NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3)
Please note: Beginning 06/01/2026 and phasing in on a rolling basis, NEBNext Multiplex Oligos provided in a 96-well plate will be updated to Eppendorf twin.tec® PCR Plates (Eppendorf #951020401) with Agilent Pierceable Aluminum Seals (Agilent #06644-001). This product has not yet been updated. Please contact NEBNext Technical Support (info@neb.com) with any questions related to these updates.
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)
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 bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact the Customized Solutions team for further information.
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
Figure 2. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) allow higher ligation efficiency.
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.
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
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
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.
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