Product Class: Kit

NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1)

Product Introduction

NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1)

NEBNext Multiplex Oligos provide adaptors and primers to enable high yield multiplex Illumina library production. The unique hairpin loop structure of the NEBNext Adaptor minimizes adaptor-dimer formation, and NEBNext index PCR primers enable index incorporation during library amplification.

For additional information on available indices and formats for multiplexing, please refer to the NEBNext Multiplex Oligos Selection Chart.

  • High efficiency adaptor ligation
  • Minimized adaptor-dimer formation
  • Includes index primers for library multiplexing

Note: NEBNext® Multiplex Oligos for Illumina® (Index Primer Set 1) (NEB #7335S/L) includes 12 indices. 

Catalog # Size Concentration
E7335S 24.0 reactions (12.0 indices)
E7335L 96.0 reactions (12.0 indices)

Product Information



Incorporating a novel hairpin loop structure, the NEBNext Adaptor ligates with increased efficiency to end-repaired, dA-tailed DNA. The loop contains a U, which is removed by treatment with USER™ Enzyme (a mix of UDG and Endo VIII), to open up the loop and make it available as a substrate for PCR. During PCR, barcodes can be incorporated by use of the NEBNext index primers, thereby enabling multiplexing.

NEBNext Oligos can be used with NEBNext products, and with other standard Illumina-compatible library preparation protocols.

Additional single- or dual-barcode primer options are available, in 12- and 96- index formats (NEB #E7500,  NEB #E7710,  NEB #E7730,  NEB #E6609,  NEB #E7600). A methylated version of the NEBNext Adaptor is also available for use with bisulfite sequencing protocols (NEB #E7535).

Figure 1: Workflow
Figure 1: Workflow.
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7335S     -20    
  • E7335L     -20    

Properties & Usage

Materials Required but not Supplied

For the list of additional materials required, please check the manual for your NEBNext Library Prep Kit.

Protocols, Manuals & Usage


  1. Refer to the manual (NEB #E7335) for a list of NEBNext library prep kits that contain protocols for use of NEBNext adaptors and primers.


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

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting


  1. Are NEBNext adaptors and primers for Illumina validated in Next Generation Sequencing Workflows?
  2. Are NEBNext adaptor and primers for Illumina compatible with NEBNext reagents for Illumina library preparation?
  3. Is USER enzyme required for library preparation using NEBNext adaptor and primers for Illumina?
  4. How much DNA can I ligate to the NEBNext adaptor for Illumina in one ligation reaction?
  5. Are these reagents available in a customized format, larger format or 96 well formats?
  6. Can USER Enzyme be purchased separately?
  7. Are the NEBNext Oligos for Illumina compatible with single read and paired end sequencing?
  8. Is the USER enzyme in our NEBNext kits identical in concentration to the standalone product?
  9. How do I know the concentration of my index primers? 
  10. Do you have a Sample Sheet with the index sequences that can be used in the Illumina Experiment Manager (IEM)?
  11. How do I use the NEB provided Sample Sheet for my specific instrument?
  12. What sequences need to be trimmed for NEBNext libraries that are sequenced on an Illumina instrument?

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.


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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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

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.