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

Development of qPCR Detection Assay for Potato Pathogen <i>Pectobacterium atrosepticum</i> Based on a Unique Target Sequence

The recent taxonomic diversification of bacterial genera Pectobacterium and Dickeya, which cause soft rot in plants, focuses attention on the need for improvement of existing methods for the detection and differentiation of these phytopathogens. This research presents a whole genome-based approach to the selection of marker sequences unique to particular species of Pectobacterium. The quantitative real-time PCR assay developed is selective in the context of all tested Pectobacterium atrosepticum strains and is able to detect fewer than 102 copies of target DNA per reaction. The presence of plant DNA extract did not affect the sensitivity of the assay

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

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

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

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