NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) are built for more streamlined and informative NGS multiplexing, more accurate determination of transcript abundance, and removal of PCR duplicates. These index adaptors contain a pair of unique dual indices (i5 and i7) and one 11 nt UMI sequence. 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 mixis supplied in a separate vial.
Combining UMI Adaptors with one of the NEBNext RNA library prep kits is simple and straightforward if you follow the protocols found in the UMI Adaptor-specific library prep kit manuals (listed below).
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760 or NEB #E7765)
NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770 or NEB #E7775)
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, index adaptors with UMIs are available for PCR-free and standard DNA-seq (NEB #E7395). For multiplexing without UMIs, NEBNext offers several options for added flexibility, including unique dual index primers (NEB #6440,NEB #6442, NEB #E6444, NEB #E6446), dual index primers (NEB #E7600andNEB #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).
The NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) workflow shown below details the library barcoding steps of the library preparation process.
Figure 1: NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) WorkflowFigure 2: NEBNext Unique Dual Index UMI Adaptors have equally efficient ligation and subsequent amplificationA. Universal Human Reference (UHR) RNA (Agilent® #740000) was used to prepare 96 libraries with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment. Library yields were quantified (Agilent Tapestation 4200) and normalized by dividing the individual library yields by the average library yield across all 96 libraries. Adaptor ligation efficiency was robust with uniformity across all 96 Unique Dual Index UMI Adaptors. Each bar represents the average of at least 2 technical replicates.B. NEBNext Unique Dual Index UMI Adaptors have uniform clustering efficiency. 96 libraries were pooled and sequenced on the Illumina NextSeq® 500 (PE70). Each library is expected to contribute 1.04% of the total sequencing reads. Individual library yields were normalized by dividing the number of actual reads by the number of expected reads. No bias was observed across the 96 Unique Dual Index UMI Adaptor libraries.Figure 3: NEBNext Unique Dual Index UMI Adaptors outperform competitor UMI adaptorsLibraries were produced using UHR RNA and the NEBNext Ultra II Directional RNA library prep kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment.A. The average library yield of triplicates is shown for three starting total RNA inputs: 10, 100, and 1,000 ng. During adaptor ligation either NEBNext Unique Dual Index UMI Adaptors or IDT xGen dual index adapters were used. Final library yields were quantified using the Agilent Tapestation 4200.B. Libraries were sequenced on the Illumina NextSeq 500 (PE70). Reads were downsampled to 5 million reads (seqtk) and aligned to the GRCh38 reference genome using HISAT 2.1. UMI information was added using fgbio (AnnotateBamWithUmis) and MarkDuplicates (Picard 2.18.2.1) was used to mark reads with UMI and determine duplication rate. NEBNext Unique Dual Index UMI Adaptor libraries produced libraries with a lower percentage of read duplicates.Figure 4: Incorporating UMIs into analysis improves the accurate quantification of duplication ratesTriplicate libraries of three input amounts (10, 100, and 1000 ng) were produced using UHR RNA and the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment and sequenced on the NextSeq 500. Reads were downsampled (seqtk) to 5 million reads and mapped to the GRCh38 reference genome (HISAT 2.1) and Fgbio (AnnotateBamWithUmis) was used to add the UMI information. Two approaches were used to calculate duplication: position and UMI information (green bars) or position alone (yellow bars) using MarkDuplicates (Picard 2.18.2.1).Figure 5: Unique Dual Index UMI Adaptors allow for the correction of bias introduced with PCR amplificationLibraries were produced using UHR RNA and the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment. Four technical replicate libraries were produced at three RNA inputs (10, 100 and 1,000 ng). Libraries were sequenced on the NextSeq 500 (PE70) and downsampled (seqtk)to 10 million reads for further analysis. To assess PCR bias, the same sequencing reads were compared using typical transcript abundance analysis (Salmon 0.9.1) against UMI deduplicated reads. UMI deduplication was performed by first mapping to the GRCh38 reference genome (HISAT 2.1) followed by Fgbio to add the UMI information. Non-duplicate reads were randomly ordered and converted to fastq before re-assessing transcript abundance using Salmon (0.9.1). The ratio of UMI-adjusted counts/raw read counts with no deduplication was calculated. Number of transcripts with at least 1 count in any library and with UMI/raw ratio ≥2 are plotted. In total, approximately 90,000 total transcripts were detected. An increased number of PCR cycles in library preparation correlates with the number of transcripts that shift upon UMI correction.Figure 6: IGV visual representation of mapped reads adjusted utilizing UMIs in MDM4 (chr1:204,548.089-204,558,681)Several isoforms of MDM4 locus showed >2X fold change in UMI/raw ratio in a 10 ng library sequenced on the NextSeq 500 (PE70). Reads that are considered to originate from unique molecules are shown in blue. Duplicate reads determined by UMI sequence and mapped read position (red bars) can be removed to improve accuracy of transcript abundance in downstream analysis.
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