The need for very high molecular weight (HMW) DNA is growing quickly as long read sequencing and other emerging long read technologies become increasingly popular. However, the extraction of HMW DNA has been a bottleneck for these applications, and rapid, efficient and affordable HMW DNA extraction solutions are highly sought after. Here, we present a novel approach to HMW DNA extraction that utilizes large glass beads and optimized buffer chemistry, resulting in a simple workflow that enables researchers to quickly purify high quality HMW DNA from cells, blood, tissue and bacteria using two dedicated Monarch Kits. Purified DNA ranges from 50 kb to several megabases and size can be tuned by varying the speed at which samples are agitated during lysis. The use of glass beads enables easy handling, extremely efficient elution, high recovery, and easy dissolving of the isolated HMW DNA.
The use of Unique Molecular Identifiers (UMIs) has become increasingly popular – offering a multitude of advantages – particularly when paired with Unique Dual Indices (UDI). Two major factors affecting sequencing accuracy are 1) duplication, arising from PCR amplification of library molecules, and 2) errors introduced during library preparation and sequencing on the flow cell. UMIs, when incorporated into library preparation, can account for and mitigate the impacts of both of these factors. UMIs improved the accuracy of low-frequency variant detection by 25-200%.
RNA-Seq is a widely used technology with a broad range of applications, including differential expression analysis and alternative splice forms identification, in normal and disease contexts as well as in developmental studies. The technology has been pushed to extremes of very low and degraded samples but still battles with the challenge of having a large dynamic range of transcript expression. Highly expressed transcripts with minimal biological interest can dominate 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 developed a user-friendly web tool to enable custom depletion of any RNAs of interest. We used this web tool and depletion method to remove rRNA from total RNA of various species, including the mosquito Aedes aegypti as well as archaea Thermococcus kodakarensis and Pyrococcus furiosus. Additionally, we used this approach to target coding RNAs in human total RNA, and supplemented an existing anti-rRNA probe set for depletion of both rRNA and the selected coding RNAs. Using strand-specific RNA sequencing we measured depletion efficiency and transcript expression. We achieved high depletion efficiency (up to 99%) for all targeted RNAs across species, while maintaining transcript abundance of non-targeted RNA. This translated into an enrichment of RNAs of interest and an increased depth of sequencing coverage.
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.
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