Sufficient Amount Of DNA Research Articles

Analysis of single nucleotide polymorphism of p16 gene in cytological samples of patients with oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC)

Backgroundpl6 (CDKN2a) play a role in tumorigenesis in some head and neck squamous cell carcinomas. Frequent homozygous deletions of the pl6 gene have been reported in many tumor cell lines including the brain, breast, osteosarcomas, melanomas, kidney, bladder, and ovary. Materials and methods5 patients without any tobacco using habit were included in group I as controls. 10 clinically and histopathologically confirmed cases of oral potentially malignant disorders (OPMDs) [oral submucous fibrosis (OSMF) −7 & leukoplakia (moderate dysplasia) −3] were included in group II. 10 clinically and histopathologically confirmed cases of OSCC were categorized as group III. Buccal scrapings were taken and analyzed for exon 1, 2, 3 of p16 and homozygous deletion in exon 2 of p16 was also detected by PCR and gel electrophoresis. ResultsPCR amplification products of exon 1, 2, 3 of p16 were found in all 25 samples which include 10 cases of OSCC, 10 cases of OPMDs and 5 controls. Homozygous deletion in exon 2 was found only in 30 % of OSCC and 20 % of OPMD. ConclusionOur study showed that cytological samples yield sufficient amount of DNA, which showed 100 % positivity with exon 1, 2, 3 of p16 gene, but the percentage of genetic alteration of p16 gene seems to be less when compared with other related studies.

Saliva-derived secondary DNA transfer on fabric: The impact of varying conditions

This study explored secondary DNA transfer involving saliva, a body fluid often encountered in forensic investigations. Various factors were examined to investigate their potential impact on the transfer of DNA from saliva stains deposited onto common types of fabric (cotton, nylon, and towel). We examined varying types of saliva moisture (wet, dry, and rehydrated) and different types of contact (controlled pressure and active/friction pressure) to quantitatively evaluate how such variables could impact transfer and possible conclusions surrounding saliva-derived DNA deposits. The transfer of DNA was generally least pronounced with more absorbent primary fabrics (cotton and towel materials) while a less absorbent primary fabric (nylon) exhibited a greater propensity for DNA transfer. There were significantly higher amounts of transferred DNA (p < 0.05) observed in wet saliva samples compared to dry and rehydrated saliva samples. Further, the use of active pressure (friction) appeared to result in more DNA transfer overall as compared to controlled pressure contact. Experiments conducted with wet saliva and active pressure (friction) demonstrated the highest likelihood of transfer, with the primary nylon and secondary towel fabric combination demonstrating the greatest average transfer percentage of 94.74 %. The variables explored in this study presented multiple combinations wherein a sufficient amount of DNA (≥ 240 pg total) was transferred to the secondary fabric, making it potentially suitable for STR-PCR amplification in our laboratory. The findings from this study indicate that the type of primary fabric receiving the saliva deposit, the type of saliva moisture, the type of secondary fabric and its moisture type, and the type of contact all have the potential to affect the quantity of DNA transferred and recovered. This study provides empirical data on the ease, and to what extent, DNA from saliva transfers between fabrics and aids DNA activity level evaluations. The significance of this research lies in its contribution to expanding our current understanding of DNA transfer involving saliva within forensic science and criminal investigations.

Whole-exome sequencing is feasible on a fresh-frozen skin sample of intravascular large B cell lymphoma

Intravascular large B-cell lymphoma (IVLBCL) is a rare aggressive extranodal non-Hodgkin lymphoma. The predominant, if not exclusive, growth of neoplastic cells within the lumina of small-sized vessels represents the hallmark of the disease. Diagnosis is challenging due to the absence of marked lymphadenopathy, the highly heterogeneous clinical presentation, and the rarity of the condition. Clinical presentation is characterized by variable combinations of nonspecific signs and symptoms (such as fever and weight loss), organ-specific focal manifestations due to altered perfusion, and hemophagocytic syndrome. The rarity of this entity and the paucity of neoplastic cells in biopsy samples hamper the study of recurrent molecular abnormalities. The purpose of this study was to explore the feasibility of a different approach to recover a sufficient amount of DNA of acceptable quality to perform next-generation sequencing studies. Here, we report the findings of whole-exome next-generation sequencing performed on a fresh-frozen cutaneous sample of IVLBCL, paired with the patient saliva used as germline DNA. To increase the cancer cell fraction, only the subcutaneous tissue was selected. With this approach, we obtained high-quality DNA and were able to identify oncogenic mutations specific for this entity and recapitulating its post-germinal center origin, even if the tumor fraction was low. Molecular studies performed on fresh-frozen cutaneous sample are feasible in IVLBCL, especially when analysis is restricted to the subcutaneous tissue. Wide adoption of this reproducible and cost-effective approach may foster further studies, which may be of help in supporting diagnosis, providing pathogenetic insights, and guiding treatment decisions.

Подбор и оптимизация методов экстракции ДНК из листьев гледичии трехколючковой (Gleditsia triacanthos L.)

Abstract. Currently, molecular genetic methods using DNA markers are increasingly used in studies of polymorphism of various populations of woody and shrubby plants. The purpose of this work was the evaluation and selection of protocols for the isolation and purification of DNA from the leaves of Gleditsia triacanthos L. for further studies using DNA labeling. Methods. Four protocols were used to isolate DNA from the leaf blade of Gleditsia triacanthos L. Anionic detergent sodium dodecyl sulfate was used in three isolation protocols for cell lysis, potassium acetate was used for purification from polysaccharides and proteins. In the fourth protocol, a cationic surfactant cetyltrimethyl ammonium bromide was used for cell lysis, the extract was purified with a mixture of chloroform-isoamyl alcohol (24 : 1). Precipitation of the isolated DNA was carried out with isopropanol. The quality of the isolated DNA was evaluated by spectrophotometry, horizontal electrophoresis and Real-time PCR with two types of primers. Results. Optimal conditions for DNA extraction from samples of Gleditsia triacanthos L. containing a large number of metabolites affecting the quality of the isolated extract were selected. By electrophoresis, it was found that both the isolation protocol with sodium dodecyl sulfate and the isolation protocol with cetyltrimethyl ammonium bromide make it possible to obtain a sufficient amount of DNA. The most purified DNA was obtained by the third protocol using sodium dodecyl sulfate and dithiotreitol and by the fourth protocol using cetyltrimethylammonium bromide. The results of PCR of the obtained samples with ITS and psbI-psbK primers indicate that a sufficient amount of product has been obtained and the reproducibility of ISSR markers. The scientific novelty of the work consists in choosing the optimal method of DNA extraction from the leaves of Gleditsia triacanthos L., which is a complex object containing a large number of potential PCR inhibitors. The protocol with sodium dodecyl sulfate and dithiotreitol made it possible to obtain DNA in the right amount and of acceptable quality.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review

Nucleic acid extraction from biological sources has evolved with time and is currently one of the most important contributors to the development of modern human molecular diagnostics. Pure and sufficient quantity of DNA and RNA from limited quantity of cells, which may be fresh or archived, holds key to successful detection of source and cause of many diseases. The challenges of cell disruption to release nucleic acids and the wide range of strategies applied to purify them by removing various enzyme inhibitors are discussed. The demographic spread of the disease of tuberculosis, hurdles faced during transportation of the clinical samples for nucleic acid extraction of Mycobacterium tuberculosis pathogen and different detection technologies supported by the extracted pathogen nucleic acid are reviewed.

Cell-Free DNA from Blood or Bone Marrow as Alternatives to Bone Marrow Cells for Molecular Diagnostics and Monitoring of Multiple Myeloma

Introduction: Molecular profiling of bone marrow (BM) tumor cells in multiple myeloma (MM) can be limited by low tumor cell content for analysis by multiple assays. We therefore evaluated the concordance of molecular profiles of cancer cells enriched from BM versus cell-free DNA (cfDNA) derived from peripheral blood (PB) and BM plasma collected at diagnosis. Use of cfDNA has the potential to enable the prioritization of scarce cancer cells for single-cell RNA-sequencing or other applications that require intact cells. Methods: The MM Molecular Monitoring (M4) Study is a prospective cohort study of newly diagnosed MM patients (pts) receiving standard-of-care treatment at 12 sites across Canada. From 27 pts in this study, we analyzed diagnostic BM-derived CD138+ enriched plasma cell (PC) DNA (n=27, 15-20 mL BM per pt), BM plasma cfDNA (n=26 same tube as BM sampling), PB cfDNA (n=27, 15-20 mL), and PB buffy coat DNA (n=27, 4-6mL) using a combination of a targeted next generation sequencing (NGS) panel (10,000X coverage) and shallow (0.1-1X) whole-genome sequencing (sWGS). PB and BM samples collected in STRECK and EDTA tubes, respectively incurred overnight shipping to a central lab for initial processing. The targeted panel includes exons from 37 genes of potential clinical relevance, all immunoglobulin (Ig) loci, and 5 MM-specific translocation hotspots. CD138+ PCs were profiled by clinical FISH for recurrent MM translocations and copy number variations (CNVs). Results: All PB and BM cfDNA samples yielded sufficient DNA quantity for analyses (&amp;gt;20ng), yet only 19/26 BM cfDNA samples passed quality control and were sequenced. Various strategies such as shearing, dilution, and the use of heparinase were not effective in salvaging the failed BM cfDNA. However, the high success rate in a separate local study (24/25 BM cfDNA samples) suggests shipping in non-cell stabilizing tubes may have contributed to failures. The average DNA yield for PB and BM cfDNA samples was 1408ng (sd = 2752ng) and 2462ng (sd = 4301ng), respectively. We estimated tumor fraction (TF) by examining the proportion of genome altered by CNVs using ichorCNA in samples with sWGS. 14/24 (58%) of PB cfDNA samples and 10/18 (56%) of BM cfDNA samples had a high TF (&amp;gt;5%). TF in PB and BM cfDNA were significantly correlated (rho = 0.85, p &amp;lt; 0.001, n = 18 pairs), but were consistently higher in PB cfDNA (p = 0.03, n = 18 pairs). We first compared mutations between BM-derived PCs and cfDNA. We found 12 distinct somatic mutations (variant allele fraction &amp;gt;1%) that were previously reported to have a pathogenic effect in 11/27 (41%) PC samples. This consisted of 10 single nucleotide variants (SNVs) and 2 deletions across NRAS, BRAF, KRAS, CYLD and PRDM1. We found 9/12 mutations in matched PB cfDNA samples [sensitivity (SN)=75%] and 4/8 mutations in matched BM cfDNA (SN = 50%). We next searched for FISH-validated chromosome rearrangements among enriched BM PCs. From these, we correctly detected 5/8 (63%) fusions using the targeted panel that were called out by clinical FISH. These were further detected in matched PB cfDNA (4/5) and BM cfDNA (2/4) samples. Recurrent MM CNVs (amplification at chromosome 1q or deletions at chromosomes 1p, 13p or 17p) were identified in 18/27 (67%) PC samples by sWGS. These CNVs were successfully detected in 8/9 matched PB cfDNA samples with high TF (by sWGS) and in 2/7 samples with low TF. In BM cfDNA, CNVs were successfully detected in 7/8 high TF samples and 1/6 low TF samples. Lastly, we evaluated BCR Ig rearrangements. We detected a clonal Ig rearrangement in 26/27 (96%) BM-derived PC samples. These clones were detected in 25/26 (96%) matched PC to PB cfDNA pairs and in 16/18 (89%) PC to BM cfDNA pairs. The proportion of fragments at Ig loci which corresponded to productive Ig rearrangements was significantly correlated with TF in PB cfDNA (rho = 0.62, p = 0.002, n = 23) but not in BM cfDNA (rho = 0.37, p = 0.14, n = 17). Conclusions: PB cfDNA yielded 78% of the aberrations detected in BM-derived PCs, whereas BM cfDNA yielded 62%. The lower rate of detection in BM cfDNA may reflect DNA contamination from dying white blood cells as a result of shipping BM samples in non-cell stabilizing preservative tubes. BM and PB cfDNA contain highly concordant CNV calls to PCs when cfDNA TF estimates were &amp;gt;5%. Tumor Ig rearrangements were more readily detected in cfDNA compared to other sources of variation and may provide a less-invasive alternative for disease monitoring.

O-225 May embryonic cfDNA in the culture media play a role as a non-invasive assessment of aneuploidy in PGT

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent blastocyst media (SBM) is a promising technique that has the potential to revolutionize current preimplantation genetic testing procedures. This technique is based in the analysis of the cell-free DNA (cfDNA) released by the embryo during the latest stages of preimplantation development. It allows for the detection of chromosomal abnormalities in embryos without the need for invasive procedures that can harm the embryo (reviewed in Navarro-Sánchez et al., 2022). Our group among others has conducted different studies in this field, and the early clinical application of the cfDNA analysis in the culture medium has been proposed as a screening tool, giving a priority score for embryo transfer based on the media results (Rubio et al., 2019, 2020). In the final analysis of our multicenter study in day-6 and day-7 blastocysts, we have been able to compare the chromosomal results of the SBM with trophectoderm (TE) biopsies in 2,187 samples, and with the inner cell mass ICM in 230 blastocysts. In this study, the analysis of the cfDNA showed a concordance rate of 87% with the ICM biopsy, which is currently considered the gold standard for PGT-A. However, it is important to continue evaluating the factors that may affect the informativity and concordance rates of cfDNA-based assays, such as the culture day when medium is collected, contamination with external and/or cumulus cell DNA, and previous manipulation of the embryos. Multivariate analysis has shown that the longer the time in culture, the higher the informativity rate, and female age had the greatest impact on concordance rates with TE biopsies. Regarding technical aspects, the SBMs with higher number of reads after sequencing resulted in higher concordance rates, reflecting the importance of analysing samples with sufficient DNA quantity. Finally, the predictive value in terms of concordance with TE biopsies was higher for aneuploid blastocyst. No significant independent correlation was observed for embryo quality. The improvements in IVF lab protocols and the use of time-lapse and short incubation protocols for vitrified-thawed blastocysts are also noteworthy, as they may further increase the concordance rates of cfDNA-based assays with blastocysts. In fact, concordance in previously vitrified-thawed blastocysts as result as high as 90%. Regarding clinical outcomes, several groups have evaluated non-invasive PGT-A with transfer of euploid embryos showing good results. In a pilot study with only 7 SETs in couples with PGT-A or PGT-SR indication, 5 pregnancies were achieved resulting in 5 livebirths (Xu et al., 2016). In a second study, in couples with recurrent spontaneous abortion or repeated implantation failure, 50 transfers of euploid media were performed resulting in a 58% clinical pregnancy rate, with 27 healthy babies born (Fang et al., 2019). Similar results were described in good prognosis patients &amp;lt;38 years of age with ongoing pregnancy rates comparable to PGT-A (61.5%), and higher than conventional IVF or ICSI (48.5%) (Franco et al., 2021). Overall, the high concordance rates with trophectoderm biopsies and inner cell mass biopsies are encouraging, and the clinical outcomes with transfer of euploid embryos are promising. It is exciting to see the progress in non-invasive preimplantation genetic testing using cfDNA in culture medium, and it will be interesting to see how this technology evolves and its potential role as a screening or diagnostic tool in the future.

Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

Establishment of a Standard Tongue Coating Collection Method for Microbiome Studies.

Objective: Recently, researchers have been focusing on characterizing the tongue coating microbiome from patients with digestive tract disease. However, to the best of our knowledge, the tongue coating collection methods have not been standardized until now. This article focuses on bridging this gap by exploring and validating the conditions suitable for the collection of tongue coating samples. Methods: One hundred forty-one healthy subjects were involved in the standardization of the tongue coating collection method. We conducted our standardization experiment by comparing different sampling tools, different preservation solutions, different scraping times, and different storage days with preservation at room temperature. The tongue coating samples from 59 normal individuals were analyzed using 16S ribosomal RNA (rRNA) gene-sequencing technology. The assessment of the quality of extracted DNA was used to verify our established method. We separated the 59 subjects into two groups (aged and younger), and the sequencing results were used to explore the age-related changes in microbiome. Results: Sterile oral swab B is suitable for the collection of tongue coating samples. To obtain a sufficient amount of DNA from a tongue coating sample, we recommend 30 times of tongue coating scraping. Normal saline, phosphate-buffered saline, and commercial preservation solution are all suitable for short-term sample storage (<1 hour). The commercial long-term preservation solution, which stores samples at room temperature (0 hour to 7 days) and can provide for fast commercial transportation, ensures the integrity of the sample DNA as well as the stability of the DNA quality. By using the established method, extracted DNA from all the 59 normal individuals' tongue coating samples passed an appropriate quality bar for microbiome studies. The average value of OD 260/280 is 1.72 ± 0.10; the average total DNA amount is 334.92 ng (±183.81 ng). The bacterial diversity of the tongue coating is increased and the bacterial community composition changes greatly in the NC group (aged normal subjects). Fusobacteriota is found as the dominant bacteria phyla in aged normal subjects with the 16S rRNA gene-sequencing technology. At the genus level, the relative abundance of Fusobacterium, Haemophilus, and Leptotrichia are significantly higher in aged individuals (all p < 0.05), and Neisseria, Streptococcus, and Porphyromonas are significantly higher in younger individuals (all p < 0.05). Conclusion: A participant-friendly tongue coating collection method for microbiome analyses can be established with good reliability and reproducibility. By taking advantage of our established method and 16S rRNA gene sequencing, significant differences were found in diversity and composition of tongue coating microbiota between aged and younger individuals, which contributes to a better understanding of the age-related composition of tongue coating microbiota.

Preserving avian blood and DNA sampled in the wild: A survey of personal experiences.

Collecting and storing biological material from wild animals in a way that does not deteriorate DNA quality for subsequent analyses is instrumental for research in ecology and evolution. Our aims were to gather reports on the effectiveness of methods commonly used by researchers for the field collection and long‐term storage of blood samples and DNA extracts from wild birds. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses but were not related to season of blood sampling, access to equipment, transporting samples, temperature, and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.

Bioaerosols in the atmosphere at two sites in Northern Europe in spring 2021: Outline of an experimental campaign

A coordinated observational and modelling campaign targeting biogenic aerosols in the air was performed during spring 2021 at two locations in Northern Europe: Helsinki (Finland) and Siauliai (Lithuania), approximately 500 km from each other in north-south direction. The campaign started on March 1, 2021 in Siauliai (12 March in Helsinki) and continued till mid-May in Siauliai (end of May in Helsinki), thus recording the transition of the atmospheric biogenic aerosols profile from winter to summer.The observations included a variety of samplers working on different principles. The core of the program was based on 2- and 2.4--hourly sampling in Helsinki and Siauliai, respectively, with sticky slides (Hirst 24-h trap in Helsinki, Rapid-E slides in Siauliai). The slides were subsequently processed extracting the DNA from the collected aerosols, which was further sequenced using the 3-rd generation sequencing technology. The core sampling was accompanied with daily and daytime sampling using standard filter collectors. The hourly aerosol concentrations at the Helsinki monitoring site were obtained with a Poleno flow cytometer, which could recognize some of the aerosol types.The sampling campaign was supported by numerical modelling. For every sample, SILAM model was applied to calculate its footprint and to predict anthropogenic and natural aerosol concentrations, at both observation sites.The first results confirmed the feasibility of the DNA collection by the applied techniques: all but one delivered sufficient amount of DNA for the following analysis, in over 40% of the cases sufficient for direct DNA sequencing without the PCR step. A substantial variability of the DNA yield has been noticed, generally not following the diurnal variations of the total-aerosol concentrations, which themselves showed variability not related to daytime. An expected upward trend of the biological material amount towards summer was observed but the day-to-day variability was large.The campaign DNA analysis produced the first high-resolution dataset of bioaerosol composition in the North-European spring. It also highlighted the deficiency of generic DNA databases in applications to atmospheric biota: about 40% of samples were not identified with standard bioinformatic methods.

Overcoming barriers to tumor genomic profiling through direct patient social media outreach.

6532 Background: Tumor genomic profiling is increasingly used to identify actionable genomic alterations as a guide to therapy selection. To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. Methods: Patients were recruited through social media outreach, engagement with disease advocacy groups, or via physician referral, with a focus on recruiting patients with histiocytosis, germ cell tumors and rare pediatric cancers. Tumor and patient-matched germline DNA were analyzed using the MSK-IMPACT targeted sequencing next generation sequencing panel with return of results to patients and their local physicians. Whole exome recapture of MSK-IMPACT DNA sequencing libraries was performed for patients with female germ cell tumors to define the genomic landscape of this rare cancer subtype. Results: 359 cancer patients expressed interest in the Make-an-IMPACT program, of whom 333 were enrolled. Tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quantity and quality for MSK-IMPACT testing. 14 histiocytosis patients have received genomically guided therapy to date, of whom 13 (93%) have had clinical benefit based on local MD response assessment with a mean treatment duration of 16.7 months (range 3-32+). Whole exome sequencing of ovarian GCTs identified a subset with fully haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%), however, 2 ovarian GCTs and squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. Conclusions: Social media outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians where they could be used to guide treatment selection. This can also open the door to diversifying and being able to study the genomic landscape in a diverse cohort.

Comparing DNA yield from fish scales following different extraction protocols

Studies on genetic diversity, adaptive potential and fitness of species have become a major tool in conservation biology. These studies require biological material containing a reliable source of DNA which can be extracted and analysed. Recently, non-invasive sampling has become the preferred sampling method of such biological material; particularly when studying endangered species. Elasmoid scales from teleost fish are an example of non-invasive samples from which DNA can successfully be extracted. This study compared different extraction protocols to find an optimal method for extracting DNA from teleost fish scales. This was done with the intent to use the protocol that yielded the highest quantity of DNA on dried, archived scales. The protocols tested in this study included (1) phenol/chloroform with a TNES-urea digestion buffer, (2) phenol/chloroform with an amniocyte digestion buffer and (3) Qiagen DNeasy Blood and Tissue Kit with variations in incubation times and temperatures of each protocol. While the phenol/chloroform with TNES-urea digestion buffer yielded significantly higher concentrations of DNA compared to the other protocols, all protocols followed in this study yielded sufficient quantities of DNA for further downstream applications. Therefore, while there are multiple viable options when selecting a DNA extraction protocol, each research project’s individual needs, requirements and resources need to be carefully considered in order to choose the most effective protocol.

Effect of formalin fixation on DNA: A Time-Based Approach

Introduction:Tissue preservation is important. From it forensic scientist may extract sufficient DNA for profiling. The most commonly used preservative found in the literature is formalin. However, it causes severe side-effects on its users as well as the environment; we need to find its replacement. Method:This study examined whether formalin could preserve soft tissues (fresh) stored at 4°C for 100 days and how DNA can be extracted from it. Qualification and Quantification of the presevred samples were done. Result:The results revealed that tissues preserved in formalin failed to generate sufficient quantity of DNA for profiling whereas tissues preserved in normal saline did so. Conclusion: The study concluded that there is a need to find an alternative to formalin which can preserve the tissue samples well and enable DNA profiling.

Minor GPI(-) Granulocyte Populations in Patients with Acquired Aplastic Anemia and Healthy Individuals Are Derived from a Few Piga-Mutated Hematopoietic Stem Progenitor Cells

[Background] Minor populations (0.003%-1.0%) of glycosylphosphatidylinositol-anchored protein-deficient granulocytes (GPI[-] Gs) are often detected in the peripheral blood (PB) of patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes and are thought to represent immune pathophysiology of bone marrow failure. We previously reported that minor GPI(-) G populations were detected in some healthy individuals (HIs) and persisted over several years at similar percentages (Katagiri T, et al. Stem Cells 2013). Several lines of evidence have suggested that small numbers of GPI(-) Gs detected in HIs are polyclonal populations mostly derived from short-lived PIGA-mutated committed progenitor cells. Minor GPI(-) G populations in AA patients may also be derived from multiple committed progenitor cells rather than from a few hematopoietic stem progenitor cells (HSPCs) with PIGA mutations. However, minor GPI(-) G populations usually persist for a long period of time at similar frequencies in AA patients, suggesting that they may instead be derived from a few HSPCs that have undergone PIGA mutations. This issue remains debated due to the inability to sequence the PIGA gene in the very few GPI(-) granulocytes available. We recently developed a sensitive method capable of detecting PIGA mutations in minor GPI(-) Gs using amplicon sequencing of GPI(-) Gs that were enriched with magnetic microbeads followed by FACS sorting. Using this method, we addressed whether minor GPI(-) G populations in AA patients and HIs are oligoclonal or polyclonal as well as which cell population they are derived from HSPCs or committed progenitor cells.[Methods] Five AA patients possessing 0.025%-0.898% GPI(-) Gs, 3 HIs who were found to have ≥0.003% (0.006%, 0.051%, and 0.059%) GPI(-) Gs during a screening of more than 200 HIs for GPI(-) Gs, 30 HIs (median: 37 years old, male/female:17/13) with 0% to 0.002% GPI(-) Gs, and 8 cord blood (CB) samples were subjected to enrichment of GPI(-) Gs for PIGA sequencing. Their leukocytes were treated with PE-labelled anti-CD55 monoclonal antibodies (mAbs) and anti-CD59 mAbs, and CD55 +CD59 + granulocytes were removed using magnetic microbeads labelled with anti-PE mAbs. CD11b +FLAER-negative granulocytes were sorted from the remaining granulocytes using FACSAria Fusion. DNA from sorted GPI(-) Gs was amplified using primers covering all exons of PIGA. Nucleotide sequences of the PIGA gene in GPI(-) Gs were determined using a next-generation sequencer.[Results] This novel enrichment method enabled the detection of only 1-4 different PIGA mutations in all 5 female AA patients (AA 1-5) with the total of different VAFs in each case reaching nearly 50% (Table 1). Limited kinds of PIGA mutations were also detected in three HIs (two males [HI 1 and 3] and one female [HI 2]). For HI 1 and HI 3, the VAFs of predominant PIGA-mutated sequences were longitudinally measurable using whole-blood DNA samples with droplet digital PCR, which showed no apparent changes in the VAF (0.020%-0.027% for HI 1 and 0.012%-0.025% for HI 3 over 4 and 6 years, respectively). The presence of mono or oligoclonal GPI(-) Gs in the 3 HIs prompted us to study 30 HIs who had been judged to be negative for minor GPI(-) G populations by a high-sensitivity flow cytometry method. The enrichment method unexpectedly identified clear CD11b highFLAER - GPI(-) G clusters in granulocytes from 24 of the 30 HIs (Figure 1a). The median number of GPI(-) Gs contained in 7 ml of PB was 31 (range, 1-136 cells). Sufficient amounts of DNA for NGS were obtained from sorted GPI(-) Gs of six subjects, and PIGA amplicon sequencing revealed 1-3 different PIGA mutations in four of the six subjects. The examination of fresh CB also revealed clear GPI(-) G clusters in four of eight samples (Figure 1b). PIGA amplicon sequencing of 79 GPI(-) Gs obtained from 1 male CB sample (CB 1) showed a sole PIGA mutation with VAFs of 95% (Figure 1c).[Conclusion] Minor GPI(-) G populations detectable in patients with AA and HIs are derived from a few PIGA-mutated HSPCs, not from committed myeloid progenitor cells, a finding that negates a hypothesis that a few PIGA-mutated HSPCs are selected from polyclonal PIGA-mutated HSPCs during transition from AA to florid PNH. Very small numbers of GPI(-) Gs are present much more frequently in HIs than previously thought and may also be derived from a few HSPCs with PIGA mutations that occur in HSPCs during the fetal stage. [Display omitted] DisclosuresTakamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Research Funding; Kyowa Kirin: Honoraria. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

Refining the evolutionary time machine: An assessment of whole genome amplification using single historical Daphnia eggs.

Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimized workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10 and 300years old. Resulting genome coverage breadth in most samples was ~70%, including those from >100-year-old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with single nucleotide polymorphisms in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300years). The mitochondrial DNA provides an additional source for evolutionary studies of these populations. We provide an optimized workflow for WGA followed by whole genome sequencing including steps to minimize exogenous DNA.

DNA profiling of single sperm cells after whole genome amplification

Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analyzing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.

Human identification from washed blood stains

BackgroundAmong the physical evidence, bloodstain is one of the most common biological pieces of evidence at the crime scene, especially in violent crimes. Bloodstains are frequently seen at the crime scene and can be easily detected due to their color and structure. Because blood and bloodstains can potentially be evidence of a crime, offenders often tend to clean or wash them to get rid of relevant evidence. Some researchers think that washed bloodstains cannot obtain enough quality DNA for DNA profiling. However, some studies have shown that bloodstains on a piece of fabric can be used for DNA profiling even after washing. This study, it is aimed to determine whether a sufficient amount of DNA can be obtained for DNA profiling used for forensic purposes from blood-stained nylon and cotton fabrics washed at different temperatures such as 40 °C, 60 °C and 90 °C.ResultsIn this study, bloodstains were created on cotton and nylon fabrics in a representative crime scene. After washing with laundry detergent at 40 °C, 60 °C and 90 °C was performed, studies were conducted in order to make bloodstains visible and obtain DNA for genetic profiling. The result is that more DNA is lost due to easy exposure to external factors in the non-absorbent nylon fabric type compared to the absorbent cotton fabric. Moreover, the rates of obtaining DNA from bloodstains on different types of fabrics washed at several temperatures are shown in this study.ConclusionsIt was determined that while a sufficient amount of DNA for the forensic genetic profiling can be obtained from cotton and nylon fabrics washed at 40 and 60 °C, a sufficient amount could not be obtained from the ones washed at 90 °C. And that shows even if bloodstained fabrics are washed at 60 °C, the fabrics still can be considered as physical evidence of the crime and can be associated with the scene, perpetrator and victim triangle.

Connectivity within isolation: dispersal, population genetics, and conservation of the rarest European damselfly

Abstract Coenagrion hylas (Trybom, 1899) has a very limited distribution in Europe, lives in very small, isolated populations, has rather specialised habitat demands, and is regarded as the rarest damselfly of Europe. Using a combination of capture‐mark‐recapture and population genetics, we aimed to evaluate the state of the populations in the Tyrolean Lech valley and to test whether exuviae from this species are usable as a DNA source. DNA was extracted from mid‐leg tibiae and exuviae and genotyped with species‐specific microsatellite markers. The results from the capture–recapture and the genetic methods were congruent. Coenagrion hylas has an unexpectedly high tendency to disperse within the valley, covering distances of up to 30 km, and lives longer than other damselflies, with an average longevity of 12 days and a maximum lifespan of at least 40 days. Low inbreeding coefficients and low ranges of genetic differentiation across sites provide evidence of panmixia, with no clear signs of inbreeding. The current population size is estimated at 1150 males based on the recapture data. We further demonstrated that exuviae deliver a sufficient amount of DNA, which will be important for future monitoring. Although C. hylas currently shows appropriate viability at most Lech valley sites, our study indicates that management measurements, such as creating stepping stone habitats, are crucial to maintain the current population status. Given the high dispersal capability of the species, such management measurements seem promising.