NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 5)
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.
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 fifth 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.
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.
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.
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.
Figure 1: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 5) 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 5) cluster evenly on the Illumina MiSeq and NovaSeq 6000.
96 libraries produced using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 5) 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 5) 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 5) 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 480 primer pairs from NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Sets 1-5) cluster evenly on the Illumina NovaSeq 6000
480 libraries produced using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805), or NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) for each primer set from the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 1-5) 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 = 0.21 %). All 480 libraries clustered efficiently and were represented at approximately the expected frequency.
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