We developed High Complexity Golden Gate Assemblies (HC-GGA) for engineering three mycobacteriophages. The phage genomes are divided into 1.7 - 5.0kb which can be carried in plasmids. The fragments can then be assembled in a one-pot reaction to generate the complete phage genomes. Through modification of plasmid parts, the system enables efficient and precise genome editing of multiple locations in the genome simultaneously, including point mutations, insertions, deletions, and domain swaps.
We present a High Complexity Golden Gate Assembly (HC-GGA) for engineering Pseudomonas phage ɸKMV. The system divides the phage genome into small, cloneable synthetic fragments, which are assembled in a one-pot reaction to generate the complete genome. Through modification of plasmid parts, the system enables efficient and precise genome editing including point mutations, insertions, deletions, and domain swaps.
RSV, a single-stranded RNA virus, is the leading cause of respiratory illness in infants. Using multiplex targeted amplification sequencing techniques, the presence of RSV in the population can be monitored for mutations that negatively impact treatment efficacy. Here, we describe a newly developed RSV sequencing approach based on amplicon-targeted sequencing for Illumina or Oxford Nanopore Technologies platforms, and featuring the NEBNext RSV Primer Module.
Formalin-fixed, paraffin-embedded samples are a challenging sample type for most NGS library prep methods. We developed a novel method, compatible with both high and low quality FFPE DNA samples, employing three new enzyme mixes, designed specifically for compatibility with FFPE samples. The NEBNext UltraShear FFPE DNA Library Prep Kit includes an enzymatic fragmentation step that improves the library yield, library metrics, and variant calling accuracy.
With the goal of streamlining the processing of a variety of samples of various input amounts, the NEBNext UltraExpress® library prep kits respond to a user-stated need for faster, more efficient, and easily automated workflows. One key optimization is the single-condition workflow, which enables the simultaneous processing of multiple different sample input amounts (within the kit’s stated input range) with a single adaptor concentration and a single recommendation for PCR cycles. These advances have resulted in a single-tube solution, incorporating master-mixed reagents, reduced incubation times, fewer clean-up steps, and the generation of less plastic waste.
DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases, such as cancer. Typically, the modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are detected by sequencing Illumina libraries generated using a gentle, enzyme-based workflow called NEBNext® EM-seq, or by the harsher bisulfite conversion. Here, we describe an enzymatic method that enables specific detection of 5hmC, using the NEBNext Enzymatic 5hmC-seq (E5hmC-seq) Kit.
NEBNext Enzymatic Methyl-seq (EM-seq) workflows involve base conversion, which can be a challenge for variant detection. In EM-seq, this challenge is overcome using bioinformatic tools. Because methylation information is only preserved on a single strand in EM-seq libraries, the other strand can be used to detect genetic variation. Using this method, we can call germline SNPs with high precision.
cfDNA and cfRNA are nucleic acid fragments found circulating in blood. These nucleic acids originate from various cell types, providing insights into the health of the system as a whole. By evaluating the change in cell-free nucleic acids over time, via serial blood draws, researchers can monitor for atypical biological processes, including cancer. When combined with the new Monarch Mag Viral DNA/RNA Extraction Kit, both the NEBNext Ultra II and the NEBNext UltraExpress library prep kits produce high-quality libraries.
The cytosine modifications 5mC and 5hmC are important regulatory marks within the genome, influencing gene expression. Older techniques of methylome analysis, like bisulfite sequencing, are harsh and can damage the input DNA, decreasing the quality of the resulting libraries. NEBNext Enzymatic Methyl-seq (EM-seq) was introduced in 2019, and brought to bear gentle, enzymatic fragmentation, minimizing damage to the DNA and improving the quality of the resulting libraries. As a further improvement on the EM-seq workflow, NEBNext Enzymatic Methyl-seq v2 has been introduced, requiring lower DNA inputs and offering improved compatibility with lower-quality samples.
Monitoring respiratory viruses through whole genome and targeted sequencing is now more important than ever before, particularly in the wake of the global pandemic. We have developed sequencing approaches for respiratory RNA viruses, including SARS-CoV-2, RSV, and Flu, to support scientists and public health laboratories to monitor these critical pathogens.
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