NEB
EXPRESSIONS
A scientific update
Issue I • 2021
Think differently about your molecular diagnostics supply chain
Feature Article
A faster workflow for the assessment of genomic loci in mice using a novel HMW DNA extraction technology upstream of Cas9 targeted sequencing
Technical Note
New reagents for RT-qPCR and featured products for mRNA synthesis
Products Supporting COVID-19 Research
New Monarch® kits deliver high yields with faster workflows
Products for HMW DNA extraction
Learn about the landscape and timeline of COVID-19 vaccine development
COVID-19 Researcher Spotlight
Australia & New Zealand New England Biolabs (Australia) PTY Telephone: 1800 934 218 (AU) Telephone: 0800 437 209 (NZ) www.nebiolabs.com.au www.nebiolabs.co.nz
Canada New England Biolabs, Ltd. Toll Free: 1-800-387-1095 www.neb.ca
China New England Biolabs (Beijing), Ltd. Telephone: 010-82378265/82378266 www.neb-china.com
USA New England Biolabs, Inc. Telephone: (978) 927-5054 Toll Free: (U.S. Orders) 1-800-632-5227 Toll Free: (U.S. Tech) 1-800-632-7799 www.neb.com
France New England Biolabs France Telephone : 0800 100 632 www.neb-online.fr
Germany & Austria New England Biolabs GmbH Free Call 0800/246 5227 (Germany) Free Call 00800/246 52277 (Austria) www.neb-online.de
Japan New England Biolabs Japan, Inc. Telephone: +81 (0)3 5669 6191 www.nebj.jp
Singapore New England Biolabs Pte. Ltd. Telephone +65 638 59623 www.neb.sg
United Kingdom New England Biolabs (UK), Ltd. Call Free 0800 318486 www.neb.uk.com
View contacts in other countries
SCIENCE
INNOVATION
INSPIRATION
1
2
3
4
5
New England Biolabs® (NEB®) partners with customers globally to address the challenges faced by innovators developing the molecular diagnostics (MDx) technologies required to address public health and pandemic preparedness.
Read Our Feature Article
Learn About Amplification-based & NGS-based Molecular Diagnostics
Contact Us
Visit OEM & Customized Solutions
By Tom Evans, Ph.D. and Salvatore V. Russello, Ph.D., New England Biolabs, Inc.
The current COVID-19 pandemic has impacted nearly every aspect of daily life. It has elevated many of the challenges faced by clinical labs, and new and innovative solutions are required to address them. One strategy to help public health professionals understand and control the spread of SARS-CoV-2 is the widespread testing of millions of people around the world. Conventional RT-qPCR based tests performed in large, centralized testing facilities have been the backbone of testing to date. Despite the rapid development of these SARS-CoV-2 assays, dozens of new modalities are being introduced to help close the gap between the number of cumulative tests that can be performed daily and the desired testing capacity required to control and track the spread of the virus. Numerous companies, diagnostic testing facilities, and academic institutes have introduced SARS-CoV-2 assays under the FDA’s Emergency Use Authorization (EUAs). Based upon recent FDA guidance, the priority of new EUAs reviewed by FDA will be on tests that increase testing accessibility or significantly increase capacity. Additionally, new SARS-CoV-2 assays can be introduced by Clinical Laboratory Improvement Amendments (CLIA) laboratories without going through the EUA process. Still, such rapid progress has not been without challenges—it has exposed weaknesses in diagnostics supply chains and has belied the need for innovation and thinking differently about how diagnostics should be developed, manufactured, and deployed. Many scientists know NEB as a trusted reagent provider to the life science community. What many do not know is that we also offer a portfolio of products that serve as critical components for a wide array of diagnostics products and services. Extensive molecular biology and enzymology experience provide NEB with the unique ability to help customers solve the challenges inherent in technology development and ultimately in scale-up and commercialization.
NEB's founder, Dr. Donald Comb, prioritized basic research ever since NEB was founded in the early 1970s, and this has influenced the product development direction of the company. Our research interests include finding new enzyme activities, engineering enzymes specifically for biotechnology applications, and understanding how enzymes behave. This level of expertise and knowledge is then harnessed by our development and production teams to create robust enzymes and optimized workflows for commercialization. For example, NEB’s expertise in amplification has resulted in an extensive portfolio of reagents for RT-qPCR and isothermal amplification, two technologies essential in today’s molecular testing landscape. In fact, many of NEB’s products have been already cited in numerous publications and EUA protocols.Currently, the gold standard for diagnostics testing is RT-qPCR, and most of today’s testing infrastructure is based on this technology. It’s highly sensitive and robust, and NEB offers a number of products in this area, as do many other suppliers. However, like any technology, it has strengths and weaknesses. For example, it requires use of expensive equipment (a thermal cycler with fluorescence detection) and, in some cases, has longer turnaround times. At NEB, our emphasis on long-term research resulted in us evaluating and working with loop-mediated isothermal amplification (LAMP), an alternate approach originally developed at the Eiken Chemical Co., Ltd. Over the last decade we combined a number of breakthroughs to make the technology even more suitable for the molecular diagnostics community. This included novel engineered DNA polymerases, a new reverse transcriptase, the ability to set up reactions at room temperature using “WarmStart®” enzymes, multiplexing, and the ability to perform carryover prevention. We also introduced a version of this technology that enables the visual detection of products amplified by LAMP and RT-LAMP. We have also published extensively in this area (1).
Leveraging NEB's research program to influence product development for SARS-CoV-2 testing
NEB is a company that scientists know and trust. We pride ourselves on being a resource—not just through our product offerings and production capabilities, but also through the support we provide to diagnostics assay development scientists—from R&D through to scale-up and commercialization. For this reason, NEB is an ideal partner as new testing technologies are moved from the bench into production. Our support starts early in the R&D process, as customers obtain information about our products and technologies through our catalog, extensive web resources and support staff, and ultimately obtain material to evaluate. As questions arise about how a product might be used in a given technology, customers can speak directly with scientists who have played a role in developing or producing these products. And in those instances where a customer wishes to optimize performance for a particular detection modality, modify a product, or request a customized packaging format, the OEM & Customized Solutions Team is brought in. We quickly assemble a cross-functional team of researchers, product developers, project managers, and logistics staff to assess and make recommen-dations as to how best to address our customers’ needs.
Partnering with you from R&D to production
Our customers range from some of the largest molecular diagnostics organizations to early stage technology companies. Their challenges differ. In some cases they are looking to build out redundan-cy in their reagent supply chain, while in others they may need NEB to work collaboratively to further develop and help bring their technology to market. That said, there are several common challenges that many customers have cited repeatedly over the past several months. These are scale, supply chain resilience, and product quality, performance, and consistency. Regardless of what technology is ultimately incorporated into a molecular diagnostics product, a consistent and reliable supply chain is essential, especially in today’s landscape. Product demand is higher than ever before, and dozens of orga-nizations are pursuing similar approaches with the need for the same reagents and associated consumables. Further, the quality and consistency of reagents can vastly impact the performance of an assay.
Identifying the pain points involved in developing diagnostics
Unlike other reagent providers of similar size and capacity, NEB has made the decision to enable science and not compete with our diagnostics customers in the markets that they serve. As one of the few privately held molecular biology tools providers, our goal is to establish partnerships that advance our customers’ science and business objectives—100% of our production capacity is earmarked for the customers we serve, and not for the manufacture of our own diagnostics products. Over the past decade, NEB has made significant investment in its manufacturing scale-up and operations, as well as in its quality systems. Our ISO 13485 facilities in Ipswich, Rowley, and Beverly, Massachusetts, have the capacity to provide reagents to enable many millions of molecular assays, whether they be for RT-qPCR or isothermal amplification. NEB was founded to serve the scientific community with humility, integrity and transparency—principles which we believe are more important now than ever. We look to our diagnostics customers as the real innovators. These customers engage with NEB scientists to understand what our products do and don’t do, to make good decisions quickly about whether or not we are a fit for their technology platform. We also provide clarity about capacity, turn-around-times and, when possible, pass along cost savings to our customers in the event that scale can be achieved to reduce the price of our products. In summary, we would like the molecular diag-nostics community to think differently about NEB and consider us as their partner for future assay development and scale-up needs.
Enabling your solution
1. Anahtar, M.N. et al (2020) Open Forum Infectious Diseases, doi.org/10.1093/ofid/ofaa631
Extensive molecular biology and enzymology experience provide NEB with the unique ability to help customers solve the challenges inherent in technology development and ultimately in scale-up and commercialization.
NEB’s expertise in amplification has resulted in an extensive portfolio of reagents for RT-qPCR and isothermal amplification, two technologies essential in today’s molecular testing landscape.
We pride ourselves on being a resource—not just through our product offerings and production capabilities, but also through the support we provide to diagnostics assay development scientists—from R&D through to scale-up and commercialization.
Regardless of what technology is ultimately incorporated into a molecular diagnostics product, a consistent and reliable supply chain is essential, especially in today’s landscape.
Learn more about how NEB can support your Molecular Diagnostics needs
Learn More
< Back to Molecular Diagnostics Home
Back to top
NEB has a long history in the development of reliable and convenient tools for amplification, and offers a large selection of products for PCR, qPCR, RT-qPCR and isothermal amplification. Our extensive expertise in this area has allowed us to develop optimized enzymes for a variety of applications, including incorporation into diagnostics. The table below summarizes some of the products available from NEB for molecular diagnostics applications. Bulk and/or custom formats are available for all products (see customization details).
Sequencing is enabling scientists to make rapid advances in epidemiology and surveillance, basic and clinical research, and diagnostics. A fast-growing number of methods are being developed to address whole genome, as well as targeted approaches. In all cases, streamlined workflows that result in high-quality, high yield libraries are critical towards optimizing your next generation sequencing (NGS) results. The table below summarizes some of the products available from NEB for molecular diagnostics applications. Bulk and/or custom formats are available for all products (see customization details).
Amplification-based & NGS-based
NGS-based
Molecular Diagnostics
Amplification-based
NEB PRODUCTS
PRODUCT NOTES
CUSTOM FORMULATIONS AVAILABLE
PCR Applications
Isothermal Applications
qPCR/ RT-qPCR
PCR/ RT-PCR
LAMP
Strand Displacement
Helicase-dependent Amplification
Other
• Sensitive, reproducible and reliable performance • Compatible with automation and reaction miniaturization • Room temperature stable for ≥ 24 hours
• Luna WarmStart RT paired with Hot Start Taq increases reaction specificity and robustness • Compatible with automation and reaction miniaturization • Room temperature stable for ≥ 24 hours • High conc. ideal for viral targets (NEB #M3019) • Includes carryover prevention (NEB #M3019)
• Novel thermostable RT
DNA, Probe • Luna® Universal Probe qPCR Master Mix (NEB #M3004) DNA, Dye • Luna Universal qPCR Master Mix (NEB #M3003)
RNA (1-step), Probe • Luna Universal One-Step RT-qPCR Kit (NEB #E3005) RNA (1-step), Dye • Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) • Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) • Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) • Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit (NEB #E3019)
RNA (2-step) • LunaScript® RT SuperMix Kit (NEB #E3010)
• ROX-free • Blue-dye-free • Lyo-compatible
• ROX-free • Blue-dye-free
• Single-tube format • 13-minute protocol • Blue-dye-free
• ~280X fidelity of Taq • Consistent, fast, reliable performance • Compatible with automation and reaction miniaturization • Room temperature stable for ≥ 24 hours
• High concentration • Glycerol free
• Amplification direct from blood
• +/- Hot Start • High concentration
• Unique aptamer-based enzyme control supports fast protocols • Compatible with automation and reaction miniaturization
Master Mixes • Q5® Hot Start High-Fidelity 2X Master Mix (NEB #M0494) • Q5 High-Fidelity 2X Master Mix (NEB #M0492) Standalone Enzyme & Buffer • Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493) • Q5 High-Fidelity DNA Polymerase (NEB #M0491)
• Hemo KlenTaq® (NEB #M0332)
• Hot Start Taq DNA Polymerase (NEB #M0495) • Hot Start Taq 2X Master Mix (NEB #M0496)
• Fast, clear pink-to-yellow visible detection of amplification • Results in approximately 30 minutes
• Simple, colorimetric detection of amplification of SARS-CoV-2 nucleic acid • Automation-compatible when coupled with absorbance plate reader
• Master mix for LAMP and RT-LAMP workflows • Supports multiple detection methods, including fluorescence and turbidity • Automation compatible
• Lyo-compatible • High concentration
• WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800) • WarmStart Colorimetric LAMP 2X Master Mix with UDG (NEB #M1804)
• SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (NEB #E2019)
• WarmStart LAMP Kit (DNA & RNA) (NEB #E1700)
• Improved reaction properties compared to wild-type Bst DNA Polymerase • Increased dUTP tolerance enables carryover prevention
• Glycerol-free • High concentration
• Bst 2.0 WarmStart DNA Polymerase (NEB #M0538) • Bst 2.0 DNA Polymerase (NEB #M0537)
• DNA binding domain fusion supports robust performance • Significantly increased RT activity up to 72°C enables single enzyme RT-LAMP
• Bst 3.0 DNA Polymerase (NEB #M0374)
• In-silico designed RT for RT-LAMP with reversibly-bound aptamer that inhibits activity below 40°C
• WarmStart RTx Reverse Transcriptase (NEB #M0380)
• High purity, high quality nicking endonuclease
• Nt.BstNBI (NEB #R0607)
• Thermostable • Improves specifically of problematic fluorescent LAMP reactions
• Requires only two primer • Produces short, discrete DNA products
• High concentration
• Tte UvrD Helicase (NEB #M1202)
• IsoAmp II Universal tHDA Kit (NEB #H0110)
• Enables low temperature isothermal applications
•Can increase yield and efficiency of amplification reactions
• Highly pure • Individual mixes available
• Custom concentration
• Bsu DNA Polymerase Large Fragment (NEB #M0330)
• T4 Gene 32 Protein (NEB #M0300)
• Deoxynucleotide (dNTP) Solution Mix (NEB #N0447)
• Unique thermolabile version is completely inactivated in typical isothermal and RT-qPCR workflows
• Antarctic Thermolabile UDG (NEB #M0372)
• Proteinase K, Molecular Biology Grade (NEB #P8107)
• Thermolabile Proteinase K (NEB #P8111)
• Glycerol-free
DNA Applications
RNA Applications
APPLICATION TYPE
NEB PRODUCT
DNA library preparation with enzymatic DNA fragmentation
• Range of packaging options including 96-well plates • Component and kit customization • Kitting • Automation-compatible • Streamlined workflows
DNA library preparation for cfDNA or pre-sheared DNA
• NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® (NEB #E7805) • NEBNext Ultra II FS DNA Module (NEB #E7810) • NEBNext Ultra II Q5® Master Mix (NEB #M0544) • NEBNext Adaptors and Primers
• NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645) • NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) • NEBNext Ultra II Ligation Module (NEB # E7595) • NEBNext Ultra II Q5 Master Mix (NEB #M0544) • NEBNext Adaptors and Primers
Depletion of abundant RNAs
RNA library preparation
• NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) • NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750) • NEBNext rRNA Depletion Kit (Bacteria) (NEB #E7850) • NEBNext RNA Depletion Core Reagent Set for customized depletion (NEB #E7865)
• NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) • NEBNext Ultra II RNA First Strand Synthesis Module (NEB #E7771) • NEBNext Ultra II Directional RNA Second Strand Synthesis Module (NEB #E7550) • NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) • NEBNext Ultra II Ligation Module (NEB #E7595) • NEBNext Ultra II Q5 Master Mix (NEB #M0544) • NEBNext Adaptors and Primers
Virus sequencing and detection
• LunaScript RT SuperMix Kit (NEB #E3010) • Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) • NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) • Blunt/TA Ligase Master Mix (NEB #M0367) • NEBNext Quick Ligation Module (NEB #E6056) • NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) • WarmStart LAMP Kit (DNA & RNA) (NEB #E1700)
Monarch® HMW DNA Extraction Kits
NEB is pleased to announce the release of a powerful new solution for high molecular weight (HMW) DNA extraction. The Monarch HMW DNA Extraction Kits employ a novel glass bead-based approach, allowing users to extract DNA into the megabase (Mb) range quickly and easily, with best-in-class yields and purity from cells, blood, tissue, bacteria and other sample types. Extracted HMW DNA is easy to dissolve and is often ready to use on the same day, significantly shortening the extraction process, which has long been a bottleneck for long read sequencing technologies.
Validated sample types:
Monarch HMW DNA Extraction Kit for Tissue:
Monarch HMW DNA Extraction Kit for Cells & Blood:
• Mouse brain • Mouse liver • Mouse muscle • Mouse kidney • Mouse tail • Mouse ear punch • Rat kidney
• E. coli • B. cereus • M. luteus • X. laevis • S. cerevisiae • C. elegans • A. aegypti
• K293 • HeLa • NIH3T3 • Jurkat • K562 (suspension cells) • HCT116
• A549 • U5Os • HepG2 • NCI-460 • SK-N-SH • Aa23
• Human • Mouse • Rat (fresh only) • Rabbit • Pig
• Horse • Cow • Rhesus money • Goat (fresh only) • Sheep (fresh only)
Cells
Mammalian Blood
Nucleated Blood
• Chicken
• Turkey
Comparison of HMW DNA Extraction Methods
Reasons to choose Monarch HMW DNA Extraction Kits
Extremely fast, user-friendly protocols utilizing a novel glass-bead-based approach
Reproducible Extraction of HMW DNA from Cells and Blood with the Monarch HMW DNA Extraction Kit
DNA extracted with Monarch HMW DNA Extraction Kit for Cells & Blood. 1 x 106 fresh HEK293 cells and 500 μl fresh human blood were used as inputs and for preps performed according to the kit instructions using the agitation speed indicated above the gel lanes. 500 ng of DNA from the replicates was resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad® CHEF-DR® III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard.
Reproducibly purify high molecular weight genomic DNA (HMW DNA) from various sample types
Use of varying agitation speeds during lysis produces tunable fragment length of extracted HMW genomic DNA from cells and blood
Preps were performed on duplicate aliquots of 1 x 106 HEK293 cells and 500 μl fresh human blood. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. Equal amounts of DNA from the replicates (cells: 500 ng; blood: 650 ng) were resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad CHEF-DR III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder and Lambda DNA-Hind III Digest (NEB #N0341 and #N3012) were used as molecular weight standards. Yield, purity ratios and DINs of the individual preps are shown in the accompanying tables.
Tune fragment length by varying agitation speeds during lysis; achieve DNA in the Megabase size range with low speeds
Monarch HMW DNA Extraction Kit produces high molecular weight DNA with excellent yields, purity, and fragment length as compared to other commercially available kits
HMW was isolated from 1x106 HEK293 cells and fresh human blood with the kits indicated in the figure legend. Blood input volumes were used as specified in manufacturers’ protocols (N: 500 μl, C: 200 μl, R: 500 μl, Q: 200 μl, P: 300 μl, Z: 200 μl). Monarch samples (lanes 1-2) were purified at maximum agitation speed during lysis (2000 rpm). Variation in fragment length of cellular DNA using the standard protocols for Monarch and Circulomics (lanes 1-2 and 3-4, respectively) results from agitation speeds during lysis (Monarch: 2000 rpm, Circulomics: 900 rpm). All other data presented are duplicate samples from each different kit and the standard protocols dedicated to blood or cells were followed. Qiagen does not provide a protocol for cultured cells; a modified version of the blood protocol was followed. Samples were eluted in 100 μl, except for Zymo which was eluted in 50 μl according to their recommendations. Yield and purity of the standard samples were analyzed on Trinean Dropsense 16 spectrophotometer (now Unchained Labs Lunatic. Reported blood sample yields were normalized per 100 μl. RNA content was determined by HPLC analysis of nucleoside content after digestion of 1 μg of eluted DNA with the Nucleoside Digestion Mix (NEB #M0649) . The optional RNase treatment was performed with the Zymo prep.
Achieve outstanding results when compared to other commercially available solutions
Single Run Sequencing Results of HEK293, Human Blood, and Mouse Kidney Samples on the Oxford Nanopore Technologies Platform
DNA used for the sequencing libraries was extracted by following the standard protocols for cultured cells and fresh mammalian blood samples, without further size selection. Mouse kidney DNA samples were extracted from fresh samples using a rotor-stator homogenizer for sample homogenization. Libraries were generated using the LSK109 ligation sequencing kit and loaded on a FLO-MIN106D flow cell. Sequencing was performed on a GridION® Mk1 for up to 48 hours, or shorter if no more data was generated by the flow cell. No additional treatment of the flow cell (e.g., flushing) was employed. In the samples that resulted in > 10 Gb of data, 800–1,000 ng of DNA library was loaded onto the flow cell for optimal sequencing performance and effective pore usage. Read lengths are indicated in bases unless otherwise noted.
Excellent performance in long read sequencing
*workflow times are estimated based on protocol protocols published as of July 2020, and internal testing or usage **Notes are based on internal testing or usage
Testimonials
Simon Lesbirel (1), Ph.D., Jeremy R. Charette (1), B.S., Chia-Lin Wei (1), Ph.D., Eric Cantor (2), Ph.D., Danielle Freedman (2), M.S., Giron Koetsier (2), Ph.D. (1) The Jackson Laboratory, Bar Harbor, ME/Farmington, CT (2) New England Biolabs, Inc., Ipswich, MA
Introduction
Methods
Results
Discussion
Conclusion
INTRODUCTION Generation of transgenic mice through random or targeted integration of DNA fragments can lead to structural variation and integration mutagenesis (1), both of which are undesirable outcomes. Due to the significant labor required for their characterization, it is estimated that only around 5% of transgenic mouse models published in the Mouse Genome Database have an annotated chromosomal location (1). Therefore, a technique capable of quickly and cost-effectively identifying chromosomal location and confirming the transgene sequence integrity is essential. Further to this, the interrogation of large loci at the base level between strains remains difficult without using whole genome sequencing. A recently described technique, Cas9 no-amplification enrichment (2), has the potential to fulfill that need. Traditionally, the genomic modifications required to generate mouse models leverage PCR-based assays and Sanger sequencing for validation. However, in many cases, the structure and the sequence of the gene or its chromosomal integration site hinder analysis by these methods. Loops and sequence repeats prevent effective assessment of DNA sequence. The CRISPR/Cas9-mediated amplification-free enrichment approach for Oxford Nanopore Technologies® sequencing is an alternative method for interrogation of loci of interest or transgene sites. The method is relatively low-cost and can enrich regions of interest over native sequences without the need for PCR amplification (Figure 1).
Successful use of the Cas9 enrichment protocol relies on using high-quality, high molecular weight (HMW) gDNA as an input material.
Successful use of the Cas9 enrichment protocol relies on using high-quality, high molecular weight (HMW) gDNA as an input material. Working with the longest possible DNA fragments increases the chance that the entire region of interest remains intact after DNA extraction. The initial Cas9 enrichment sequencing workflow implemented within the Genome Technologies group at The Jackson Laboratory was dependent on phenol/chloroform DNA extraction, which initially fulfilled the requirements. However, while the phenol/chloroform-based workflow is effective for ultralong sequencing, it proved to be laborious and time consuming when applied to the Cas9 enrichment protocol. The sample lysis, phenol extraction and DNA precipitation take approximately one full day. Subsequently, this method requires up to 3 days of “rest time” to allow the isolated HMW gDNA to return to solution, resulting in the whole extraction process taking several days. Accordingly, the Cas9 enrichment can be started around day 5 (Figure 8). In addition, the increased frequency of extractions produced excessive amounts of hazardous waste. Therefore, we sought a faster and more environmentally friendly DNA extraction alternative.
In standard ligation-based whole genome sequencing approaches, desired loci/transgenes will be sequenced only once or a few times per nanopore sequencing run, but not with enough coverage to collect reliable sequence information. The Cas9 no-amplification enrichment workflow allows for specific enrichment of targeted regions by reducing undesired fragments from the sequencing process via dephosphorylation of their phosphate ends. Lacking terminal 5´ phosphate groups, they do not participate in adapter ligation. The target region, however, is subsequently cleaved using a Cas9-sgRNA (single guide RNA) ribonucleoprotein complex (RNP) making it accessible for sequencing adapter ligation. The resulting libraries allow for enriched sequence generation from the region of interest against a minimal background of genomic DNA sequences typically resulting from off-target Cas9 cleavage and non-specific adapter ligation. Furthermore, multiple sgRNAs can be used to enrich a variety of targets in a single library, thereby increasing efficiency and decreasing cost (3). At The Jackson Laboratory, one of our priorities has been to establish assays to standardize analysis for routine assessment of genomic alterations such as targeted mutagenesis and transgene integrations. Cas9 enrichment has proven to be an effective approach. Simultaneous Cas9 enrichment analysis of 2 to 4 targeted sequences has now been established as a standard workflow, with targeted regions typically being around 5 kb in size and sometimes up to 30 kb.
In this work, we leveraged the novel glass bead-based approach employed by the New England Biolabs Monarch® HMW DNA Extraction Kits to significantly reduce the time required for generating HMW gDNA from mouse tissue samples.
In this work, we leveraged the novel glass bead-based approach employed by the New England Biolabs Monarch® HMW DNA Extraction Kits to significantly reduce the time required for generating HMW gDNA from mouse tissue samples. With this new approach, the HMW DNA extraction process from tissues is complete in about 90 minutes, and DNA is ready to use shortly after, thereby significantly reducing the overall time required to perform the Cas9 enrichment workflow. Yield, purity, and integrity of the isolated HMW DNA is compared to phenol-extracted DNA and its efficient use in the optimized Cas9 sequencing workflow is demonstrated. Sequencing results are presented describing several case studies: locus analysis for mouse strain comparison, targeted mutagenesis sites and several transgenes and their insertion sites, including loci that proved difficult to sequence in earlier attempts.
FIGURE 1: Overview of the Cas9 no-amplification enrichment library prep workflow
METHODS Description of samples and inserts Transgenic mice with homozygous and heterozygous inserts as well as targeted mutations and endogenous regions of the genome (5 kb to 30 kb) were analyzed (Table 1). Liver and kidney serve as good sources of HMW DNA and were therefore chosen as target organs. Organs were harvested in accordance with the ethical standards at The Jackson Laboratory, and samples were flash frozen in liquid nitrogen and pulverized prior to extraction. HMW DNA extraction with phenol/chloroform HMW gDNA was extracted using a modified phenol/chloroform extraction protocol (4). 10–20 mg of tissue was pulverized on dry ice, incubated in 10 ml lysis buffer (100 mM NaCl; 10 mM Tris, pH 8.0; 25 mM EDTA, pH 8.0; 0.5% SDS; 0.02 mg/ml RNase A) at 37°C for 1 hour. After addition of Proteinase K (0.1 mg/ml final concentration), samples were incubated at 50°C for 3 hours to complete tissue digestion. DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1, v/v), phase separated twice using Qiagen® MaXtract™ High Density tubes, centrifuging at 3000 x g for 10 minutes, precipitated in ice cold ethanol, washed twice in 70% ethanol, and resuspended in 400 µl TE low. The purified DNA rested at 4°C for 2–3 days to ensure complete dissolving. HMW DNA extraction using Monarch HMW DNA Extraction Kits HMW genomic DNA from mouse tissue samples was isolated using the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060) using the recommended protocols.For extraction from mouse liver samples, the recommended addition of NaCl to a final concentration of 2.2M before bead binding was included in the tissue protocol. Agitation speed used during lysis was either 500 rpm and 1700–2000 rpm to generate longer and shorter fragments, respectively. Following extraction, if DNA was not homogenously dissolved, DNA was prepared for use by heating to 50°C for 10 minutes with occasional pipetting up and down with a P200 wide bore tip. If extractions were done at the end of the day, DNA samples were stored at 4°C overnight prior to analysis and library prep on the following day.
FIGURE 2: Depiction of sgRNA targeting design for homozygous and heterozygous transgene insertions
TABLE 1: Description of target regions investigated
DNA measurement and analysis A rapid screen of DNA integrity (DIN) was carried out by TapeStation® 4200 (Agilent Technologies) using Genomic DNA ScreenTapes® and reagents (Agilent Technologies #5067-5365). Rapid assessment of the amount of HMW DNA (>50 kb) was carried out by region analysis of the DNA signals on the TapeStation. DNA Purity was assessed by Nanodrop® 2000 and yield was analyzed with both Nanodrop and Qubit® dsDNA BR Assay Kit and the Qubit Fluorometer 3.0 (Thermo Scientific). For concentration measurement, 1 µl of DNA was taken from the top, middle, and bottom of the HMW DNA sample and averaged. Cas9 enrichment library generation The Cas9 sequencing protocol was carried out using Oxford Nanopore’s Ligation Sequencing Kit (SQK-LSK109) coupled with the New England Biolabs NEBNext® Companion Module (NEB #E7180). Alternatively, this protocol can be conducted using a specific Cas9 Sequencing Kit (Oxford Nanopore Technologies SQK-CS9109). The corresponding Oxford Nanopore Technologies protocol was followed for library construction with minor modifications as described below. sgRNAs were designed to avoid common SNP sites (5) and were sourced from Integrated DNA Technologies (IDT), along with the described Cas9 enzyme and duplex buffer. Typical sgRNA design for homozygous and heterozygous inserts is shown in Figure 2. A few protocol modifications were implemented to improve the relatively low efficiency of the Cas9 library preparation caused by the high viscosity of the HMW samples: • Cas9-cleavage was carried out for 1 hour • Sequencing adaptor ligation was carried out for 1 hour instead of 10 minutes • Elution incubation time was 30 minutes instead of 10 minutes, and elution was carried out in 13 µl (1 µl for measurement, 12 µl for sequencing)
Oxford Nanopore Technologies sequencing Samples were sequenced on MinION R9.4.1 flow cells for 24 hours on either MinION MK1B or GridION Mk1 (Oxford Nanopore Technologies). Samples were run as single runs or multiplexed with up to 4 targets. Flow cells were reused 2 to 4 times after washing every 24 hours using the protocol and reagents from Oxford Nanopore Technologies (Flow Cell Wash Kit, EXP-WSH003).Sequencing data analysisBase calling was carried out using GUPPY (v3.2.10). The resulting fastq files were aligned to a reference sequence using minimap2 (v2.17). Custom reference sequences were constructed for transgene insertions sites using the Mus musculus reference genome (MM10) and corresponding insert sequence. Alignment results were subject to MapQ score filtering using Samtools (v1.11). Subsequent coverage depth for on-target reads was generated using Bedtools (v2.29.2). On-target reads were visualized in Integrative Genomics Viewer (IGV).
RESULTS Assessment of DNA concentration, purity and fragment length The HMW DNA that was used for initial Cas9 enrichment studies was isolated using phenol/chloroform extraction. For later studies, the Monarch HMW DNA Extraction Kit for Tissue was introduced. Yield and purity data are shown for DNA extracted from liver and kidney samples with phenol/chloroform (Table 2) or Monarch (Table 3). Phenol/chloroform-extracted samples Concentration and purity measurements by Nanodrop and Qubit (Table 2) revealed that based on spectrophotometric readings, around 1.6 µg DNA per mg tissue was isolated for liver and 4-6 µg per mg tissue for kidney. However, Qubit values were significantly lower, particularly for the liver samples. Purity grade was intermediate; though A260/A280 purity ratios were in the normal range (1.83-1.85), the A260/A230 ratios were lower than optimal, with 1.8-2.1 for kidney and only 1.1-1.3 for liver.
TABLE 2: Yield and purity analysis of phenol/chloroform-extracted HMW DNA samples HMW DNA samples were analyzed on a Nanodrop 2000 (Thermo Scientific) to determine the concentration and purity ratios. Additionally, fluorometric concentration assessment was carried out using a Qubit dsDNA BR Assay Kit and a Qubit 3.0 fluorometer (both Thermo Scientific). In each case 1 µl samples were taken from top, middle and bottom of the solution. Averages of the three were calculated and displayed in the table.
FIGURE 3: Example of the HMW DNA % >50 kb region analysis of a Genomic DNA ScreenTape run 1 µl HMW DNA samples was loaded on Genomic DNA ScreenTapes using accompanying reagents and run on a TapeStation 4200 device (all Agilent Technologies). At the end of each run a value for % of DNA >50 kb was determined by using the region analysis tools and integrating the signal area above 50 kb. An example of Monarch sample 6 is shown here.
Monarch HMW DNA Extraction Kit-extracted samples Concentration and purity measurements were assessed by Nanodrop and Qubit (Table 3). Yields of initial samples were somewhat lower than expected, but good yields (1.4 µg/mg for liver and 2–3 µg/mg for kidney) were obtained after becoming familiar with the protocol. Consistent high purity was observed among all extracted samples as A260/A280 purity ratios were greater than 1.83 and A260/A230 ratios were >2.1.
Rapid assessment of DNA integrity For a quick assessment of the fragment length of the phenol/chloroform- and the Monarch- extracted DNA samples, samples were run on the TapeStation using Genomic DNA ScreenTapes. With the help of the region analysis tool the percentage of HMW DNA (DNA > 50 kb) was determined. Overall, the percentages were 69% for the phenol/chloroform samples and around 81% for the Monarch samples (Table 4). A typical example of such region analysis is shown in Figure 3. DIN values were comparable for both extraction methods.
Cas9-targeted sequencing metrics and coverage Cas9-targeted sequencing was carried out on a variety of genomic loci, including transgenic insertions and sites of targeted mutagenesis. Sequencing coverage metrics are shown in Table 5. Although the percentage of on-target reads is low compared to total reads generated, the mean coverage of targeted regions is far greater than the whole genome coverage generated from each run, as shown in Table 6.
TABLE 3: Yield and purity analysis of HMW DNA samples extracted with the Monarch HMW DNA Extraction Kit HMW DNA samples were analyzed on a Nanodrop 2000 to determine the concentration and the purity ratios. Additionally, fluorometric concentration assessment was carried out using a Qubit dsDNA BR Assay Kit and a Qubit 3.0 fluorometer. In each case 1 µl samples were taken from top, middle and bottom of the solution. Averages of the three were calculated and are displayed in the table. DNA samples from kidney purified using 500 rpm agitation speed during lysis showed some loss of yield; the reduced gDNA yield from 500 rpm agitated samples can be mitigated by increasing bead binding incubation from 5 minutes (yellow) to 8 minutes (green).
TABLE 4: HMW DNA samples isolated using the Monarch Kit demonstrate longer fragment legnths than phenol/chloroform extraction 1 µl HMW DNA samples were loaded on Genomic DNA ScreenTapes using accompanying reagents and run on a TapeStation 4200 device (Agilent Technologies) to produce DNA Integrity (DIN) values. At the end of each run a % value of DNA >50 kb was determined by using the region analysis tools and integrating the signal area above 50 kb.
TABLE 5: Cas9-targeted sequencing coverage metrics Alignment and coverage data were generated for each mouse line using nanopore targeted sequencing metrics, sequencing data, and dedicated software applications as described in the methods section.
TABLE 6: Comparison of on-target coverage versus whole genome coverage On-target coverage was compared to the whole genome coverage for mouse line 10 and 11 to demonstrate the on-target coverage enrichment.
Run metrics from the multiple 24-hour targeted sequencing runs are summarized in Table 7. Samples 1, 10 and 11 were run on Oxford Nanopore Technologies GridION platform, while all others were run on a MinION. Due to the low number of target regions, only a small proportion of DNA ligates with the sequencing adaptors, resulting in low sequencing data overall. Accordingly, only 10-15% pore usage is observed (Figure 4).
Targeted sequencing case study results Targeted sequencing for rapid strain comparison CRISPR/Cas9 enrichment sequencing was employed to investigate sequence variation at the MX1 locus in its entirety by targeting 2 kb up- and downstream. This assay was applied to the common laboratory strain C57BL/6J (Sample 10) and a wild-derived strain CAST/EiJ (Sample 11). The resulting data confirm a known 3.5kb deletion in C57BL/6J spanning exons 8 to 12. The deletion appears as an insertion in our CAST/EiJ (Sample 11) alignment due to the mouse reference being constructed with C57BL/6J. The resulting capture sequencing generated 80X coverage over a 22.5 kb region in CAST/EiJ and 230X coverage over a 19 kb region in C57BL/6J (Figure 5).
FIGURE 4: Typical pore usage of Cas9 targeted run Screenshots taken from the GridION X5 software during the sequencing of Sample 10 and Sample 11. After 24 hours sequencing of Sample 10 (A), the flow cell was nuclease washed, primed, and loaded with Sample 11 and ran for a further 24 hours (B).
TABLE 7: Cas9 capture sequencing MinION and GridION run metrics Run metrics from the multiple 24-hour targeted sequencing runs are summarized. Samples 1, 10 and 11 were run on Oxford Nanopore Technologies GridION platform, while all others were run on a MinION.
CONCLUSION Overall, the Cas9 enrichment approach is a powerful tool for interrogation of genomic loci. Having a rapid high-quality method like Monarch for HMW DNA extraction enables a significant reduction in the workflow time and facilitates troubleshooting efforts without adding several days of work. Looking forward, we aim to develop Cas9 enrichment-based transgene screening approaches that do not require the sacrifice of the animals, as they currently do when HMW DNA needs to be isolated from organ tissue. Future tests will, therefore, focus on using ear punch or tail clip tissue as input material for HMW DNA extraction and optimizing Cas9 enrichment sequencing for low input samples.
FIGURE 5: Comparison of MX1 in Mouse Lines C57BL/6J and CAST/EiJ (A) Sample 10: 230X coverage depth over a 19 kb region of C57BL/6J spanning the entire MX1 locus aligned to MM10. (B) Sample 11: 80X coverage of a 22.5 kb region from CAST/EiJ spanning the MX1 locus and identifying the 3.5 kb not present in C57BL/6J aligned to MM10 (C) Reads covering MX1 C57BL/6J when aligned to CAST/EiJ.
Analysis of targeted mutagenesis sites Cas9 targeted sequencing was also employed to validate the integrity of targeted mutations within multiple mouse strains (Samples 1 and 8). In Sample 1, which harbors a 5 kb insert, 2 insertions were detected, indicated in purple in Figure 6. In Sample 8, significantly longer than expected reads were obtained (up to 95 kb) from the targeted region of 13 kb. Analysis of transgene insert regions Figure 7 demonstrates reads collected from mouse lines 4 and 5 with sequencing results including a homozygous 30 kb insert and a heterozygous 5 kb insert. The typical reads obtained with homozygous and heterozygous transgene inserts are illustrated in Figure 2. Similar coverage plots were obtained with Sample 9, a 5 kb insertion with 74X coverage (data not shown). Generating reads from Samples 6 and 7 (heterozygous 5 kb insert) was not successful with ultra HMW DNA. Therefore, the Monarch HMW DNA extraction was repeated with larger input amounts and lysis was carried out at maximal agitation speed (2000 rpm) to reduce the size of the HMW DNA fragments and the accompanying viscosity. This modification yielded DNA that resulted in a usable 20X/23X coverage of the regions of interest (data not shown).
FIGURE 6: Coverage depth and associated reads validating samples with targeted mutations (A) Sample 8: homozygous 5 kb region covering a floxed exon. Sequencing generated 70X converge over the region of interest. (B) Sample 1: 13 kb region of interest for the validation of a targeted mutation. Capture sequencing generated a coverage depth of 100X over region of interest. Reads from 13 to 95 kb in length were generated.
FIGURE 7: Analysis of 30 kb homozygous transgene insertion and 5 kb heterozygous transgene insertions (A) Sample 5: Homozygous 30 kb insert; coverage across the region of interest was 44X. (B) Sample 4: a mouse line with a 5 kb heterozygous insertion, coverage depth of 92x. Note: mean coverage depth does not include endogenous chromosomal reads located at the 5´ and 3´ ends of the transgenic insertion.
DISCUSSION Significant time savings and increased flexibility with Monarch HMW DNA Extraction Kit Until recently, Cas9 enrichment workflows have been described using phenol/chloroform extracted HMW DNA when working with animal tissues. The drawbacks of this extraction approach are numerous, including significant handling, hazardous chemicals, and excessive time required for dissolving. Because this method requires several days to obtain usable DNA, it is not conducive to rapid transgene/strain analysis and limits the flexibility needed for troubleshooting or tweaking of parameters. The new Monarch HMW DNA extraction workflow provided DNA with high yields, high purity, and high DNA integrity which was ready to use in only a few hours. The Monarch-extracted DNA performed well in Cas9 sequencing and resulted in a significant time savings in the Cas9 enrichment sequencing workflow of up to 3 days (Figure 8).
FIGURE 8: Comparing the Cas9 sequencing workflow duration for phenol/chloroform and Monarch HMW DNA extraction
The Monarch workflow also enables tunable fragment length generation by having the user change the agitation speed of the thermal mixer during lysis, empowering the user to adjust the size of the DNA to optimize conditions for the relevant downstream application. This property proved useful, as it allowed for troubleshooting of experiments that failed with highly viscous ultra-high molecular weight DNA which had been isolated after lysis at a low agitation speed (500 rpm). A repeat of the same Cas9 sequencing experiment with less viscous samples that were purified with 2000 rpm agitation speed during lysis, led to sufficient coverage of the region of interest (Samples 6 and 7, described more in detail below). Monarch extraction outperforms phenol/chloroform in most metrics DNA yield The Monarch protocols provide good yields for both mouse tissue types investigated. Yields per mg tissue for liver samples were similar for both extraction methods at around 1.5 µg/mg tissue. Actual yields with phenol may be significantly lower if Qubit values are taken as standard. For kidney, yields per mg were high with both methods, with phenol giving the largest overall yield. Troubleshooting of the lower Monarch yields observed with 4 kidney samples prepared with low agitation speeds during lysis revealed that for such samples, increasing the length of the binding step in the vertical rotating mixer is recommended in the protocol. This extended binding time resulted in higher yields in the next set of DNA extractions from kidney tissue. Although phenol preps by nature offer more flexibility regarding the maximal sample input amounts, the total yields obtained using the Monarch kit were good and sufficient for several experiments. Moreover, recent Cas9 sequencing experiments performed with Monarch-purified DNA indicate that high coverage in Cas9 experiments can be achieved with significantly lower DNA input amounts (data not shown). DNA quality Samples isolated using the Monarch kit were higher purity than those isolated with phenol/chloroform, particularly with liver samples (A260/A230 with phenol ranged 1.1-1.3). Also, the rapid DNA integrity analysis on the TapeStation indicated that DNA isolated using the Monarch kit had a higher proportion of DNA > 50 kb when compared with that isolated by phenol extraction. Consequently, using Monarch as an alternative to phenol extraction is not only a faster approach, but may also result in better performance in long read sequencing applications.
Targeted sequencing results Cas9-enrichment for comparison of wild-derived and common inbred laboratory strains It has been known for some time that most inbred laboratory mice do not possess genetically-mediated resistance to Myxoviruses (6,7) . The interferon-inducible protein responsible for this resistance is encoded by MX1 located on Chromosome 16. Several inbred mouse strains have acquired a large deletion or nonsense mutations in MX1 due to a founder effect resulting in a non-functional protein (8). The absence of a functional Mx1 protein in C57BL/6J mice has been linked to an increased susceptibility to Influenza A virus infection. In contrast, recently wild-derived strains, including CAST/EiJ contain a functional MX1 allele that renders them highly resilient to viral infections. The high coverage depth generated by Cas9 targeted sequencing of the entire MX1 locus identified the large 3.5 kb deletion present in the common laboratory mouse C57BL/6J (Figure 5, Samples 10 and 11). Analysis of transgene-targeted mutagenesis sites Figure 6A, Sample 8 illustrates the power of this technology for validating mouse models. Targeted mutagenesis was carried out to introduce loxP sites flanking exon 4 of our gene of interest. To assess the integrity of the loxP sites and the surrounding chromosomal context, sgRNAs were designed up and downstream to excise a 5 kb fragment. Cas9 enrichment was carried out, producing 70X coverage of the region of interest. Sequence alignment revealed the presence of two unexpectedly large inserts – one 180 bp and another 80 bp (Figure 6A). These larger-than-expected insertions are suspected to be a result of plasmid DNA integration along with the loxP sequence. Figure 6B, Sample 1, depicts the targeted sequencing of a 13 kb region at the terminal end of the gene of interest, covering exons 25–29. The model in question was subject to targeted mutagenesis within this region. Cas9 capture sequencing was employed to assess the success of the genetic modification. The subsequent sequencing yielded a mean of 100X coverage over our target region whilst generating reads 13-95 kb in length. Larger insertions than expected were revealed, indicating the requirement for further investigation into this region. The Cas9 targeted sequencing approach saved considerable time as traditional Sanger-based methods did not yield any insights into this particular locus condition.
The new Monarch HMW DNA extraction workflow provided DNA with high yields, high purity, and high DNA integrity which was ready to use in only a few hours.
The Cas9 targeted sequencing approach saved considerable time as traditional Sanger-based methods did not yield any insights into this particular locus condition.
Cas9-enrichment is a powerful tool for assessment of transgene insertion sites The efficiency of this method is highlighted in Figure 7, showing analysis of Sample 5 (homozygous 30 kb insert) and Sample 4 (heterozygous 5 kb insert). Sample 5 contains a large insert and delivered 44X coverage over the region of interest. In Samples 4 and 9, the Cas9 enrichment approach was used to check the integrity of the insertion site as well as the transgene sequence. The sgRNAs were designed 1 kb up and downstream of the transgene, and the enrichment approach worked well with a 92X and 74X coverage, respectively. A slight overrepresentation of the shorter allele not containing the transgene was observed. Sample 6 and 7 (both heterozygous 5 kb inserts) proved to be difficult targets; even with the Cas9 enrichment approach, it was challenging to generate reads. Monarch HMW DNA extraction was repeated with larger input amounts and lysis was carried out at maximal agitation speed (2000 rpm) to reduce the size of the HMW DNA fragments and the accompanying viscosity. This modification enabled 20X/23X coverage of the region of interest, respectively, sufficient for the application. This example provides a compelling case of how having access to a rapid extraction method enables troubleshooting experiments without significant time loss.
The Monarch HMW DNA Extraction Kit enabled a reduction of the total Cas9 enrichment workflow time by 3 days. In addition to the significantly faster DNA extraction workflow, better solubility of the Monarch purified HMW DNA resulted in significant time savings.
The HMW DNA isolated with the Monarch kit consistently was of high yield, superior quality and longer fragment length; thus, this approach proved to be a powerful alternative to phenol chloroform extractions.
Tunable fragment length of the Monarch-isolated HMW DNA is a valuable feature when troubleshooting targeted sequencing results.
Coupling the Monarch HMW DNA extraction procedure with an optimized Cas9 no-amp enrichment library prep protocol has resulted in the successful establishment of a highly efficient standard procedure for the analysis of transgene insertion sites in mice by Genome Technologies at The Jackson Laboratory.
References 1. Goodwin, L.O. et al. (2019) Genome Research. https://doi.org/10.1101/gr.233866.117. 2. Gilpatrick, T. et al. (2020) Nature Biotechnology 38, 433–438. View 3. Oxford Nanopore Technologies (2020). “Cas9 Targeted Sequencing Before Start Checklist Cas9 Targeted Sequencing,” 1–6. 4. Gong, L. et al. (2019) Journal of Visualized Experiments: JoVE. https://doi.org/10.3791/58954. 5. Pacific Biosciences. 2019 “Reference Guide – Designing CRISPR-Cas9 RNA Oligonucleotides for the No-Amp Targeted Sequencing Procedure.” Pacific Biosciences 01 (101): 1–10. 6. Lindenmann, J. et al. (1962) The Resistance of A2G Mice to Myxoviruses. 7. Tumpey, T.M., et al. (2007) Journal of Virology 81, 10818–10821. PMID:17652381 8. Staeheli, P, et al. (1988). Molecular and Cellular Biology. 8, 4518-4523. PMID: 2903437
View Technical Note
COVID-19 Researcher Spotlight: mRNA Vaccine Development
In our recent COVID-19 Researcher Spotlight, Lydia Morrison chatted with NEB Senior Scientist Bijoyita Roy about the landscape and timeline of COVID-19 vaccine development, focusing on the mRNA vaccine platform and how NEB is helping improve mRNA vaccine development and production. You can read an excerpt from our interview with Bijoyita below. You can access the COVID-19 Researcher Spotlight Series to watch the entire interview and to learn about two of the vaccine candidates that were recently approved by the FDA.
Watch Full Interview
Lydia Morrison: mRNA vaccines are relatively new. What are the advantages of that platform? Bijoyita Roy: Messenger RNA is transient in expression, and its expression is rapid. It’s easy to titer and it can also be synthesized, standardized and easily scaled. So, these are some of the most prominent advantages of the mRNA based platform. Now, mRNA based vaccines are emerging as an alternative to conventional vaccine approaches. So, the idea here is really simple. You make an RNA or a messenger RNA in a tube, and you introduce it into a cell to hijack the cells machinery, to make any protein you are interested in. And once it is delivered in the cell, it gets translated by the cellular machinery resulting in the synthesis of the protein antigens. Now these antigens are then recognized by the immune system and you see immune responses. So, what’s happening here is, the cell itself acts as a bioreactor to make any protein of interest. So all you really need is the DNA sequence to make the RNA that you are interested in. So, you use the same process for any protein, and that makes this entire platform really lucrative, and it streamlines a lot of early development and discovery work. As an example, once the sequence of SARS-CoV-2 genome was released, the DNA sequence of interest that could potentially be used as a vaccine target was generated in a matter of few days, and the mRNA molecule that could actually encode for the spike protein was actually synthesized in a matter of weeks. Lydia Morrison: So what are the similarities and differences between the two FDA approved vaccines? Bijoyita Roy: The main similarity is that both of these vaccines contain the genetic instructions for building a specific Coronavirus protein, the spike protein. So, when injected into the cell the vaccine causes them to make spike proteins, which then gets released into the body, and it involves an immune response from the immune system. What is really interesting is that for the two independent vaccines, the mRNA sequence is very different, but the mechanism of action for both the Pfizer – BioNTech, as well as the Moderna vaccines, is exactly the same and they are showing similar efficacy.
Browse Other Spotlights
Doing COVID-19 related research?
Let us know what you are working on, so we can keep you informed on new COVID-19 related products and publications available from NEB.
Stay Informed
Upcoming episode
Lydia Morrison and Senior Application Scientist, Bjoern Textor of NEB Germany, will talk to Bas Oude Munnink of Erasmus MC in the Netherlands about the impact of multiple circulating strains of SARS-CoV-2 and what he's learned from his work on COVID-19 that could aid in identifying and stopping outbreaks of COVID-19 or other pathogens in the future.
Bas Oude Munnink
Bijoyita Roy
Supporting COVID-19 Research
As the SARS-CoV-2 virus continues to impact our communities, our manufacturing and distribution teams continue to be fully operational, and we are working closely with our suppliers and distribution partners to ensure uninterrupted access to our products and technical support. NEB’s products are available for research purposes only. However, we are supplying and supporting customers who are working diligently to develop better diagnostic tools and vaccines for the SARS-CoV-2 virus, and are ready to supply additional customers with the reagents they need to validate and develop them as diagnostic tools for lab-based or point-of-care settings.
Visit www.neb.com/COVID-19 to learn more about our growing selection of products available for these applications:
Featured Products
The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit is a research use only (RUO) kit optimized for real-time qualitative detection of SARS-CoV-2 nucleic acid using hydrolysis probes.The kit features a primer/probe mix specific to two regions of the SARS-CoV-2 virus N gene [based on sequences provided by the Centers for Disease Control and Prevention (CDC)]. The probes have been modified to contain different fluorophores (N1: HEX; N2: FAM) to enable multiplexing. An internal control primer and probe set, designed to amplify the human RNase P gene, is also included in the primer mix. The reverse primer of this target has been modified from the CDC design to target an exon/exon boundary to reduce background amplification from possible contaminating genomic DNA.
Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit
The Luna Probe One-Step RT-qPCR 4X Mix with UDG supports robust, sensitive detection and quantitation of up to 5 targets in a multiplexed reaction. It is supplied at a 4X concentration and enables higher amounts of sample input, which is relevant for applications where RNA present in low abundance is of interest, such as pathogen detection. The Dual WarmStart/Hot Start enzyme formulation enables room temperature setup and stability for up to 24 hours. The single tube master mix format includes thermolabile UDG and dUTP for reduced risk of carryover contamination.
Luna Probe One-Step RT-qPCR 4X Mix with UDG
Compatible with low reaction volumes including 384-well plate formats
Increase sensitivity with Luna Probe One-Step RT-qPCR 4X Mix with UDG allowing for more sample input
Reduce background amplification from genomic DNA by use of a modified RNase P Internal Control reverse primer to target an exon-exon boundary
Multiplex detection of 2019-nCoV_N1 and 2019-nCoV_N2 targets and human RNase P gene enables high throughput workflows
NEB’s portfolio of research-grade and GMP-grade* reagents enables bench-scale to commercial-scale mRNA manufacturing. Our optimized HiScribe™ kits enable convenient in vitro transcription (IVT) workflows. When it is time to scale up and optimize reaction components, our standalone reagents are readily available in formats matching our GMP-grade offering, enabling a seamless transition to large-scale therapeutic mRNA manufacturing.
From research to therapeutic production, NEB’s in vitro transcription portfolio will meet your needs
"GMP-grade" is a branding term NEB uses to describe reagents manufactured at NEB’s Rowley facility. The Rowley facility was designed to manufacture reagents under more rigorous infrastructure and process controls to achieve more stringent product specifications and customer requirements. Reagents manufactured at NEB’s Rowley facility are manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards. However, at this time, NEB does not manufacture or sell products known as Active Pharmaceutical Ingredients (APIs), nor does NEB manufacture its products in compliance with all of the Current Good Manufacturing Practice regulations.
Enabling gram-scale RNA synthesis
NEB manufactures and inventories the following enzyme specificities at GMP-grade, meeting customer needs with short lead times:
Learn about how the Innovative Genomics Institute at University of California, Berkeley has designed a novel SARS-CoV-2 assay using this product.
View PDF
View NEB products being used in COVID-19 related research
Visit www.neb.com/COVID19 to:
View citations and EUA protocols utilizing NEB products
Check out our COVID-19 Researcher Spotlight podcast/video series
Tell us about your own COVID-19 related research
Using the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, up to 94 different samples can be assessed in a single 96-well plate. Anticipated results for each sample type are shown (in each fluorophore channel).
Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit components
The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit detects two regions of the N gene and the human RNase P gene in a single reaction
A. The two SARS-CoV-2 sequences are based on those provided by the CDC, but modified to contain different fluorophores (N1: HEX, N2: FAM). B. The RNase P internal control includes a Cy5 labeled probe and a re-designed reverse primer. This primer spans an exon-exon junction to avoid amplification of human genomic DNA which contains a 2.4 kb intron.
The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit demonstrates a lower limit of detection than TaqPath™1-Step RT-qPCR Master Mix, CG
LOD comparison using: Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit for multiplex RT-qPCR targeting 2019-nCoV_N1 target (HEX) and 2019-nCoV_N2 target (FAM), according to reaction and cycling conditions provided in the E3019 product manual, and TaqPath 1-Step RT-qPCR Master Mix, CG for singleplex RT-qPCR targeting 2019-nCoV_ N1 (FAM) and 2019-nCoV_N2 (FAM), according to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel guidelines. Performance was evaluated using Synthetic Twist SARS-CoV-2 RNA Control 2 diluted in 10 ng of Jurkat total RNA. Data was collected on an Applied Biosystems® 7500 Fast real-time instrument (96-well, 20 µl reactions). Under these conditions, the Luna Kit has an LOD of 5 copies/reaction for both targets while the LOD using TaqPath is 10 copies/reaction for these targets.
A new product choice for one-step RT-qPCR assays
Robust amplification and detection of different viral RNA with Luna Probe One-Step RT-qPCR 4X Mix with UDG
Multiplex detection (5 targets) with the Luna Probe One-Step RT-qPCR 4X Mix with UDG
Multiplex RT-qPCR was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG over a 5-log range of Jurkat total RNA (100 ng to 10 pg) on a Bio-Rad CFX96 real-time instrument. Amplification standard curves and efficiencies for each of the 5 human targets are shown. Reactions (20 μl) included primers and probes at 200 nM each, and followed the product recommended cycling conditions. All five targets were detected linearly in the multiplex reactions with strong efficiency and R2 values.
The Luna Probe One-Step RT-qPCR 4X Mix with UDG outperforms comparable products
One-step RT-qPCR was tested on 8 RT-qPCR targets (indicated by color) varying in abundance, length, and %GC. Data was collected on multiple days by two users according to manufacturer’s recommendations using the Applied Biosystems™ QuantStudio™ 6 real-time PCR system. Results were evaluated for efficiency, low input detection and lack of non-template amplification (where ΔCq = average Cq of non-template control – average Cq of lowest input). In addition, consistency, reproducibility and overall curve quality were assessed based on metrics described previously (Quality Score). Although both products performed reasonably well, NEB’s Luna Probe RT-qPCR 4X Mix with UDG outperformed the TaqPath 1-Step RT-qPCR Master Mix, CG, as evidenced by the higher number of experiments whose results fell in the green box.
RT-qPCR targeting SARS-CoV-2 (N1 target) and H. influenza H1N1 (HA target) was performed using the Luna Probe One-Step RT-qPCR Mix with UDG. Performance in 20 μl reactions was evaluated in two real-time instruments over a 5-log range of (A) 10–100,000 copies Synthetic SARS-CoV-2 RNA Control 2 (Twist Biosciences, SKU: 102024) diluted in 10 ng of Jurkat total RNA (BioChain, #R1255815-50) and (B) 12.4–124,000 copies Influenza A (H1N1) RNA (ATCC®VR-95DQ™) diluted in 10 ng Jurkat total RNA. Sensitive, linear performance can be observed in the amplification of both viral targets.
