NEB Scientific Posters
Your search returned 67 results.
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EM-seq™ Enables Accurate and Precise Methylome Analysis of Challenging DNA Samples (2019)
Cell-free DNA (cfDNA) is gaining popularity as a noninvasive biomarker of disease. Most often, cfDNA is recovered from exosomes and other microvesicles released into body fluids (e.g., blood, urine, tears, etc.), providing an indicator of an organism’s health and/or disease. As methylation status has been shown to influence the progression of certain diseases, including cancer, analyzing the methylome of circulating cfDNA is an essential step.
Historically, the method of choice for methylome analysis was bisulfite sequencing, a method that leaves significant DNA damage in its wake. The NEBNext® Enzymatic Methyl-seq Kit (EM-seq™) enables higher quality library generation and improved sequence coverage, without added GC bias. To learn more about EM-seq after reviewing this poster, check out the tech note on this topic. -
NEBNext Direct® Custom Ready Panels Overcome Challenges Associated with Targeted Re-sequencing (2019)
As research questions change, it may become more appropriate for a researcher to conduct more-targeted analysis of the genome, necessitating deeper sequencing with a gene panel approach. Gene panels are most commonly custom assembled, but this can be time consuming and expensive.
The NEBNext Direct® Custom Ready Panels are a collection of predesigned and premade baits specific to ~850 genes with active relevance to disease research. To obtain a Custom Ready panel, one must simply select the genes they’d like to analyze as a custom panel in a fraction of the time it takes to develop one from scratch. To learn more about how NEBNext Direct Custom Ready Panels work, or to search for your genes of interest, visit the product page.
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A Single-tube, Low Input Protocol for Long Read Sequencing (2019)
Long read sequencing has become more popular with the advent of new technologies that support it. Both the PacBio® Sequel and Oxford Nanopore MinION™ offer platforms for long read sequencing, enabling simpler genome assembly, sequencing through complex regions, and identifying structural variants.
To fully exploit long read sequencing, researchers require a robust and reliable option for generating full-length cDNA from the source mRNA. The NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module is well-suited for upstream cDNA generation prior to a long read sequencing method. For additional details, please visit the product page. -
A Robust, Streamlined, Enzyme-based DNA Library Preparation Method Amenable to a Wide Range of DNA Inputs (2019)
Precision Medicine holds great promise for human health and disease treatment but, in order to deliver on that promise, the techniques used to analyze human samples must first ensure reliable, high-quality, and accurate data in a high-throughput fashion. Library prep protocols for Next Generation Sequencing (NGS) have traditionally required costly DNA fragmentation equipment and several transfer and clean-up steps that increase the time required and potential for errors.
The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® addresses these challenges with a novel enzymatic fragmentation step, integrated into the Ultra II DNA kit, and requiring fewer clean-up steps. FS fragmentation is time dependent, but independent of input amounts, GC composition, and DNA storage buffer. For additional details on these findings, as well as notes on library yield and GC coverage, download this poster. -
Improving Transcriptome Profiling for Single Cell and Low Input RNA (2019)
The increased availability and sensitivity of transcriptomic analyses have changed the way that people think about cell population studies. Analyzing a T-flask of cells or a tube of blood was once standard practice, as smaller samples were not easily or reliably obtained. Now, at the dawn of single-cell transcriptomics, it’s possible to assess transcriptomes on a single-cell basis, ensuring that rare events and cell subtypes are captured in their true proportion to the sample.
This poster describes a method for full-length transcript sequencing, with a wide range of input types including RNA (UHR; 2 pg – 200 ng), cultured cell lines (single cells), and mouse primary cells (single cells). Among other findings, two populations of cells were identified arising from mouse (8 weeks old) mammary glands, which were traceable back to the basal and luminal developmental lineages. Learn more when you download this poster. -
Highly Multiplexed, Targeted Sequencing for Genotyping Maize with the NEBNext Direct Approach (2019)
argeted next-generation sequencing of molecular markers is a desirable approach to genotype crops for marker-assisted breeding. These methods offer several advantages over other genotyping approaches, including the ability to interrogate thousands of variant sites with a single assay while providing additional information on nearby sequences. However, NGS-based genotyping is typically more expensive than traditional genotyping methods, and for marker-assisted selection, many samples need to be screened to identify individuals to cross. Thus, it is important that the approach used to prepare samples for sequencing is high-throughput and that the cost per sample is low. Here we present the NEBNext Direct Genotyping Solution, a novel, capture-by-hybridization method that allows for processing of up to 9216 samples in a single 96-well plate.
To demonstrate the capabilities of this approach, we applied the NEBNext Direct Genotyping Solution to genotype maize genomic DNA. We developed a panel of over 4600 legacy SNPs from the Panzea project and tested the ability of the panel to evenly enrich targets from 25 ng of maize DNA. Additionally, because the baits were individually synthesized, subsets of the panel could be rapidly generated to reduce sequencing costs when fewer targets were required. To demonstrate this ability, we selected a 100 marker subpool from the larger bait set and observed consistent coverage of the selected targets while maintaining the high specificity and uniformity of the panel. With this one day, highly multiplexed protocol, hundreds of samples could be processed in a high-throughput manner, making this approach ideal for genomic selection in maize.
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Uncovering the Cannabis sativa Methylome Through Enzymatic Methyl-seq (2019)
The medical and agricultural value of Cannabis is undeniable, and research is just now beginning to fully interrogate the plant and its products. Due to the base composition of the Cannabis genome (66% AT-rich), obtaining high-quality methylome analysis with whole genome bisulfite analysis (WGBS) has been challenging. WGBS is known to cause DNA damage that skews post-WGBS base composition (83% AT-rich).
NEBNext® Enzymatic Methyl-seq (EM-seq®) is an alternative to WGBS that does not require harsh conditions to accurately generate high-quality libraries, without losing sample integrity or details about methyl marks (both 5mC and 5hmC). With EM-seq, it becomes possible to analyze Cannabis plant tissues with greater accuracy and with less risk for DNA damage. For additional details on this study, please download this poster. -
High-Throughput Screening of 2300 Genetic Markers in S.lycopersicum using the NEBNext Direct Multiplexed Genotyping Approach (2019)
Targeted DNA sequencing is rapidly being adopted for the molecular screening of markers during selective crop breeding. For these applications, the need for cost-effective and high-throughput technologies to process large numbers of samples is imperative. Here we describe a novel capture- by-hybridization method for targeted genotyping of crops. This simple workflow allows processing of up to 9216 samples in a single 96-well plate in one day and is easily automated.
The NEBNext Direct Genotyping Solution can target 100 to 5000 markers from up to 96 samples within a single hybridization. Here we developed a panel targeting 2300 SNPs in the tomato crop, Solanum lycopersicum. Baits were placed within 75 nucleotides of the targeted SNPs, allowing for an efficient sequencing run of 75 bases of target sequencing, 8 bases of sample barcode, 8 bases of hybridization barcode, and 12 bases of a unique molecular identifier (UMI) for filtering PCR duplicates. After an initial screening of the panel, the bait concentrations were adjusted by performance to ensure uniform coverage of the targets. The optimized panel resulted in greater than 90% of the sequencing reads mapping to targeted regions and highly uniform coverage. As a result, this approach reduced the cost and increased the throughput of crop sequencing while generating robust data to reliably genotype multiple varieties of S. lycopersicum. -
NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples (2019)
Next generation sequencing (NGS) is currently an important tool used in many fields to answer biological questions. DNA fragmentation is the critical initial step in the construction of high quality NGS libraries, however, current fragmentation methods create a bottleneck in library preparation throughput. To meet this challenge, we have developed a robust library construction method (NEBNext Ultra II FS) that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This method eliminates the need for expensive equipment to fragment DNA; moreover, the optimized workflow reduces the numerous cleanup and liquid transfer steps, reducing the time, cost, and errors associated with library construction.
The robustness of the Ultra II FS DNA library preparation workflow was tested using genomic DNA from a variety of sources including the model organism Arabidopsis thaliana, the less- documented genome of Cannabis sativa, and Sus scrofa (pig). Libraries were prepared from a range of DNA inputs to achieve different insert sizes with or without PCR amplification. All libraries were sequenced, reads aligned to the appropriate reference genome, and quality metrics generated using Picard tools. Compared with the traditional, mechanical shearing based library preparation method, Ultra II FS is significantly easier to automate, has higher library conversion rate and similar or superior sequencing quality. We further discuss several applications of Ultra II FS in plant and animal research, including genome assembly and sample quality control. -
Application of a Novel, Targeted Sequencing-Based Genotyping Approach for Cost Effective Marker Assessment in O. sativa (2019)
Decreases in sequencing costs have increased the availability of public SNP databases while necessitating development of targeted genotyping assays for use in marker assisted genomic selection for a variety of crop species. The NEBNext Direct Genotyping Solution is a novel, hybridization-based target enrichment approach that has been optimized for use in genotyping applications to increase the number of assays that can be performed in a single reaction, while providing sequencing coverage depth suitable for SNP identification. The approach enables high-levels of multiplexing of both isolates and markers, allowing enrichment of hundreds of thousands of SNP targets in a single hybridization reaction, and the protocol is easily completed in a single day.
We developed a panel covering the 1,996 single nucleotide polymorphisms previously identified as markers for polymorphism detection in O. Sativa. Here, we demonstrate the application of this panel to cost-effectively enrich defined SNP markers in a highly specific and uniform manner prior to next-generation sequencing.
