Advances in sequencing instruments and chemistries continue to evolve, and new sequencers are being released at an unprecedented pace by both the current market leader, Illumina, and challengers, including MGI. Lower run costs, faster run times, and ease of use offered by new instruments often makes switching an appealing and reasonable choice. However, data quality, error rates, and error types vary among sequencers, even from the same manufacturer. In this study, we evaluated the design of 5 sets of 96 unique dual index primers using diverse library types, and characterized the variation in clustering efficiency, error rates, and error types, in addition to abundance levels.
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.
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.
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.
Nucleic acid diagnostic tests for SARS-CoV-2, whether based on RT-qPCR, RT-LAMP, RPA, or other amplification technology, all depend on primers. While the SARS-CoV-2 genome seems to be less variable than some other retroviral genomes, variants with potential effects on amplification efficiency have arisen and become prevalent in local areas. We developed a streaming analysis method to identify variants that may affect specific primers and a website to allow interested users to register primer loci.
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 sequencing is a powerful tool for the study of gene regulation and function. Applications of RNA-seq have expanded to include low-RNA input library prep methods. In this study, we show that the incorporation of UMIs into RNA-seq analysis allows for a more accurate calculation of transcript abundance.
The use of Unique Molecular Identifiers (UMIs) has become increasingly popular, offering a multitude of advantages, especially when paired with unique dual indexing (UDI). By incorporating UMIs into UDI adaptors, we have assessed their effect on the accuracy of quantitative sequencing assays. We demonstrate that the sensitivity of variant detection is improved with UMI consensus calling.
The impact of the novel coronavirus SARS-CoV-2 on the global community has produced a critical need to develop reliable and accurate protocols for sequencing emergent pathogens. Here, we present refined protocols and reagents for the sequencing of SARS-CoV-2 on Illumina sequencing platforms with protocols based on the ARTIC consortium methods.
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.
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