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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Precision Medicine holds great promise for human health and disease treatment but, in order to deliver on that promise, the techniques used to analyze human samples must first ensure reliable, high-quality, and accurate data in a high-throughput fashion. Library prep protocols for Next Generation Sequencing (NGS) have traditionally required costly DNA fragmentation equipment and several transfer and clean-up steps that increase the time required and potential for errors.
The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® addresses these challenges with a novel enzymatic fragmentation step, integrated into the Ultra II DNA kit, and requiring fewer clean-up steps. FS fragmentation is time dependent, but independent of input amounts, GC composition, and DNA storage buffer. For additional details on these findings, as well as notes on library yield and GC coverage, download this poster.
As research questions change, it may become more appropriate for a researcher to conduct more-targeted analysis of the genome, necessitating deeper sequencing with a gene panel approach. Gene panels are most commonly custom assembled, but this can be time consuming and expensive.
The NEBNext Direct® Custom Ready Panels are a collection of predesigned and premade baits specific to ~850 genes with active relevance to disease research. To obtain a Custom Ready panel, one must simply select the genes they’d like to analyze as a custom panel in a fraction of the time it takes to develop one from scratch. To learn more about how NEBNext Direct Custom Ready Panels work, or to search for your genes of interest, visit the product page.
Cell-free DNA (cfDNA) is gaining popularity as a noninvasive biomarker of disease. Most often, cfDNA is recovered from exosomes and other microvesicles released into body fluids (e.g., blood, urine, tears, etc.), providing an indicator of an organism’s health and/or disease. As methylation status has been shown to influence the progression of certain diseases, including cancer, analyzing the methylome of circulating cfDNA is an essential step.
Historically, the method of choice for methylome analysis was bisulfite sequencing, a method that leaves significant DNA damage in its wake. The NEBNext® Enzymatic Methyl-seq Kit (EM-seq™) enables higher quality library generation and improved sequence coverage, without added GC bias. To learn more about EM-seq after reviewing this poster, check out the tech note on this topic.
Long read sequencing has become more popular with the advent of new technologies that support it. Both the PacBio® Sequel and Oxford Nanopore MinION™ offer platforms for long read sequencing, enabling simpler genome assembly, sequencing through complex regions, and identifying structural variants.
To fully exploit long read sequencing, researchers require a robust and reliable option for generating full-length cDNA from the source mRNA. The NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module is well-suited for upstream cDNA generation prior to a long read sequencing method. For additional details, please visit the product page.
DNA expression is tuned, both in nature and in the laboratory, with the application or removal of epigenetic marks, the most common of which being the methylation of cytosine residues. Historically, cytosine methylation at the single-base level has been detected by bisulfite sequencing, where sodium bisulfite is used to convert all unmethylated cytosines to uracils. This treatment is harsh, however, commonly leading to damage and even fragmentation of the very DNA meant to be sequenced; therefore, whole-genome bisulfite sequencing (WGBS) has substantial drawbacks.
Developed to address this challenge, NEBNext® Enzymatic Methyl-seq (EM-seq™) relies on a gentler, enzyme-based process for conversion of unmethylated cytosines, but not 5mC or 5hmC, to uracils. This poster introduces some of the ways in which EM-seq provides superior-quality sequencing metrics, including uniformity of coverage and detection sensitivity. For additional details, refer to the EM-seq product page.
The increased availability and sensitivity of transcriptomic analyses have changed the way that people think about cell population studies. Analyzing a T-flask of cells or a tube of blood was once standard practice, as smaller samples were not easily or reliably obtained. Now, at the dawn of single-cell transcriptomics, it’s possible to assess transcriptomes on a single-cell basis, ensuring that rare events and cell subtypes are captured in their true proportion to the sample.
This poster describes a method for full-length transcript sequencing, with a wide range of input types including RNA (UHR; 2 pg – 200 ng), cultured cell lines (single cells), and mouse primary cells (single cells). Among other findings, two populations of cells were identified arising from mouse (8 weeks old) mammary glands, which were traceable back to the basal and luminal developmental lineages. Learn more when you download this poster.
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