One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics

Background Accessible chromatin landscape allows binding of transcription factors, and remodeling of promoter and enhancer elements during development. Chromatin accessibility along with integrated multiomics approaches have been used for determining molecular subtypes of cancer in patient samples. Results One-pot Universal NicE-seq (One-pot UniNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. One-pot UniNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in as low as 25 fixed cells. Accessible chromatin profile is more efficient on formaldehyde-fixed cells using one-pot UniNicE-seq compared to Tn5 transposon mediated methods, demonstrating its versatility. Conclusion One-pot UniNicE-seq allows the entire process of accessible chromatin labeling and enrichment in one pot at 4% formaldehyde cross-linking conditions. It doesn’t require enzyme titration, compared to other technologies, since accessible chromatin is labelled with 5mC incorporation and deter degradation by nicking enzyme, thus opening the possibility for automation.

Optimization of Enzymatic Fragmentation is Crucial To Maximize Genome Coverage: A Comparison of Library Preparation Methods for Illumina Sequencing

Background Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 µg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits. Results We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits` fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection. Conclusions The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Directed homoenzymatic fragmentation of the plant protopectin complex: Assessment criteria

Introduction. The functional basis of protopectin complex can be represented as a network of regions that consist of homogalacturonan sequences and a base of rhamnogalacturonans-I, i.e. rhamnosyl-containing branching sites. Enzymatic isolation of these regions is possible only at a certain minimal native degree of polymerization. The research objective was to develop a system of criteria for assessing the potential applicability of the enzymatic transformation of plant protopectin complex. Study objects and methods. The research featured the polymerization degree of the homogalacturonan regions of the protopectin complex and produced a system of assessment criteria for the enzymatic fragmentation potential of the protopectin complex. The theoretical calculations were based on the values of the mass fractions of rhamnosyl and galacturonide residues in plant cell walls. The result was a new polymerization degree analytical function. Results and discussion. The ratio of the mass fractions of rhamnosyl and galacturonide residues in the water-insoluble plant tissue served as a dimensionless criterion of applicability. The rational condition for the dimensionless criterion of applicability was based on the fundamental constraint for homogalacturonan regions in the protopectin complex. It was expressed by a fundamental inequation. The rational area for determining the numerical values of the applicability criterion was presented as . The functional dependence was reduced to a two-dimensional criteria space as “width of rhamnosyl branches vs. the criterion of applicability”, where each pectin-containing raw material was given a single uniquely defined position. The boundary conditions for the criteria space were determined analytically. Conclusion. The new approach offers an assessment of the enzymatic fragmentation potential of the plant protopectin complex by homoenzyme preparations. The approach is in fact the second stage of the decision tree in the science-based technology for pectin and its products.

ISOLATION AND ENZYMATIC FRAGMENTATION OF GENOMIC DNA FROM UNCULTURED MICROBIAL SYMBIONTS OF Theonella sp. SPONGE AS PREREQUISITE FOR METAGENOMIC LIBRARY CONSTRUCTION

Microbial symbionts of sponges are well known as the sources of numerous bioactive products which are of potential industrial and pharmaceutical importance, including secondary metabolites, enzymes, lipids and heteropolysaccharides.