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Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group.

Presentation of poster 556A at TAGC 2020 Online. Single PDF file.

Advances in next generation sequencing (NGS) platforms have outpaced those in library preparation. While hundreds to thousands of NGS libraries can be sequenced in a single run of an Illumina instrument, a single library is constructed using a multi-step procedure requiring expensive consumables and specialized equipment. To overcome these limitations and increase the ease and throughput of library construction, we have developed a robust, streamlined library construction method that integrates enzyme-based DNA fragmentation with end repair and dA-tailing. The NEBNext Ultra II FS DNA Library Prep Kit eliminates the need for specialized equipment to fragment DNA and reduces the number of steps in the library construction protocol. This method produces high quality libraries from gDNA isolated from organisms whose genomes vary widely in GC content, as well as amplicons and DNA purified from blood. In addition, the FS kit generates libraries with substantially higher yields than those using mechanically sheared DNA, enabling greatly reduced input requirements. Libraries constructed using the FS kit from inputs as low as 100 pg of human gDNA for amplified libraries and 100 ng for PCR free, show similar coverage uniformity and GC bias compared to libraries constructed with 100 ng of mechanically sheared DNA. The ability to generate high quality libraries from low amounts of starting material and a broad range of inputs will help advance the widespread implementation of NGS in both basic science and the clinic.

As the quantity of data generated per next generation sequencing (NGS) run increases and the time required per run decreases, the ability to quickly produce and track large numbers of libraries is becoming increasingly important. In addition, the ability to produce high quality libraries from limited starting material and multiple sample types, including FFPE is essential. To overcome these challenges and to minimize the bottleneck of NGS library prep, we have developed a fast, streamlined DNA library preparation method using novel reagents and adaptors. This method accommodates a wide range of sample input quantities and types including genomic, ChIP and fragmented DNA (e.g. FFPE). Data analysis of libraries constructed from as little as 250 pg of ChIP DNA show high complexity and significant overlap of target peaks with libraries made from10 ng of DNA. We have extended the utility of this library prep method by developing additional adaptor and primer reagents. These include a dual barcoding approach that is compatible with Illumina library prep and our novel NEBNext adaptor. This approach enables multiplexing of up to 96 different samples, which can be used to increase the number of samples per flow cell, and/or to identify specific samples/libraries in a lab. Together, the simple, streamlined workflow and dual barcode approach, significantly reduces the turn-around time, enabling high throughput processing of samples for clinical analysis and large scale genomics studies.

Massively parallel next generation cDNA sequencing (RNA-Seq), has allowed many advances in the characterization and quantification of transcriptomes. In addition to enabling the detection of non-canonical transcription start sites and termination sites, alternative splice isoforms, transcript mutations and edits can be identified. Additionally, the ability to obtain information on the originating strand is useful for many reasons including for example: identification of antisense transcripts, determination of the transcribed strand of noncoding RNAs, and determination of expression levels of coding or noncoding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of a RNA-seq experiment. However, standard methods for sequencing RNA do not provide information on the DNA strand from which the RNA strand was transcribed, and methods for strand-specific library preparation can be inefficient and time-consuming. Our objective was to address this challenge by developing a streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information while maintaining even coverage of transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differential tagging of the first strand cDNA ends. We have also extended the utility of this method by developing additional adaptor and primer reagents, including a dual barcoding approach that allows for multiplexing up to 96 samples. As a result, we have enabled highly multiplexed, strand-specific mRNA sequencing, as well as whole transcriptome sequencing (Total RNA-seq) from ribosomal-depleted samples, enabling the discovery of a much broader picture of expression dynamics including discovery of antisense transcripts. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful technique that allows for sensitive digital quantification of transcript levels. Moreover, RNA-Seq enables the detection of non-canonical transcription start sites and termination sites, alternative splice isoforms and transcript mutation and edition. Standard “next-generation” RNA-sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. In this approach, the synthesis of randomly primed double-stranded cDNA followed by addition of adaptors for sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Our objective was to address this need by developing a novel streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information while maintaining even coverage of transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differential tagging of the first strand cDNA ends. As a result, we have enabled strand specific mRNA sequencing, as well as whole transcriptome sequencing (Total RNA-Seq) from ribosomal-depleted samples. Total RNA-Seq provides a much broader picture of expression dynamics including discovery of antisense transcripts. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.