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HomeNEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1)
NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1)
For details on how to combine UMI adaptors with our NEBNext library prep kits, click the product information tab below.
NEBNext® Multiplex Oligos for Illumina® are an essential piece of the NEBNext suite of library preparation products, 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 unique molecular identifier (UMI) sequence to enable 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.
NEBNext Unique Dual Index UMI Adaptors for RNA-seq (NEB # E7416) are also available, as well as single, dual, and unique dual index primers, plus an optimized adaptor.
NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1) 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.
Combining UMI Adaptors DNA Set 1 with one of the NEBNext DNA library prep kits is simple and straightforward if you follow the protocols found in the library prep kit manuals. Quickly find the correct manual by selecting which library prep kit/module you plan to use:
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, 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, and NEB #E6446), 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).
Figure 1. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) Workflow
Figure 2. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) 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 the suppliers shown using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E6177), without PCR amplification. After ligation, two rounds of SPRIselect clean-up steps were performed, and libraries were quantified using the NEBNext Library Quant Kit (NEB #E7630). B) 90 Libraries were prepared in a 96 well plate with 100 ng NA19240 genomic DNA using the Ultra II FS DNA Library Prep Kit, 30 different adaptors each from the suppliers shown and without PCR amplification. Two bead clean-up steps were performed, followed by qPCR quantification.
Figure 3. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) allow more-sensitive, low-frequency variant detection.
AcroMetrix Oncology Hotspot Control DNA (Thermo Scientific® #969056) was used as the mutation DNA source (>500 mutations with 5-35% allele frequency) and mixed with NA19240 genomic DNA at various ratios to generate a serial range of allele frequencies. 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 the Illumina NovaSeq®, reads were downsampled to 110 million and mapped to hg38 with BWA MEM (0.7.17). Mapped reads were analyzed by MarkDuplicates (Picard 2.20.6) without utilizing UMI sequences 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 UMIs 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 frequencies, the more benefit provided by UMIs in SNV detection.
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