NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3)

Catalog # Concentration Size List Price Quantity Your Price
E6444L 384 reactions $3,035.00
$2,731.50
E6444S 96 reactions $844.00
$759.60
Catalog # Size List Price Your Price
E6444L 384 reactions $3,035.00
$2,731.50
E6444S 96 reactions $844.00
$759.60
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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 third 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 3) may be combined with sets 1, 2, 4 and 5 for 480 barcodes
 
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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 #E6446.

Figure 1: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 3) 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 3) 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 3) 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: Use of NEBNext Adaptor and Unique Dual Index Primer Pairs substantially increases library yields and minimizes adaptor-dimers



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 3) 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. 


Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  NEBNext® Adaptor for Illumina® E6612AVIAL -20 1 x 0.96 ml 15 µM
  USER® Enzyme E6610AVIAL -20 1 x 0.288 ml
  NEBNext® Unique Dual Index Primer Set 3 E6445AVIAL -20 1 x 10 μl/well 10 µM
  NEBNext® Adaptor for Illumina® E6612AVIAL -20 4 x 0.96 ml 15 µM
  USER® Enzyme E6610AAVIAL -20 2 x 0.576 ml
  NEBNext® Unique Dual Index Primer Set 3 E6445AVIAL -20 4 x 10 μl/well 10 µM
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Quality Control Assay
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.
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