NEXTFLEX Unique Dual Index Barcodes (8NT index, 1-24)

For labs interested in increasing their multiplexing capabilities, a set of 1536 NEXTFLEX Unique Dual Index Barcodes is also available.

Index architecture and error robustness

The NEXTFLEX Unique Dual Index Barcodes contain full length adapters with unique dual 8NT index pairs. Every index in the set is separated by ≥ 3 Hamming distance. This design allows detection of single-base substitution errors during demultiplexing, limits index collisions and lowers false assignment on patterned flow cells where index hopping can occur. When using the NEXTFLEX Unique dual index pairs (i7 + i5) a read is assigned only when both indices match the same dedicated pair, greatly reducing misassignment from single-end index hopping or low-level contamination.

Figure 1. Dual unique indexing removes residual misassignment on MiSeq® sequencer. Eight libraries with NEXTFLEX 8 nt UDIs show low off-diagonal cross assignment (≤0.02%) when demultiplexed by i7 only (left). Demultiplexing with the paired unique i7 + i5 indices (right) yields 100% correct assignment for all samples (no detectable misassignment).

Demultiplexing confidence and data quality 

A ≥3 base Hamming distance between indices makes it extremely unlikely that a single base miscall converts one valid index into another. Standard demultiplexing tools (bcl-convert, bcl2fastq, DRAGEN) can therefore confidently assign reads with one mismatch without risking misassignment. This preserves usable read counts, maintains balanced sample representation, and reduces cross-sample index bleed in applications such as rare variant detection, low-input RNA sequencing, and metagenomics.

Workflow integration and flexibility

NEXTFLEX Adapters can act as drop-in replacements for any TruSeq-style library prep with T-overhang ligation steps in both PCR-free and amplified library preps. Our plate format supports partial use while maintaining performance of remaining wells (with proper storage), accommodating both high-throughput batches and occasional low-plex runs.

Barcode design, quality control and traceability

Sequence-based QC verifies index purity with spike-in libraries so that each barcode maps only to its intended sequence, while proprietary contamination-reduction steps lowers index crosstalk to ~0.1%. Indices are engineered with a ≥3 base Hamming distance, avoid homopolymers longer than two bases, and are color balanced even in low-plex pools, design choices that limit misassignment from sequencing errors, reduce base-calling bias, and maintain balanced signal. Automated plating with controlled volumes supports consistent handling and traceability. Together these design and manufacturing controls deliver high usable read percentages with very low misassigned reads, supporting sensitive low-frequency variant detection.

Scalability, Universal blockers, and UMIs

Start with 24 or 96 unique dual indices, scale to 384, then up to 1,536 while keeping the same ≥3 base spacing so demultiplex parameters stay unchanged. NEXTFLEX® Universal Blockers   block non-specific hybridization between adapter sequences, enhancing specificity and reducing sequencing costs. For workflows needing molecular counting or error suppression, NEXTFLEX® UDI-UMI adapters  layer a molecular identifier onto the UDI framework to enable duplicate collapsing and more accurate quantitation.

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