Something old, something new, a DNA preparation procedure for long read genomic sequencing

Extracting high molecular weight DNA is critical for successful long read DNA sequencing. Here we present a DNA preparation procedure combining CTAB DNA extraction protocol and a short read eliminator kit, which consistently produces high yields of high molecular weight DNA in the freshwater microcrustacean Daphnia. This method only requires common chemicals and equipment, providing researchers a viable alternative to DNA extraction kits that often require specialized tools.

Comparative Investigation of the Suitability of DNA Preparation Methods for Microbiome Analysis of Complex Clinical Samples

Efficient DNA preparation is essential for accurate and reproducible microbial data acquisition. In this study an in-house method (NWU lysis system), involving a lysis micro tube (LMT) was employed to determine its suitability for safe, rapid and accurate characterisation of the bacterial microbiome associated with clinical samples in comparison to two available commercial DNA extraction kits. During the experimental setup, it was confirmed that the LMT is suitable for downstream bacterial microbiome analyses with the incorporation of short read sequencing; but due to the fragmented nature of the DNA its suitability is highly dependent on the sequencing technology applied downstream. The study also served to demonstrate that shearing of DNA can have a significant impact on downstream microbiome analyses, based on the sequencing technology used; and that care must be taken especially if long read sequencing is to be used employed.

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Exonuclease combinations reduce noises in 3D genomics technologies

Chromosome conformation-capture technologies are widely used in 3D genomics; however, experimentally, such methods have high-noise limitations and, therefore, require significant bioinformatics efforts to extract reliable distal interactions. Miscellaneous undesired linear DNAs, present during proximity-ligation, represent a main noise source, which needs to be minimized or eliminated. In this study, different exonuclease combinations were tested to remove linear DNA fragments from a circularized DNA preparation. This method efficiently removed linear DNAs, raised the proportion of annulation and increased the valid-pairs ratio from ∼40% to ∼80% for enhanced interaction detection in standard Hi-C. This strategy is applicable for development of various 3D genomics technologies, or optimization of Hi-C sequencing efficiency.

EVALUATING THE EFFECTIVENESS OF THE TUMOR-INITIATING STEM CELLS ERADICATION STRATEGY ON THE EXAMPLE OF HUMAN GLIOBLASTOMA CELL LINE U87

The effectiveness of the new therapeutic approach aimed at the destruction of the cancer cell community was evaluated on the immortalized human glioblastoma cell line U87. Initially, the reference elements of a new strategy for eradication of tumor-initiating stem cells were characterized. The main points of the strategy were as follows. 1) Evaluation of the TAMRA+ (tumor-initiating stem cells) presence in the population of U87 culture cells. 2) Determination of the reference time points of the DNA interstrand cross-links repair, induced by cross-linking cytostatics mitomycin C. 3) Determination of the day after initiation of therapy when TAMRA+ cells accumulate and are synchronously present in the G1/S sensitive for the treatment phase of the cell cycle. Based on the data obtained, a therapeutic regimen aimed at eradicating TAMRA+ cells (tumor-initiating stem cells) was identified. Treatment of the culture was carried out by cross-linking cytostatic mitomycin C and complex DNA preparation. Transplantation experiments showed that high experimental doses of mitomycin C (20 μg/ml) totally destroy transplantation potential of U87 cells. Diminution of mitomycin C to therapeutic doses (5 μg/ ml) clearly demonstrated effect of complex double-stranded DNA preparation to reducing of U87 tumorigenic potential. These fully confirmed the concept of developed cancer treatment technology.