Highly expressed transcripts with minimal biological interest can dominate RNA-seq readouts, masking detection of more informative low abundance transcripts. Here, we present an improved method to enrich for RNAs of interest by eliminating abundant, typically unwanted, RNAs. We conclude that the reduction of abundant transcripts for RNA-Seq studies significantly increases the ability to detect true biological variation that could not be detected in nondepleted samples. The method described here is a reliable and simple solution that greatly improves sensitivity in transcriptome studies and is amenable to high-throughput automation.
Next-generation sequencing is increasingly being adopted for genetic screening and clinical diagnostics.
To prevent false reporting of results, it is imperative that patient samples are tracked throughout sample processing and data analysis. A reliable method to track sample identity throughout a workflow is to monitor single nucleotide polymorphisms (SNPs) that are highly discriminatory across individuals. In order to incorporate a routine sample tracking method into diagnostic workflows, the method should be reliable, high-throughput, and cost-effective. The NEBNext Direct® Genotyping Solution offers a convenient and reliable method to ensure that data integrity is maintained in a diagnostic workflow.
NEBNext® Enzymatic Methyl-seq (EM-seq™) offers several improvements over traditional sodium bisulfite-based methylome analysis, owing – in large part – to the gentler, enzyme-based workflow. Less DNA damage enables longer reads with less sequencing depth. This poster summarizes the use of EM-seq with traditionally challenging sample types, cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded DNA (FFPE DNA).
DNA methylation is important for gene regulation. The ability to accurately identify 5-methylcytosine(5mC) and 5-hydroxymethylcytosine (5hmC) gives us greater insight into potential gene regulatory mechanisms. Bisulfite sequencing (BS) is traditionally used to detect methylated Cs, however, BS does have its drawbacks. DNA is commonly damaged and degraded by the chemical bisulfite reaction resulting in libraries that demonstrate high GC bias and are enriched for methylated regions. To overcome these limitations, we developed an enzymatic approach, NEBNext® Enzymatic Methyl-seq (EM-seq™), for methylation detection that minimizes DNA damage, resulting in longer fragments and minimal GC bias, here demonstrated with Arabidopsis thaliana and Cannabis sativa DNA.
Fragmentation is a bottleneck in the standard NGS workflow. The NEBNext® Ultra™ II FS DNA Library Prep Kit addresses this challenge with one-step enzymatic fragmentation, end-repair, and dA-tailing. Samples of plant tissue can be difficult to completely fragment without bias due to their molecular structure, but the Ultra II FS DNA kits enable robust library prep from Arabidopsis thaliana, Oryza sativa, and Zea mays samples.
The NEBExpress™ Cell-Free E. coli Protein Synthesis System is a high-performing, versatile and robust cell-free protein synthesis system developed by genetic engineering E. coli, optimizing a reaction buffer, and employing stringent manufacturing practices. This system was developed for coupled in vitro transcription and translation reactions for a variety of applications such as high throughput protein screening and engineering, as well as synthetic biology.
The NEBExpress™ Cell-Free E. coli Protein Synthesis System has been developed for coupled in vitro transcription and translation reactions resulting in high yields of proteins of various sizes (up to 230 kDa) and origins. A genetically engineered E.coli strain ensures stability of template DNA, RNA, and protein product. The Cell-free E.coli Protein Synthesis System is compatible with PURExpress Disulfide Bond Enhancer for better folding, and NEBExpress™GamS Nuclease Inhibitor for enhanced yield from linear templates. The reaction buffer formulation is compatible with SDS-PAGE (no acetone or TCA precipitation needed) and protein synthesis can be sustained for 10 hours at 37 °C or up to 24 hours at lower temperatures. Reproducible batches of lysate are produced, using highly stringent biomanufacturing processes and quality standards.
In metazoan cell nuclei, chromatin is functionally divided into transcriptionally active (euchromatin) or inactive (heterochromatin) regions. These heterochromatin regions constitute large chromatin domains that are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to organize chromosomes inside the nucleus and are enriched for repressive histone modifications. Genome-wide profiling of heterochromatin, especially LADs, is often challenging and warrants a simpler and direct method. Here we developed a new method, Protect-seq, aimed at identifying regions of heterochromatin via resistance to nuclease degradation followed by next-generation sequencing. We performed Protect-seq on the human colon cancer cell line HCT-116 and observed overlap with previously curated LADs. We provide evidence that these protected regions are enriched for the repressive histone modification H3K9me3 and to a lesser extent H3K9me2 and H3K27me3. Moreover, the loss of H3K9me3 in human cells leads to an increase in chromatin accessibility. In sum, we demonstrate a novel technique to identify nuclease inaccessible regions of the genome and our data is consistent with the model that repressive chromatin domains are compacted and targeted to the nuclear lamina, likely via HP1 proteins, which act as scaffolds to maintain chromatin architecture.
Mansoneliasis is caused by infection with the parasites Mansonella perstans, M. ozzardi and M. streptocerca and is transmitted by insects such as biting midges and black flies. Immunosuppression caused by the parasitic infection may lead to worsening of other medical conditions. Mansoneliasis patients are often co-infected with multiple filarial parasites and anti-helminthic treatment is complicated. In this study, a bioinformatic filtering approach identified new diagnostic biomarkers, which were used to develop sensitive and species-specific LAMP assays that were validated on both patient and insect samples for point-of-care diagnostics.
Mansoneliasis is a widespread, yet neglected, filariasis of humans caused by infection with Mansonella perstans, M. ozzardi and M. streptocerca. Transmission to humans is via midge and black fly insect vectors whose endosymbiont is a member of a unique Wolbachia supergroup that is from both insect and filarial hosts. In this study, draft genome sequences of M. perstans, M. ozzardi and Wolbachia were obtained. This will provide insight into the biology and evolution of some of the most neglected filarial parasites.
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