Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination

Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing.

Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends

Algorithms for automated detection of hook effect-bearing amplification curves

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect. Other detection chemistries also exhibit this negative trend, however the underlying molecular mechanisms are different.In this study we present two approaches to automatically detect hook effect-like curvatures based on linear (hookreg) and nonlinear regression (hookregNL). As the hook effect is typical for qPCR data, both algorithms can be employed for the automated identification of regular structured qPCR curves. Therefore, our algorithms streamline quality control, but can also be used for assay optimization or machine learning. Keywords: Hook effect, QPCR, TaqMan, EvaGreen, SybrGreen, Linear, Nonlinear, RDML, Automatio

Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA

<div><p>Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.</p></div

SARS-CoV-2 Subunit Virus-like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study

Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of “Betuvax-CoV-2”, a vaccine developed as a subunit vaccine containing a recombinant RBD-Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant (“Betuspheres”). The study aimed to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, “Betuvax-CoV-2” was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway

Lorenz curves of PicoPlex and iDOP-PCR WGA samples.

<p>A Lorenz curve gives the cumulative fraction of reads as a function of the cumulative fraction of genome. Perfectly uniform coverage would result in a diagonal line (black). PicoPlex (red curve) and iDOP-PCR (blue curve) generate similar deviations from the diagonal as a result of biased coverage. All samples were sequenced at 8x depth.</p

Comparison by NGS the parameters of PicoPlex and iDOP-PCR whole-genome amplification of single-genome-copies.

<p>Comparison by NGS the parameters of PicoPlex and iDOP-PCR whole-genome amplification of single-genome-copies.</p

Multiplex STR genotyping of WGA samples and non-amplified gDNA.

<p>Multiplex STR genotyping of WGA samples and non-amplified gDNA.</p

Electrophoretic analysis of WGA libraries by Agilent 2200 TapeStation Instrument.

<p>The libraries were obtained by DOP-PCR (lane 1), PicoPlex (lane 2), and iDOP-PCR (lane 3) from 15 pg of human gDNA. <b>M</b>–DNA marker “Genomic DNA ScreenTape”.</p