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  1. Application of High Complexity Golden Gate Assembly to Rapid Engineering of Bacteriophage Genomes

    Data-optimized Assembly Design (DAD) enhances Golden Gate Assembly (GGA) by using precise ligation fidelity measurements to select high-accuracy fusion sites. This approach replaces traditional GGA methods, enabling the efficient assembly of multiple parts while minimizing errors. We have developed a set of tools to apply DAD to the design of complex assemblies of up to 36 parts and 50kB final size in a single reaction. Combined with optimized reagents, DAD yields sequence-accurate constructs ready for use without further processing. The small parts are easy to produce via PCR or DNA synthesis and can be manipulated in E. coli plasmid systems, supporting viral mutagenesis and gene swaps. These principles facilitate modular assembly of bacteriophage genomes for high-throughput pathogen research.

  2. Enhanced Rolling Circle Amplification Performance with a Newly Engineered phi29 DNA Polymerase

    Isothermal DNA amplification techniques exponentially increase the amount of DNA in a sample without the constraint of thermocycling. NEB offers a wide range of products for isothermal amplification, including reagents for rolling circle amplification (RCA). The most widely used DNA polymerase for RCA is phi29, which possesses high processivity and fidelity, and strong strand-displacement activity. We recently engineered an improved version of this polymerase, phi29-XT, that shares the same positive characteristics of the wild-type phi29 but also generates a higher yield in less time than the wild-type enzyme. Additionally, while wild-type phi29 works optimally at 30°C, phi29-XT performs best at 42°C. phi29-XT has been used successfully downstream of DNA assembly technologies, such as GoldenGate and NEBuilder HiFi Assembly, to perform high-throughput cell- and vector-free protein expression. Furthermore, the phi29-XT RCA kit also enabled high-throughput fosmid amplification directly from bacterial cells, followed by ONT sequencing for de novo fosmid DNA assembly. Together, these applications demonstrate the utility of the phi29-XT and the RCA kit for isothermal amplification of circular DNA templates.

  3. Utilizing Lyophilized LAMP Reagents in Rapid Molecular Assays

    Loop-mediated isothermal amplification (LAMP) has become a widely used method for detecting target nucleic acids (DNA and RNA) as it offers a robust and simple alternative to PCR. LAMP is particularly well suited to point-of-care (POC) applications such as COVID-19 diagnostics because only a single incubation temperature is required for nucleic acid amplification, and the technology is compatible with simple detection strategies, including colorimetric or lateral flow readouts. To further increase the utility of LAMP in POC molecular diagnostic workflows, eliminating cold chain requirements for reagent shipment and storage is desired and can often be accomplished by lyophilization. This study investigates single and multiplex detection of several viral infectious diseases using lyophilized LAMP/RT-LAMP reagents.

  4. E5hmC-seq™: Detection of 5hmC at single base resolution

    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.

  5. Streamlined, Express DNA Library Preparation Methods Meet Requirements of High-throughput Library Construction

    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.

  6. NEBNext UltraExpress™ RNA: A fast and flexible workflow for stranded RNA-seq library preparation

    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.

  7. NEBNext® solutions for challenging methods and samples

    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.

  8. Genomes of the Wolbachia endosymbionts from the human filarial parasites Mansonella perstans and Mansonella ozzardi reveal multiple origins of nematode-Wolbachia symbiosis

    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 WolbachiaWolbachia 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.

  9. Genome sequences of the human filarial parasites Mansonella perstans and Mansonella ozzardi

    Mansonelliasis is a widespread yet neglected filariasis of humans, caused by infection with any of the three filarial species: Mansonella perstansM. ozzardi and M. streptocerca.

    The goal of the current study was to obtain whole genome sequences of M. perstans and M. ozzardiM. 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.

  10. Increasing the sensitivity of transcriptome profiling in eukaryote and blood samples by depleting abundant RNAs

    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.

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