Product Class: Other

NEBNext® RSV Primer Module


Catalog #E9642

Product Introduction

NEBNext® RSV Primer Module is intended for use in cDNA synthesis, amplification, and library preparation upstream of Respiratory Syncytial Virus (RSV) sequencing. The Primer Module can be used upstream of both Illumina® and Oxford Nanopore Technologies® sequencing, when paired with LunaScript Multiplex One-Step RT-PCR Kit and a compatible library prep solution (e.g., NEBNext UltraExpress FS DNA Library Prep Kit or ONT library prep reagents).

  • Full-length coverage of RSV A and RSV B
  • Intentionally designed, balanced, and functionally optimized primer sequences
  • Flexibility to use with Illumina® or Oxford Nanopore Technologies® platforms
  • Timely RSV variant surveillance with NEB’s Primer Monitor tool
  • Primer sequence information can be found in the NEBNext GitHub Repository

Note: This product is intended for research-use only. 

Product Information

Description

RSV is a virus that commonly infects infants and the elderly, causing severe respiratory symptoms, while leading to milder illness in older children and young adults. For laboratories interested in sequencing the RSV genome, the NEBNext RSV Primer Module contains a curated set of primer mixes for use in RT-PCR upstream of NGS library preparation. Downstream, the libraries are compatible with Illumina or ONT chemistries. Refer to the product manual for the required materials not supplied for your sequencing platform of choice.

The NEBNext RSV Primer Module has undergone extensive QCs to ensure that the primer mixes are balanced, well-aligned with modern strains of RSV, and free of cross contamination. In-house testing includes the full recommended workflow, combining RT-PCR, NGS library prep, and sequencing followed by analysis of sequencing metrics.

Due to its modular nature, and compatibility with a range of downstream sequencing platforms, the NEBNext RSV Primer Module provides flexibility for maximal ease of use. Not only is the NEBNext RSV Primer Module compatible with both Illumina and ONT sequencers, the workflow is automation friendly.  

Full information on the primer sequences included can be found at https://github.com/nebiolabs/rsvab-sequencing. To access up-to-date information on sequence variants that are impacting established primer sets, visit primer-monitor.neb.com.


Figure 1: NEBNext® RSV Primer Module is designed to generate overlapping amplicons across the Respiratory Syncytial Virus (RSV) genome

The NEBNext® RSV primer scheme consists of pan-RSV primers that target both RSV A and RSV B, as well as subtype-specific primers that recognize either RSV A or RSV B. Each primer mix consists of several primer pairs.

Figure 2: Workflow for use of NEBNext® RSV Primer Module for RT-PCR upstream of RSV library prep and sequencing

The NEBNext® RSV Primer Module is designed to be paired with the LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555) for streamlined cDNA synthesis and targeted amplification, followed by library construction using either NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340) or Oxford Nanopore Technologies® Native Barcoding Kit.

Figure 3: NEBNext® RSV Primer Module coupled with LunaScript® Multiplex One-Step RT-PCR Kit provides robust RSV amplicon yields


Overlapping amplicons were generated from 100–10,000 total copies of viral gRNA templates, with 1 ng Universal Human Reference RNA (UHR) background, using dual multiplexed RSV-targeting primer pools. The gRNA templates were RSV A gRNA (ATCC® VR1540), RSV B gRNA (ATCC VR-1580), a 1:1 [RSV A : RSV B] gRNA mix, or a 1:1:1:1 [RSV A : RSV B : Influenza A : SARS-CoV-2] mix.
A. 1/10th diluted amplicons were run on TapeStation® using DS 5000 HS reagents without a cleanup. TapeStation traces show expected amplicon peaks within the 400 to 1,400 bp size range.
B. Average amplicon yields (n=3) within 400 to 1400 bp window determined via TapeStation Analysis of diluted post-RT-PCR amplicons.

Figure 4: High amplicon coverage of control templates containing RSV A and/or RSV B is attained with NEBNext® RSV Primer Module’s Pan-RSV, RSV A or RSV B specific genome targets using Illumina® sequencing


Overlapping cDNA amplicons were generated from 100–10,000 total copies of viral gRNA templates, with 1 ng Universal Human Reference RNA (UHR) background, using dual multiplexed RSV targeting primer pools. The gRNA templates were RSV A gRNA (ATCC® VR-1540), RSVB gRNA (ATCC VR-1580), a 1:1 [RSV A : RSV B] gRNA mix, or a 1:1:1:1 [RSV A : RSV B : Influenza A : SARS-CoV-2] mix. The NTC template consisted of Nuclease-Free Water. Following targeted amplification, libraries were constructed with the NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340) and sequenced on a NextSeq® 500. 1M read pairs were sampled with seqtk and aligned with Bowtie 2 to a composite RSV A, RSV B, Influenza A, SARS-CoV-2, and human reference genome. Heat map of the average number of reads mapping to amplicons targeting either Pan-RSV, RSV A-specific, or RSV B-specific regions across all RSV and Multi-viral gRNA templates utilized is shown.

Figure 5: NEBNext® RSV Primer Module generates high overall genome coverage of RSV A and RSV B with Illumina® sequencing


Overlapping amplicons were generated from 100–10,000 total copies of viral gRNA templates, with 1 ng Universal Human Reference RNA (UHR) background, using dual multiplexed RSV targeting primer pools. The gRNA templates were RSV A gRNA (ATCC® VR-1540), RSV B gRNA (ATCC VR-1580), a 1:1 [RSV A : RSV B] gRNA mix, or a 1:1:1:1 [RSV A : RSV B: Influenza A : SARS-CoV-2] mix. Following targeted amplification, libraries were constructed with the NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340) and sequenced on a NextSeq® 500. 1M read pairs were sampled with seqtk and aligned with Bowtie 2 to a composite RSV A, RSV B, Influenza A, SARS-CoV-2, and human reference genome. Integrative Genome Viewer visualization of read coverage across RSV A (A) or RSV B genomes (B) (0–1,000 log scale) is shown.

Figure 6: High amplicon coverage of control templates containing RSV A and/or RSV B are attained with NEBNext® RSV Primer Module’s Pan-RSV, RSV A, or RSV B specific genome targets using Oxford Nanopore Technologies® sequencing


Overlapping amplicons were generated from 100-10,000 total copies of viral gRNA templates, with 1 ng Universal Human Reference RNA (UHR) background, using dual multiplexed RSV targeting primer pools. The gRNA templates were RSV A gRNA (ATCC® VR-1540), RSV B gRNA (ATCC VR-1580), a 1:1 [RSV A : RSV B] gRNA mix, or a 1:1:1:1 [RSV A : RSV B : Influenza A : SARS-CoV-2] mix. The NTC template consisted of Nuclease-Free Water. Following targeted amplification, ONT® libraries were constructed with NEB reagents and ONT’s Native Barcoding Kit V14. Libraries were loaded onto a MinION® Flow Cell (R.10.4.1) and sequenced on a GridION®. Sequencing reads were aligned with MiniMap2 to a composite RSV A, RSV B, Influenza A, SARS-CoV-2, and human reference genome. Heat map of the average number of reads mapping to amplicons targeting either Pan-RSV, RSV A-specific or RSV B-specific regions across all RSV and Multi-viral gRNA templates utilized is shown.

Figure 7: NEBNext® RSV Primer module generates high genome coverage of RSV A and RSV B control templates with Oxford Nanopore Technologies® sequencing

Overlapping amplicons were generated from 100–10,000 total copies of viral gRNA templates, with 1 ng Universal Human Reference RNA (UHR) background, using dual multiplexed RSV targeting primer pools. The gRNA templates were RSV A gRNA (ATCC® VR-1540), RSV B gRNA (ATCC VR-1580), a 1:1 [RSV A : RSV B] gRNA mix, or a 1:1:1:1 [RSV A : RSV B : Influenza A : SARS-CoV-2] mix. Following targeted amplification, ONT® libraries were constructed with NEB® reagents and ONT’s Native Barcoding Kit V14. Libraries were loaded onto a MinION® Flow Cell (R.10.4.1) and sequenced on a GridION®. Sequencing reads were aligned with MiniMap2 to a composite RSV A, RSV B, Influenza A, SARS-CoV-2, and human reference genome. Integrative Genome Viewer visualization of read coverage across RSV A (A) or RSV B (B) genomes (0–1,000 linear scale) is shown. 

Figure 8: NEBNext® RSV Primer Module generates high RSV genome coverage from clinical samples

The NEBNext® RSV Primer Module and LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555) were used to generate amplicons from IRBapproved clinical (nasopharyngeal swab) samples. Amplicon sequencing libraries were made for the Oxford Nanopore Technologies® platform (MinION® Flow Cell R10.4.1; high-accuracy basecalling). Each sample initially tested positive for RSV via the BioFire® assay; Ct values shown are from subsequent qRT-PCR assays. The qRT-PCR, targeted whole-genome RT-PCR, library prep, and sequencing of an equal volume library pool of these clinical samples was performed by collaborators at the Applied Physics Laboratory at Johns Hopkins University. Sequencing reads were aligned to a composite reference containing Human (T2T), RSV A (ATCC® VR-1540, and RSV B (ATCC VR-1580) genomes, with MiniMap2. A. Percent of reads that aligned to the human, RSV A, or RSV B reference for each sample; all samples were identified as RSV A. B. Integrative Genome Viewer plot of RSV A genome coverage (0–1,000 log scale) for each sample.

This product is related to the following categories:
Respiratory Syncytial Virus (RSV) Sequencing,
Infectious Diseases & ARTIC Sequencing,
This product can be used in the following applications:
Polymerases for NGS Library Preparation

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for the NEBNext RSV Primer Module?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

FAQs & Troubleshooting

FAQs

  1. Which additional reagents are needed for cDNA synthesis and amplification with the NEBNext RSV Primer Module (NEB #E9642)?
  2. Are the NEBNext RSV primer sequences (NEB #E9642) available?
  3. Which RSV strains can be targeted with the NEBNext RSV primers (NEB #E9642)?
  4. Is it possible to target only RSV A or RSV B with the primer mixes included in the NEBNext RSV Primer Module (NEB #E9642)?
  5. Which library prep reagents and protocols can be used for sequencing amplicons made with the NEBNext RSV Primer Module (NEB #E9642)?
  6. Is there a recommended Ct value-range for RSV inputs?
  7. What is the minimum read depth needed when using the NEBNext RSV Primer Module (NEB #E9642)?
  8. What sample types are supported when using the NEBNext RSV Primer Module (NEB #E9642)?
  9. What extraction methods can be used to isolate RSV gRNA ahead of RT-PCR?
  10. Are there recommendations for cleaning up RSV amplicons prior to library prep when using the NEBNext RSV Primer Module (NEB #E9642)?
  11. Are there recommended RSV cDNA amplicon input (after using the NEBNext RSV Primer Module (NEB #E9642)) for Oxford Nanopore Technologies End Prep reactions for low-plexity or high-plexity library prep?
  12. Can I minimize non-specific barcode ligation amongst pooled samples, after using the NEBNext RSV Primer Module (NEB #E9642)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.