As the use and throughput of RNA sequencing continues to increase, there is a growing need for faster, more streamlined workflows that generate high-quality libraries. We have developed a kit with a 3-hour library prep protocol that enables the creation of high-quality directional RNA libraries in a single day when paired with poly(A) mRNA enrichment or rRNA depletion kits. The NEBNext UltraExpress RNA Library Prep Kit uses a single adaptor concentration and PCR cycle number for all RNA inputs (25 - 250 ng total RNA) and incorporates master mixed reagents, reduced incubation times and fewer cleanup steps, thereby reducing the total time and consumables used.
DNA methylation, crucial in development and diseases like cancer, involves epigenetic regulation through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Traditional detection methods, such as NEBNext® EM-seq™, cannot differentiate between these two cytosine forms. The new NEBNext Enzymatic 5hmC-seq (E5hmC-seq™) method overcomes this limitation by specifically identifying 5hmC. It glucosylates 5hmC to prevent deamination, allowing clear discrimination from 5mC and cytosine. Tested on human brain DNA, E5hmC-seq shows accurate 5hmC measurement with minimal GC bias, maintaining data quality. This advancement in identifying 5hmC is pivotal for understanding its role in cellular processes and diseases.
NEBNext UltraShear® and NEBNext UltraShear FFPE DNA Library Prep Kit excel in preparing DNA libraries, especially from complex samples like formalin-fixed, paraffin-embedded (FFPE) DNA. These solutions effectively fragment genomic DNA, enhancing library yields and sequencing metrics. Their use, particularly with the NEBNext FFPE DNA Repair v2 Mix, improves yield and coverage. Comparative studies with Covaris® ME220 demonstrate NEBNext UltraShear’s comparable methylation detection but with higher yields. The kit significantly reduces unmapped, chimeric, and foldback reads, lowering artificial mutation frequencies, and outperforms other kits in hybrid capture libraries for on-target coverage.
The importance of DNA sequencing as a laboratory technique continues to grow, and with it, the importance of faster, more streamlined workflows that generate high-quality libraries. The NEBNext UltraExpress DNA Library Prep Kit, for pre-sheared DNA and the NEBNext UltraExpress FS DNA Library Prep Kit, for intact DNA, take you from sample to library in under two hours. The streamlined workflow uses a single adaptor concentration and PCR cycle number for all inputs (10-200 ng) and incorporates master mixed reagents, reduced incubation times and fewer cleanup steps.
Wolbachia are Gram-negative, obligate intracellular bacteria in some filarial nematodes and about 60% of arthropods. The filarial parasites Mansonella perstans and Mansonella ozzardi harbor Wolbachia. Wolbachia have been consistently detected in M. ozzardi, but in M. perstans, the presence of Wolbachia may be isolate-dependent. Phylogenetically, the majority of Wolbachia from filarial parasites cluster into supergroups C, D and J, while arthropod Wolbachia are clustered in supergroups A, B, E, H and S. Wolbachia from Mansonella (wMpe, wMoz) are different from other filarial Wolbachia as they are placed in supergroup F, with Wolbachia from insects such as the bed bug. We present here the genomes of wMpe and wMoz, representing the first genomes from filarial Wolbachia of supergroup F. We also present two new genomes of arthropod Wolbachia from supergroup F.
Mansonelliasis is a widespread yet neglected filariasis of humans, caused by infection with any of the three filarial species: Mansonella perstans, M. ozzardi and M. streptocerca.
The goal of the current study was to obtain whole genome sequences of M. perstans and M. ozzardi. M. perstans infections are endemic in Central and West Africa, and in a few areas of South America. M. ozzardi infections are highly prevalent in South America and the Caribbean islands. Transmission to humans is via insect vectors - biting midges of Culicoides spp. for M. perstans, and Culicoides midges as well as black flies of Simulium spp. for M. ozzardi.
The large dynamic range of transcript expression within a total RNA sample presents a challenge in whole transcriptome sequencing. Highly expressed transcripts with minimal biological interest can dominate readouts, masking detection of more informative lower abundance transcripts. Here, we present a method to enrich for RNAs of interest by eliminating unwanted RNAs before sequencing.
DNA methylation is one of the most important epigenetic regulatory mechanisms. 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 cytosines, but the chemical-based conversion of cytosines to uracils leads to DNA damage that subsequently translates to shorter DNA insert sizes and biases in the data. To overcome these drawbacks, we developed NEBNext Enzymatic Methyl-seq (EM-seq™), an enzymatic approach to detecting cytosine methylation.
Currently, in the U.S. alone, there are nearly 25 million confirmed cases of COVID-19 and over 400,000 deaths. Thus, developing fast, reliable, and accurate methods for sequencing SARS-CoV-2 has become a worldwide necessity. Based on the ARTIC Network’s nanopore sequencing protocols for real-time detection of viral outbreaks, we have assembled an all-in-one kit that can be used in conjunction with the Oxford Nanopore Technologies Native Barcoding Kit/Ligation Sequencing Kit for sequencing and surveillance of infection.
RNA-seq is a widely used technology with a broad range of applications. However, it is not always feasible for unique prokaryotic species or other interesting organisms, where tools to study them can frequently lag. Here, we present a robust method to enrich for RNAs of interest by eliminating rRNAs in diverse bacterial species. We further introduce an approach to customize RNA depletion, eliminating specific RNAs in any organism not well covered by a pre-optimized kit.
Help us celebrate our 50th anniversary! We have hidden 1,000 golden butterflies and are waiting for you to find them. They can be anywhere that you find NEB! Beginning April 15th, be sure to visit our website and tables at tradeshows and events you are attending. Visit our social media channels frequently for tips on where we have hidden the butterflies – and once you find one, either click or scan the code to be eligible for a 50th anniversary prize pack, as well as a grand prize trip to NEB headquarters in Ipswich, MA!.
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