Product Class: Kit

NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 4)

Product Introduction

For optimal workflow flexibility, NEBNext® Multiplex Oligos are available separately from library preparation kits, providing adaptors and primers for high-yield multiplex Illumina® library production. The NEBNext Adaptor has a unique hairpin loop structure that minimizes adaptor-dimer formation, while NEBNext index PCR primers enable index (barcode) incorporation during library amplification.  This fourth set of unique-dual indices (UDIs) includes 96 pre-mixed unique pairs of index primers (i5 and i7), packaged in a single-use 96-well plate with a pierceable foil seal.

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

  • Enables complete filtering of index-hopped reads
  • Highly efficient adaptor ligation
  • Minimized adaptor-dimer formation
  • Includes index primers for library multiplexing
  • This set (Set 4) may be combined with sets 1, 2, 3 and 5 for 480 barcodes
 
Catalog # Size Concentration
E6446S 96.0 reactions
E6446L 384.0 reactions

Product Information

Description

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. This kit includes 96 pre-mixed unique pairs of i5 and i7 index primers, packaged in a single-use 96-well plate with a pierceable foil seal.

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

Additional dual barcode options (NEB #E7600, NEB #E7780), and single primer options are also available, in 12- and 96- index formats (NEB #E7335, NEB #E7500, NEB #E7710, NEB #E7730, NEB #E6609). A methylated version of the NEBNext Adaptor is also available for use with bisulfite sequencing protocols (NEB #E7535). 


Workflow:

Designed for use in library prep for DNA, ChIP DNA and RNA (but not Small RNA), the NEBNext Adaptors enable high-efficiency adaptor ligation and high library yields, with minimized adaptor-dimer formation. 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 combination 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. The 96 8-base index primer pairs included in this kit are pre-mixed and are packaged in a single-use 96-well plate with a pierceable foil seal.

For multiplexing with 384 indices, combine this set with NEB #E6440, NEB #E6442, and NEB #E6444.

Figure 1: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 4) Workflow





Figure 2: Use of NEBNext Adaptor and Unique Dual Index Primer Pairs substantially increases library yields and minimizes adaptor-dimers



16 libraries were prepared with 100 ng of Human NA19240 genomic DNA (Coriell Institute), using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805). Adaptors and primers were from either the IDT® for Illumina –TruSeq® DNA UD Indexes (Illumina #20022370), or the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs), NEB #E6440. After 4 PCR cycles, libraries were quantified on an Agilent® TapeStation® 4000.

A) Average library yields for the 8 libraries made using each workflow show 60% higher yield when the NEBNext Adaptor and Unique Dual Index Primer Pairs are used.

B) TapeStation traces of 16 libraries show the presence of adaptor-dimer (indicated by the orange arrow) with the libraries made using IDT for Illumina adaptors and primers, which is absent in the libraries prepared using NEBNext adaptors and primers, which also have higher yields.



Figure 3: Libraries amplified with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 4) cluster evenly on the Illumina NovaSeq™ 6000



96 libraries produced using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 4) were pooled at equimolar concentrations and sequenced on the Illumina MiSeq® and NovaSeq® 6000 instruments. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 1.04 %). All 96 libraries clustered efficiently and were represented at approximately the expected frequency on both platforms. 


Figure 4: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 4) amplify libraries with equal efficiency



Human NA19240 genomic DNA (Coriell Institute) was used to prepare 96 libraries using either the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805), Covaris-sheared DNA with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), or the NEBNext Ultra II DNA Library Prep Kit combined with bisulfite conversion. Universal Human Reference RNA was used to prepare 96 libraries using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760). Libraries were PCR-amplified using the NEBNext Multiplex Oligos (96 Unique Dual Index Primer Pairs Set 4) to produce libraries containing unique i5 and i7 indices. Library yields were quantified (Agilent® TapeStation® 4200) and normalized by summing the total yield of all 96 libraries and calculating the contribution from each library (expected fraction per library = 1.04%). Library amplification efficiency was robust with each library prep method, and efficiency was uniform across all 96 unique index primer combinations. Each bar represents the average of at least 2 technical replicates.  


Figure 5: Libraries amplified with 384 primer pairs from NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 1-4) cluster evenly on the Illumina NovaSeq™ 6000



384 libraries produced using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 1-4) were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq® 6000 instrument. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 0.26 %). All 384 libraries clustered efficiently and were represented at approximately the expected frequency. 


This product is related to the following categories:
NEBNext® Multiplex Oligos (Adaptors & Primers) Products,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E6446S     -20    
      NEBNext® Adaptor for Illumina® E6612AVIAL -20 1 x 0.96 ml 15 µM
      USER® Enzyme E6610AVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Unique Dual Index Primer Set 4 E6447AVIAL -20 1 x 10 μl/well 10 µM
  • E6446L     -20    
      NEBNext® Adaptor for Illumina® E6612AVIAL -20 4 x 0.96 ml 15 µM
      USER® Enzyme E6610AAVIAL -20 2 x 0.576 ml Not Applicable
      NEBNext® Unique Dual Index Primer Set 4 E6447AVIAL -20 4 x 10 μl/well 10 µM

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs)? (NEB #E6440, #E6442, #E6444, #E6446, #E6448)

Manuals

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

FAQs

  1. Are NEBNext adaptors and primers for Illumina validated in Next Generation Sequencing Workflows?
  2. What is the concentration of the adaptor and primers in the NEBNext Multiplex Oligos kits?
  3. Can I use the 96-well primer plate to do my PCR reactions when using the NEBNext® Multiplex Oligos for MGI® (Dual Index Primer Pairs Set 1) (NEB #E9640)?
  4. How many reactions worth of primer are in each well?
  5. Is the NEBNext adaptor methylated?
  6. Can I perform single read runs and still get both index sequences? Can I run both single read and paired-end recipes with dual-indexed libraries?
  7. Are dual indexed libraries compatible with single end sequencing?
  8. How many indices are available with NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (NEB #E6440, E6442, E6444, E6446, and E6448)?
  9. Can the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB #E6440, #E6442, #E6444, #E6446, and #E6448) be used to address "index hopping" with Illumina patterned flow cells?
  10. Do you have Sample Sheets for use with the NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 4)?
  11. What should I do if the sample sheet from the NEB.com website is not compatible with the software version I am using?
  12. Which sets of NEBNext Unique Dual Index Primer Pairs can be combined in a single Illumina sequencing run?
  13. 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.

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]

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 busdev@neb.com.

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.

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