Product Class: Kit

NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3)


Catalog #E7876

Product Introduction

NEBNext® Multiplex Oligos for Illumina® are an essential component of the NEBNext suite of library preparation products, and are available in several different configurations for use with NEBNext products or other standard Illumina-compatible library preparation protocols. This set was designed and optimized for use in DNA sequencing workflows, including PCR-free. These unique dual index adaptors also include a 12-nt unique molecular identifier (UMI) sequence to enable either the PCR-free sample preparation for NGS, or the identification and removal of PCR errors or duplicates from amplified libraries. Supplied as 96 unique, pre-annealed index adaptors with UMIs, packaged in a single-use 96-well plate with a pierceable foil seal. Universal primers are included for applications requiring PCR amplification.

Additional NEBNext Unique Dual Index UMI Adaptors for DNA-seq are available (NEB #E7395, #E7874, #E7878) for multiplexing up to 384 samples. NEBNext Unique Dual Index UMI Adaptors for RNA-seq (NEB # E7416) are also available.

  • Index adaptors enable PCR-free library preparation
  • Increased sample identification specificity
  • Superior and reliable ligation efficiency
  • More-sensitive detection of low-frequency single-nucleotide variants (SNVs)
  • Supports multiplexing for higher throughputs

Product Information

Description

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) are built for more streamlined and informative NGS multiplexing, tracking the sequencing reaction at the individual molecule level and enabling bioinformatic removal of misassigned reads for optimal accuracy. These index adaptors contain a pair of unique dual indices (i5 and i7) and one UMI sequence, making the resulting library multiplex-ready, with or without PCR. This set includes 96 unique dual index UMI adaptors, packaged in a single-use 96-well plate with a pierceable foil seal. The universal primer mix is supplied in a separate vial.

The NEBNext line of Multiplex Oligos can be used with NEBNext sample prep products or other standard Illumina-compatible library preparation protocols. In addition to this set, there are three other sets of NEBNext Unique Dual Index UMI Adaptors for DNA (NEB #E7395, #E7874, #E7878) for multiplexing of up to 384 samples; additional index adaptors with UMIs are available for RNA-seq (NEB #E7416). For multiplexing without UMIs, NEBNext offers several options for added flexibility, including unique dual index primers (NEB #6440, NEB #6442, NEB #E6444, NEB #E6446, and NEB #E6448), dual index primers (NEB #E7600 and NEB #E7780), and single primer sets (in 12- and 96-index formats; NEB #E7335, NEB #E7500, NEB #E7710, NEB #E7730, NEB #E6609), as well as an option compatible with EM-seq™ and bisulfite sequencing (NEB #E7140). 

For larger volume requirements,  customized and/or bulk packaging is available through our Customized Solutions Team. Please contact us at custom@neb.com.


NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) enable both PCR-free and amplification-based workflows for DNA library preparation. Refer to the below diagram for the differences between the two options.


Figure 1. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) Workflow

UMI Worflows


Figure 2. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) allow higher ligation efficiency.

UMI Sets

A. Libraries were prepared with 100, 250, and 500 ng inputs of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and unique dual index adaptors from different suppliers using the NEBNext Ultra™ II FS DNA Library Prep Kit (NEB #E7805) without PCR amplification. The adaptors used were NEBNext Unique Dual Index UMI Adaptrs, xGen® Dual Index UMI Adapters (IDT®) and Nextflex® Unique Dual index Barcodes 1-96 (Bioo Scientific®). Two rounds of SPRIselect bead-based cleanup steps were performed post-ligation, and libraries were quantified by qPCR (NEBNext Library Quant Kit, NEB #E7630).  
B. 90 libraries were prepared with 100 ng NA19240 genomic DNA using the Ultra II FS DNA Library Prep Kit without PCR amplification in a 96-well plate. For the 90 libraries, 30 different adaptors each from NEB, IDT, and Bioo were used for ligation. Libraries were cleaned up and quantitated by qPCR. 


Figure 3. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) allow more-sensitive, low-frequency variant detection.

UMI Sets

AcroMetrix® Oncology Hotspot Control DNA (Thermo Scientific®) was used as a mutation DNA source (> 500 mutations with 5-35% allele frequency) and mixed with NA19240 genomic DNA at various ratios to generate a range of allele frequencies (5%-35%, 2.5%-17.5% and 1%-7%). Libraries were constructed using NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) with NEBNext Unique Dual Index UMI Adaptors, and multiplex hybrid capture was performed on all samples using a customized panel for 152 genes from Twist Bioscience®. Libraries were sequenced on a NovaSeq® 6000 (PE140), and reads were downsampled to 110 Million (Seqtk Sample) and mapped to GRCh38 with BWA MEM (0.7.17). Mapped reads were analyzed by MarkDuplicates (Picard 2.20.6) without utilizing UMI information or by building UMI consensus sequences (Fgbio 0.8.1). The final BAM files were used to call somatic variants with Strelka2 (2.9.10).
A. The number of total and correct SNV calls increased when using UMI information for duplicate removal and consensus sequence-based error correction. 
B. The sensitivity of variant detection was improved with UMI consensus calling. The lower the allele frequency, the more benefit provided by UMI information in SNV detection.


Figure 4. NEBNext Unique Dual Index UMI Adaptors DNA Set 3 provides consistent library yields



96 Libraries were each prepared with 50 ng input of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and the NEBNext Unique Dual Index UMI Adaptors DNA Set 3 using the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) in a 96 well plate. Library cleanup was followed by qPCR quantification. Library concentrations ranged from 7.3 nM to 3.4 nM with a 2.1-fold difference across all 96 adaptors.


Figure 5.  NEBNext Unique Dual Index UMI Adaptors DNA Set 3 provides consistent clustering efficiency on MiSeq® and NovaSeq 6000

E7876

Fig Desc: 96 libraries prepared using the NEBnext Unique Dual Index UMI Adaptors DNA Set 2 and the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) 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 faction per library = 1.04%). All 96 libraries clustered efficiently and were represented at approximately the expected frequency on both platforms.


Figure 6.  All libraries prepared with 384 NEBNext Unique Dual Index UMI Adaptors cluster evenly on the Illumina NovaSeq 6000

NEBNext 

384 libraries prepared using the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 and the NEBNext Ultra II FS PCR-free Library Prep Kit (NEB #E6177) were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq 6000 instrument. The total number of reads from all libraries was 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
  • E7876S     -20    
      NEBNext® UMI DNA Adaptor Plate Set 3 E7877AVIAL -20 1 x 2.5 μl/well 20 µM
      NEBNext® Primer Mix E7397AVIAL -20 1 x 0.48 ml Not Applicable
      NEBNext® UMI Adaptor Dilution Buffer E7398AVIAL -20 2 x 2.5 ml Not Applicable
  • E7876L     -20    
      NEBNext® UMI DNA Adaptor Plate Set 3 E7877AVIAL -20 4 x 2.5 μl/well 20 µM
      NEBNext® Primer Mix E7397AAVIAL -20 2 x 0.96 ml Not Applicable
      NEBNext® UMI Adaptor Dilution Buffer E7398AAVIAL -20 1 x 20 ml Not Applicable

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® DNA Library Prep reagents?

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. What is the difference between NEBNext Unique Dual Index UMI Adaptors and NEBNext Adaptors for use with multiplex primers?
  2. What are the main applications for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
  3. How long are the UMIs and where are they on the adaptors?
  4. How are UMIs sequenced, and where can I find them?
  5. Can I skip UMI sequencing if I don’t need the UMIs for my applications?
  6. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with NEBNext reagents for Illumina library preparation?
  7. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with commercially available library prep reagents?
  8. How many reactions worth of the UMI adaptors are in each well?
  9. Are the adaptors methylated?
  10. What is the concentration of the adaptors and PCR primer mix in the NEBNext Multiplex Oligos (Unique Dual Index UMI Adaptors DNA Sets 1-4)?
  11. How many indices are available with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets1-4)? 
  12. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 validated in next generation sequencing workflows?
  13. Are the NEBNext UMI Adaptors compatible with single-end and paired-end sequencing?
  14. Do I need to spike in custom sequencing primers when sequencing libraries made with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets 1-4) on the Illumina platforms? 
  15. 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?
  16. Where can I find the sample sheets for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
  17. 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.