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

Index architecture for accurate demultiplexing

The NEXTFLEX Unique Dual Index (UDI) Barcodes use full-length adapters with dual 8 nt i7 + i5 indices. Each index is separated by a ≥ 3-base Hamming distance, so standard demultiplexing tools (bcl-convert, bcl2fastq, DRAGEN) can allow a single mismatch without misassigning reads. Reads are assigned only when both indices match the same dedicated pair, which limits index collisions, suppresses index hopping on patterned flow cells, and reduces cross-sample index bleed from low-level contamination. This design delivers higher confidence demultiplexing, preserves usable read counts, and maintains balanced sample representation for sensitive applications such as rare variant detection, low-input RNA sequencing, and metagenomics.

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).

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, & 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.

Cellular & organismal transcriptomics / functional genomics

• Estermann, M. (2020). Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. preLights. https://doi.org/10.1242/prelights.18906
• Veenvliet, J. V., Bolondi, A., Kretzmer, H., Haut, L., Scholze-Wittler, M., Schifferl, D., … Herrmann, B. G. (2020). Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. Science, 370(6522), eaba4937. https://doi.org/10.1126/science.aba4937
• Hirose, K., Chang, S., Yu, H., Wang, J., Barca, E., Chen, X., … Huang, G. N. (2019). Loss of a novel striated muscle-enriched mitochondrial protein Coq10a enhances postnatal cardiac hypertrophic growth. bioRxiv. https://doi.org/10.1101/755793
• León, K. E., Buj, R., Lesko, E., Dahl, E. S., Chen, C.-W., Imamura, Y., … Aird, K. M. (2020). DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A. bioRxiv. https://doi.org/10.1101/2020.08.21.258020
• Dubey, S. K., Dubey, R., Jung, K., Hernandez, A. G., & Kleinman, M. E. (2025). Deciphering age-related transcriptomic changes in the mouse retinal pigment epithelium. Aging (Albany NY), 17(3), 657–684. https://doi.org/10.18632/aging.206219
• Pujari, A. N., & Cullen, P. J. (2024). Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae. G3 (Bethesda), 14(6), jkae072. https://doi.org/10.1093/g3journal/jkae072
• Miura, H., Takahashi, S., & Shibata, T. (2020). Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing. Nature Protocols, 15(12), 4058–4100. https://doi.org/10.1038/s41596-020-0378-5
• Gardner, E. J., Prigmore, E., Gallone, D., Danecek, P., Samocha, K. E., Handsaker, J., … Hurles, M. E. (2019). Contribution of retrotransposition to developmental disorders. Nature Communications, 10(1), 4630. https://doi.org/10.1038/s41467-019-12520-y

Microbial, environmental, and pathogen genomics / metagenomics

• Adolfi, A., Gantz, V. M., Jasinskiene, N., Lee, H.-F., Hwang, K., Terradas, G., … James, A. A. (2020). Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi. Nature Communications, 11, 5553. https://doi.org/10.1038/s41467-020-19426-0
• Gaeta, N. C., Bean, E., Miles, A. M., de Carvalho, D. U. O. G., Alemán, M. A. R., Carvalho, J. S., … Ganda, E. (2020). A cross-sectional study of dairy cattle metagenomes reveals increased antimicrobial resistance in animals farmed in a heavy metal contaminated environment. Frontiers in Microbiology, 11, 590325. https://doi.org/10.3389/fmicb.2020.590325
• Kant, P., Brohier, N., Mann, R., Rigano, L., Taylor, R., Wong-Bajracharya, J., … Constable, F. (2023). High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach. Microbiology Resource Announcements, 12(11), e00536-23. https://doi.org/10.1128/MRA.00536-23
• Arras, S. D. M., Sibaeva, N., Catchpole, R. J., Horinouchi, N., Si, D., Rickerby, A. M., … Poole, A. M. (2023). Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of parasitism and endosymbiosis. eLife, 12, e83845. https://doi.org/10.7554/eLife.83845
• Larkin, A. A., Brock, M. L., Fagan, A. J., Moreno, A. R., Gerace, S. D., Lees, L. E., … Martiny, A. C. (2025). Climate-driven succession in marine microbiome biodiversity and biogeochemical function. Nature Communications, 16(1), 3926. https://doi.org/10.1038/s41467-025-59382-1
• Klauer, R. R., Silvestri, R., White, H., Hayes, R. D., Riley, R., Lipzen, A., … Blenner, M. A. (2024). Hydrophobins from Aspergillus mediate fungal interactions with microplastics. bioRxiv. https://doi.org/10.1101/2024.11.05.622132
• Starr, T. N., Greaney, A. J., Hilton, S. K., Crawford, K. H. D., Navarro, M. J., Bowen, J. E., … Bloom, J. D. (2020). Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding. bioRxiv. https://doi.org/10.1101/2020.06.17.157982