Pyrosequencing enhancement for better detection limit and sequencing homopolymers.

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.

Zygotic resetting of the HISTONE 3 variant repertoire participates in epigenetic reprogramming in Arabidopsis.

In most eukaryotes, the HISTONE 3 family comprises several variants distinguished by their amino acid sequence, localization, and correlation with transcriptional activity. Transgenerational inheritance of epigenetic information carried by histones is still unclear. In addition to covalent histone modifications, the mosaic distribution of H3 variants onto chromatin has been proposed to provide a new level of epigenetic information. To study the transmission of patterns of H3 variants through generations, we combined transcriptional profiling and live imaging of the 13 H3 variants encoded by the Arabidopsis plant genome. In comparison with somatic cells, only a restricted number of H3 variants are present in male and female gametes. Upon fertilization, H3 variants contributed by both gametes are actively removed from the zygote chromatin. The somatic H3 composition is restored in the embryo by de novo synthesis of H3 variants. A survey of Arabidopsis homologs of animal H3 chaperones suggests that removal of parental H3 from the zygote nucleus relies on a new mechanism. Our results suggest that reprogramming of parental genomes in the zygote limits the inheritance of epigenetic information carried by H3 variants across generations.