NEXTFLEX UDI-UMI Barcodes (1-8)

Integrated UDI + 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 fragment or RNA-derived cDNA 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 label molecules for UMI-based grouping and analysis.

The 9 bp UMI, also known as a molecular barcode, is attached to each DNA fragment or RNA-derived cDNA fragment before amplification. During analysis, reads are grouped using the UMI and mapping context, with correction for UMI errors. Families with sufficient read support can be used for consensus-based error correction. Molecular-family counts can help assess captured diversity, but UMI collisions and sampling must be considered. UMI tagging does not correct losses or errors introduced before tagging. Rare-variant sensitivity must be validated for the assay and sample type. For analysis guidance, review the common bioinformatic challenges with UDI-UMI data we outline here.

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. Collision rates depend on the number of original molecules sharing the same grouping context and the observed UMI distribution. 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, showing the per-position nucleotide composition in these tested libraries.

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

UMI-family consensus can reduce random sequencing errors and some errors introduced after tagging when sufficient family support is available. PCR-free preparation avoids library-amplification errors, but PCR-free compatibility alone does not establish consensus-based error correction: a singleton UMI family cannot provide multi-read consensus support. Rare-variant sensitivity depends on the assay, sample input, molecular coverage, and analysis and must be validated. See our deep dive on the power of UMIs in cancer research for data examples