Integrated UD + UMI architecture

Each kit arrives as a 96 well plate, every well holding a full length adapter pair with a 10 bp Unique Dual Index (UDI) and a 9 bp Unique Molecular Identifier (UMI). The barcode is compatible with standard TruSeq® workflows. Placing distinct 10 base UDI on both ends of every fragment reduces misassignment and index hopping rates below 0.1-0.3 % on Illumina® and Element® flow cells.

Figure 1. Example of a library containing NEXTFLEX UDI-UMI Adapters, where Insert = DNA or RNA fragment from a sample, P5 and P7 = Flow cell binding sites for Illumina® platforms, SP1 and SP2 = Binding sites for sequencing primers, i5 and i7 = Short pair of 10 bp sequences used to identify a particular sample (UDI), and UMI = 9 bp sequence used to uniquely tag each molecule within a library.

The 9 bp UMI, also known as a molecular barcode, is attached to each original DNA or RNA fragment prior to amplification. During analysis, reads that share the same UMI and map to identical genomic coordinates are grouped together. These groups are collapsed into a consensus sequence, removing PCR duplicates and correcting random sequencing errors. If you’re setting up your analysis pipeline, review the common bioinformatic challenges with UDI-UMI data we outline here. Because every starting molecule carries a distinct tag, counting unique UMIs after deduplication provides a direct measure of library complexity (i.e., how many unique molecules were captured versus how many were merely re sequenced). This molecular level view supports confident detection of low frequency variants down to 0.1% in ctDNA and low input projects, while ensuring that sequencing depth is translated into true biological coverage rather than inflated duplicate counts.

Each UMI is a 9 nt sequence drawn from a roughly equimolar mix of A, T, C, G bases yielding 4⁹ ≈ 262 k unique tags. The large number of tags keeps UMI-collision rates low, even in high depth runs, while the uniform base composition also improves cluster calling and phasing on Illumina® and Element® flow cells, ensuring that UMI information is read accurately for downstream duplicate removal and variant calling.

Figure 2. Balanced nucleotide composition across the 9 bp Unique Molecular Identifier (UMI) in 24 NEXTFLEX UDI UMI libraries prepared from 0.1–1000 ng human gDNA with the NEXTFLEX Rapid XP v2 DNA seq Kit and sequenced on an Illumina® MiSeq™ system, demonstrating high UMI diversity and stable library complexity from low to high input DNA.

Quantitative sequencing

When starting from low input, FFPE or GC skewed samples, extra PCR cycles can magnify polymerase bias and inflate duplicate counts, making alignment based deduplication unreliable, especially for highly expressed transcripts. By reducing overrepresentation from PCR, the NEXTFLEX UDI-UMI adapters improve the linear correlation between true and observed expression.

Rare variant analysis

Ultra low frequency variants (down to 0.1 % VAF) sit at the noise floor of conventional sequencing. NEXTFLEX UDI-UMI consensus reads strip away random polymerase and sequencing errors, while PCR free prep eliminates the polymerase step altogether, preventing artefacts that can masquerade as real variants. The combination delivers confident rare variant calls in ctDNA, targeted panels, low input RNA samples, and heterogeneous tumor biopsies even at the deep coverages typical of these sample types. See our deep dive on the power of UMIs in cancer research for data examples