62.11%). Further, 93.22% of the contaminants in cell lines established in laboratories of China were HeLa cells or a possible hybrid of HeLa with an unknown cell line. Results from these misidentified lines have been published in thousands of potentially erroneous articles and may have distorted the findings visible to the scientific community. False lines have been used in drug screening, potentially leading to unusable or even harmful therapeutic strategies. We also noted the causes of contamination and provided suggestions for remediation.