Short Tandem Repeat Profiles Research Articles

Investigation of Linear Amplification Using Abasic Site-Containing Primers Coupled to Routine STR Typing for LT-DNA Analysis.

Obtaining a full short tandem repeat (STR) profile from a low template DNA (LT-DNA) still presents a challenge for conventional methods due to significant stochastic effects and polymerase slippage. A novel amplification method with a lower cost and higher accuracy is required to improve the DNA amount. Previous studies suggested that DNA polymerases without bypass activity could not perform processive DNA synthesis beyond abasic sites in vitro and our results showed a lack of bypass activity for Phusion, Pfu and KAPA DNA polymerases in this study. Based on this feature, we developed a novel linear amplification method, termed Linear Aamplification for double-stranded DNA using primers with abasic sites near 3′ end (abLAFD), to limit the replication error. The amplification efficiency was evaluated by qPCR analysis with a result of approximately a 130-fold increase in target DNA. In a LT-DNA analysis, the abLAFD method can be employed as a pre-PCR. Similar to nested PCRs, primer sets used for the abLAFD method were designed as external primers suitable for commercial multiplex STR amplification assays. The practical performance of the abLAFD method was evaluated by coupling it to a routine PP21 STR analysis using 50 pg and 25 pg DNA. Compared to reference profiles, all abLAFD profiles showed significantly recovered alleles, increased average peak height and heterozygote balance with a comparable stutter ratio. Altogether, our results support the theory that the abLAFD method is a promising strategy coupled to STR typing for forensic LT-DNA analysis.

Practical forensic use of kinship determination using high-density SNP profiling based on a microarray platform, focusing on low-quantity DNA

Instead of traditional short tandem repeat (STR) profiling, the genetic genealogy method, which uses hundreds of thousands of single nucleotide polymorphisms (SNPs) spread across genome-wide, has emerged as a powerful kinship determination tool and recently attracted great attention in forensic genetics.In this study, we explored the tolerance and viability of kinship discrimination based on a high-density SNP profile for forensic DNA, especially focusing on low-quantity DNA. Using the Affymetrix Genome-Wide Human SNP Array 6.0 platform (Thermo Fisher Scientific), the influence of low-quantity DNA on SNP genotype determination was evaluated. The low-quantity DNA samples failed once every few samples, the generated SNP profile had low data quality. Our investigation revealed that the SNP profile with low data quality contained many genotyping errors in which the SNP genotype changed from homozygote to heterozygote. The kinship discrimination analysis using KING software was directly influenced by these genotyping errors, which was confirmed that some unrelated pairs were mis-specified as 4th-degree relatives. We confirmed that the false heterozygous SNPs resulted in an inflation of kinship coefficient and a decrease of non-shared allele between a tested pair.To eliminate the influence of these genotyping errors and acquire an accurate kinship discrimination result, we developed a novel method to select only the robust SNPs, which stably give the genotype determination with high accuracy even in SNP profiles with low data quality. The application of our novel method led to the improved results of kinship discrimination up to the same level as in the SNP profile with high data quality.In addition, this study demonstrated the advantage of kinship analysis using a high-density SNP profile in the forensic field. It is well known that likelihood ratio calculation based on autosomal STR profile, which is the most commonly applied approach, has difficulty in gaining true kinship analysis results, especially when the relationship between the tested two individuals is more biologically distant. We showed the kinship discrimination analysis with a high-density SNP profile is more suitable for the case without close relatives, using the real case data. Although further study with larger samples will be necessary, this study indicated that practical forensic use of kinship determination with a high-density SNP profile would bring benefits to the forensic field.

DSMZCellDive: Diving into high-throughput cell line data

Human and animal cell lines serve as model systems in a wide range of life sciences such as cancer and infection research or drug screening. Reproducible data are highly dependent on authenticated, contaminant-free cell lines, no better delivered than by the official and certified biorepositories. Offering a web portal to high-throughput information on these model systems will facilitate working with and comparing to these references by data otherwise dispersed at different sources. We here provide DSMZCellDive to access a comprehensive data source on human and animal cell lines, freely available at celldive.dsmz.de. A wide variety of data sources are generated such as RNA-seq transcriptome data and STR (short tandem repeats) profiles. Several starting points ease entering the database via browsing, searching or visualising. This web tool is designed for further expansion on meta and high-throughput data to be generated in future. Explicated examples for the power of this novel tool include analysis of B-cell differentiation markers, homeo-oncogene expression, and measurement of genomic loss of heterozygosities by an enlarged STR panel of 17 loci. Sharing the data on cell lines by the biorepository itself will be of benefit to the scientific community since it (1) supports the selection of appropriate model cell lines, (2) ensures reliability, (3) avoids misleading data, (4) saves on additional experimentals,and (5) serves as reference for genomic and gene expression data.

DSMZCellDive: Diving into high-throughput cell line data

Human and animal cell lines serve as model systems in a wide range of life sciences such as cancer and infection research or drug screening. Reproducible data are highly dependent on authenticated, contaminant-free cell lines, no better delivered than by the official and certified biorepositories. Offering a web portal to high-throughput information on these model systems will facilitate working with and comparing to these references by data otherwise dispersed at different sources. We here provide DSMZCellDive to access a comprehensive data source on human and animal cell lines, freely available at celldive.dsmz.de. A wide variety of data sources are generated such as RNA-seq transcriptome data and STR (short tandem repeats) profiles. Several starting points ease entering the database via browsing, searching or visualising. This web tool is designed for further expansion on meta and high-throughput data to be generated in future. Explicated examples for the power of this novel tool include analysis of B-cell differentiation markers, homeo-oncogene expression, and measurement of genomic loss of heterozygosities by an enlarged STR panel of 17 loci. Sharing the data on cell lines by the biorepository itself will be of benefit to the scientific community since it (1) supports the selection of appropriate model cell lines, (2) ensures reliability, (3) avoids misleading data, (4) saves on additional experimentals, and (5) serves as reference for genomic and gene expression data.

Establishment and characterization of novel autologous pair cell lines from two Indian non‑habitual tongue carcinoma patients.

Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India due to very high percentage of patients with habits of smoking and chewing tobacco and associated products. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To understand disease progression in an improved way, knowledge of cross‑talk between tumor stroma and the tumor cells becomes indispensable. Patient‑derived invitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, particularly the cancer‑associated fibroblasts (CAFs). In the present study, two novel autologous pairs were immortalized spontaneously from non‑habitual, HPV‑positive patients, who presented with OTSCC. The epithelial and fibroblast cell lines had typical polygonal and spindle‑shaped morphology, respectively. Positive staining with epithelial specific Pan‑cytokeratin (PanCK) and fibroblast specific protein (FSP‑1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. An increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF‑conditioned medium proved the 'CAF‑ness' of the established fibroblast cells. The established cultures are the first of their kind which would serve as a useful platform in understanding the tumor‑stroma cross‑talk in tongue cancer progression.

Evaluating the Impact of Dropout and Genotyping Error on SNP-Based Kinship Analysis With Forensic Samples.

Technological advances in sequencing and single nucleotide polymorphism (SNP) genotyping microarray technology have facilitated advances in forensic analysis beyond short tandem repeat (STR) profiling, enabling the identification of unknown DNA samples and distant relationships. Forensic genetic genealogy (FGG) has facilitated the identification of distant relatives of both unidentified remains and unknown donors of crime scene DNA, invigorating the use of biological samples to resolve open cases. Forensic samples are often degraded or contain only trace amounts of DNA. In this study, the accuracy of genome-wide relatedness methods and identity by descent (IBD) segment approaches was evaluated in the presence of challenges commonly encountered with forensic data: missing data and genotyping error. Pedigree whole-genome simulations were used to estimate the genotypes of thousands of individuals with known relationships using multiple populations with different biogeographic ancestral origins. Simulations were also performed with varying error rates and types. Using these data, the performance of different methods for quantifying relatedness was benchmarked across these scenarios. When the genotyping error was low (<1%), IBD segment methods outperformed genome-wide relatedness methods for close relationships and are more accurate at distant relationship inference. However, with an increasing genotyping error (1–5%), methods that do not rely on IBD segment detection are more robust and outperform IBD segment methods. The reduced call rate had little impact on either class of methods. These results have implications for the use of dense SNP data in forensic genomics for distant kinship analysis and FGG, especially when the sample quality is low.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90°C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

Abstract 3079: Development of an automated platform for screening patient-derived organoid models

Abstract One of the major challenges in preclinical cancer therapeutic development is establishing physiologically relevant in vitro assays that correlate with the in vivo responses of patient tumors to anticancer agents. By incorporating tumor cell heterogeneity and three-dimensional morphological features, patient-derived organoids provide an improved in vivo relevancy compared to established tumor cell lines grown as monolayers. However, organoids grow while embedded in an extracellular matrix material with complex media formulations, which poses challenges for culture scale-up and automated drug screening methods. Organoid models derived from a variety of human solid tumor types are distributed by the National Cancer Institute’s Patient-Derived Models Repository program (https://pdmr.cancer.gov). A panel of human patient-derived colon adenocarcinoma organoid models was assembled to evaluate an automated high-throughput screening (HTS) platform. The organoid panel members were characterized for their reproducible growth and expansion capacity in culture, recovery from cryopreservation, and amenability to operations associated with HTS. Short tandem repeat profiling was performed regularly throughout the process to authenticate each sample. Among the organoid models, variations were observed in morphology (assessed by brightfield imaging) and growth rate (measured by population doublings). Most models expanded well in culture for greater than sixty days and all models demonstrated a sufficient recovery from cryopreservation. The aims in adapting organoid cultures to a HTS platform included minimizing the operational complexity, maximizing the process throughput, and maintaining high organoid viability. Assay conditions for all panel members were selected in conjunction with automated methods, instrumentation, and endpoint measurements. Details such as the optimal sample preparation steps, media formulation, and inoculation density varied among the organoid models. However, other aspects such as liquid handling procedures for organoid inoculation and drug delivery, microwell plate type, assay duration, and endpoint measurements were selected for their suitability to all organoid models tested. Using a custom-designed automated screening system, the refined methods were validated by screening the panel of patient-derived colon adenocarcinoma organoids against a library of oncology drugs approved by the United States Food and Drug Administration (https://dtp.cancer.gov/organization/dscb/obtaining/default.htm). Assay performance metrics and pharmacological data demonstrate the robust performance of this organoid screening platform. Future efforts will establish additional patient-derived organoid panels for expanded HTS using this platform. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Siddhartha Paul, Curtis Hose, Eric Jones, Erik Harris, John Connelly, Petreena Campbell, Mariaestela Ortiz, Thomas S. Dexheimer, Thomas Silvers, Penny Sellers Brady, Julie Grams, Tiffany Nikirk Rohrer, Karen Martin, Patricia Ramsey, Lori Bowles, Annamaria Rapisarda, Ralph E. Parchment, Beverly A. Teicher, James H. Doroshow, Nathan P. Coussens. Development of an automated platform for screening patient-derived organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3079.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

A survey of the effects of common illicit drugs on forensic DNA analysis

Profiling of DNA associated with illicit drug packages and paraphernalia is a common investigative tool. In addition, research is being conducted regarding the analysis of trace DNA present within illicit drugs and on capsules. The application of trace DNA analysis to illicit drugs has the potential to identify individuals involved in their manufacture and distribution. However, the inhibitory effects of illicit drugs and related compounds on downstream DNA analysis has not yet been investigated. If drug-induced polymerase chain reaction (PCR) inhibition occurs, the quality or informativeness of the resultant DNA profile may be impacted. In this study, the effects of a range of drugs, diluents, adulterants, and synthetic precursors on both quantitative PCR (qPCR) data and short tandem repeat (STR) DNA profiling results were examined. Twenty-two compounds representative of drug compounds and adulterants which may be encountered in drug seizures were spiked with 1 ng/μL and 0.05 ng/μL of control DNA and underwent DNA quantification using Quantifiler™ Trio. A subset of 13 compounds, including the majority that indicated potential inhibition in Quantifiler™ Trio, underwent STR profiling with VeriFiler™ Plus to determine if inhibition also occurred at this stage. The effect of diluting the DNA extract on the extent of inhibition of STR profiling was also investigated. Internal PCR controls within the qPCR were not a reliable indicator of inhibition, although suppression of the short and long autosomal fragments was observed in the presence of many compounds, and four compounds gave inconclusive results. STR internal quality controls indicated inhibition in 5 of the 13 compounds, however, profiles were affected by the presence of 11 of the 13 compounds in various ways such as a decreased average relative fluorescence units (RFU), drop out of certain alleles (some based on allele size range of locus) leading to a decreased likelihood ratio (LR), an increase in the proportion of stutter peaks and the presence of split or shoulder peaks. All profiles improved following a dilution of the compound in the PCR and allowing the generation of LR values in excess of 1 × 1025, indicating inhibition occurred rather than DNA degradation. The data obtained show that removal of some of these compounds is required through an effective DNA extraction process for successful downstream trace DNA profiling. Upon successful PCR, the resultant DNA profiles provide the opportunity for opening new investigative avenues for law enforcement agencies.

Noninvasive isolation of transcervical trophoblast cells for fetal identification.

To identify trophoblastic cells retrieved from the cervix at a gestational age (GA) of 5-9 weeks by a noninvasive modality in fetuses. Transcervical cells (TCCs) were noninvasively extracted by a cytobrush using the Papanicolaou sampling method. TCCs were immunostained with antihuman leukocyte antigen (HLA)-G and anticytokeratin (CK)-7 antibodies to identify trophoblastic cells. Maternal finger blood, gestational sacs, and 20 trophoblastic cells collected by a laser-guided microscopic single-cell capture system were examined and compared by short tandem repeat (STR) genotyping. Forty-nine pregnant women with GA of 5-9 weeks and six nonpregnant healthy women were included in the study. Trophoblastic cells were identified in 37 (75.5%) TCC samples, among which 34 (69.4%) were eligible for STR genotyping analysis. No trophoblastic cells were identified in nonpregnant healthy women. The STR genotyping analyses revealed 24 female and 10 male fetuses. TCC trophoblastic cells exhibited the same STR profiles as gestational sac and maternal blood in all samples, which indicated that the TCC trophoblastic cells originated from fetuses. This primary study validated that trophoblastic cells from TCCs at GA 5-9 weeks originated from the fetus. Further studies are needed to verify whether this method can be used for early noninvasive prenatal diagnosis and paternity testing.

DSMZCellDive: Diving into high-throughput cell line data.

Human and animal cell lines serve as model systems in a wide range of life sciences such as cancer and infection research or drug screening. Reproducible data are highly dependent on authenticated, contaminant-free cell lines, no better delivered than by the official and certified biorepositories. Offering a web portal to high-throughput information on these model systems will facilitate working with and comparing to these references by data otherwise dispersed at different sources. We here provide DSMZCellDive to access a comprehensive data source on human and animal cell lines, freely available at celldive.dsmz.de. A wide variety of data sources are generated such as RNA-seq transcriptome data and STR (short tandem repeats) profiles. Several starting points ease entering the database via browsing, searching or visualising. This web tool is designed for further expansion on meta and high-throughput data to be generated in future. Explicated examples for the power of this novel tool include analysis of B-cell differentiation markers, homeo-oncogene expression, and measurement of genomic loss of heterozygosities by an enlarged STR panel of 17 loci. Sharing the data on cell lines by the biorepository itself will be of benefit to the scientific community since it (1) supports the selection of appropriate model cell lines, (2) ensures reliability, (3) avoids misleading data, (4) saves on additional experimentals,and (5) serves as reference for genomic and gene expression data.

Carrier RNA is a key factor affecting fully integrated short tandem repeats profiling in challenging forensic samples models

BackgroundShort tandem repeats (STRs) are used today to provide discriminatory power for DNA fingerprinting. The present results showed that different factors may affect STR profiles in challenging samples including DNA quantity, DNA quality, PCR inhibitors and storage time. In the present study, blood stain samples were applied on two types of fabrics (black cotton and denim) to compare the efficiency of two different DNA-extraction methods (automated magnetic based beads method (EZ1), and manual organic method), with and without adding carrier RNA molecules, and to assess the quality and quantity of the extracted DNA and their capabilities for producing reportable STR-profiles in the presence of PCR inhibitors at two different storage times.ResultsCarrier RNA caused a dramatic increase in DNA recovery from black cotton or denim using EZ1 in contrast to organic method. EZ1 was found to be preferred than organic, especially when a time passed over, while organic method was preferred when samples are available in small quantities. In addition, using carrier RNA within the organic method steps showed no improvement in STR profiling. EZ1 with carrier RNA was preferred for bloodstained samples on fabrics with textile dyes (black dye or denim indigo), especially when stored for a long time.ConclusionsDenim was found to be more problematic than black cotton due to presence of challenging inhibitors (indigo dye). DNA concentration, storage time and types of fabrics are key factors for choosing the appropriate extraction method for reportable STR profile. Using EZ1 with carrier RNA gives less dropout profile than not using it, or when using organic method even in presence or absence of carrier RNA. Anyway, innovation of more sensitive, more robust analytical protocols could result in a better understanding of these inhibitory samples.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/μL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

Establishment of four head and neck squamous cell carcinoma cell lines: importance of reference DNA for accurate genomic characterisation.

There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines. A cell culture based, in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours. Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent). Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls.

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system.

The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)‐based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all‐encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.

Establishment and characterization of a novel highly malignant lung cancer cell line ZX2021H derived from a metastatic lymph node lesion.

BackgroundLung cancer is a highly malignant tumor with a poor prognosis. The establishment of faithful ex vivo cell models is essential for investigating the metastatic mechanisms and developing new anticancer agents. In this study, we established and characterized a novel lung cancer cell line derived from metastatic lymph node tissue from a Chinese patient.MethodsA primary culture of metastatic lymph node tissue from a patient with lung cancer was used to establish a cell line. The phenotypic characteristics of the cell line were characterized by colony‐formation, CCK8, and Transwell assays, and xenografting. The genetic characteristics were evaluated by chromosome analysis, short tandem repeat (STR) profiling, and quantitative real time‐polymerase chain reaction (qRT‐PCR).ResultsA novel lung cancer cell line, named ZX2021H, was successfully established from a metastatic lymph node lesion from a lung cancer patient. The cell line exhibited high capacities for proliferation and invasion, as validated by its phenotypic and genetic characteristics. This cell line had a unique STR profile and karyotype analysis revealed numerical and structural chromosome abnormalities. The growth rate of in vivo xenografted tumors established using ZX2021H cells was higher than that using H1299 cells. The cell stemness‐related gene SOX2 was overexpressed in ZX2021 compared with H1299 cells, as determined by qRT‐PCR.ConclusionWe successfully established a novel, highly malignant lung cancer cell line, ZX2021H, derived from metastatic lymph node tissue from a Chinese lung cancer patient. This cell line may provide an ideal cell model for further studies of the molecular mechanisms underlying lung cancer metastasis and for the development of new anticancer agents.

QueSTR probes: Quencher-labeled RNase H2-dependent probes for Short Tandem Repeat genotyping

Forensic Short Tandem Repeat (STR) genotyping is almost exclusively performed by capillary electrophoresis (CE) in specialized laboratories. As an alternative to CE, and to enable miniaturized lab-on-a-chip STR profiling, we developed the QueSTR probes, a hybridization-based genotyping assay that relies on the recognition and cleavage of an RNA:DNA duplex by the RNase H2 enzyme. For each STR allele to be genotyped, a matching DNA probe containing one RNA moiety is designed. After performing asymmetric STR PCR, a hybridization curve analysis indicates the matching probe(s), and thus indicates the allele(s) present in the sample. Accurate genotyping of 13 samples was obtained using the QueSTR probes for three CODIS core loci (D16S539, D7S820, and TH01). A probe corresponding to the TH01 9.3 allele was included to demonstrate accurate genotyping, even in the presence of a partial repeat. The QueSTR probes are a valuable option to miniaturize STR genotyping in lab-on-a-chip devices that cannot harbor a CE analysis.