Multiplexing samples in a single NGS run on Illumina platforms is now routine. Multiplexing saves time and money by sequencing many libraries together. However, multiplexing introduces a big challenge: index misassignment. Here, reads from one sample get incorrectly labeled as originating from another sample. This usually happens due to index hopping or unanticipated barcode cross-talk, and could result in contaminated data leading to false conclusions. For high-throughput sequencing experiments, even a 1% rate of index misassignment could mean that a substantial amount of reads belong to another sample. This is specifically problematic for sensitive applications such as detecting low-frequency mutation or viral pathogens, where index misassignment could potentially lead to false positive results.
Unique Dual Index (UDI) primer sets are a solution to virtually eliminate index misassignment. With UDI primer sets, each sample is assigned two unique index codes (one on each end of the fragment). They ensure that any hopping or contamination event is detected during demultiplexing.
The issue: Index hopping and cross-talk in multiplexed sequencing
Index hopping, barcode misreads and primer contamination are forms of index cross-talk.
Index hopping
When multiple libraries are pooled, each library is tagged with a short DNA index sequence, also called barcode, to identify its reads later. Traditionally, a single index per library was used, or dual indices that were reused across samples in different combinations. However, during sequencing, a phenomenon called index hopping can occur. Here, adapter or index from one library bind to another library’s DNA during cluster generation. It’s relatively rare, but can occur in about 0.1–2% of reads.
Barcode misreads and primer contamination
Here, leftover primers in the library prep that weren’t fully cleaned could anneal at off-target sequences, or sequencing errors in the index read can convert one sample’s barcode into another’s. The result is a proportion of reads that are labeled with the wrong sample index. A contamination of 0.5% of the reads may sound small, but can confound analyses of high-sensitivity applications
How unique dual indexing solves index cross-talk issues
Unique dual indexing addresses index hopping and cross-talk by using two independent index sequences per sample, and ensuring each index combination is used for one sample only.
In practice, this means each library gets a unique Index 1 (i7) and Index 2 (i5) sequence. If a hopped index from one library somehow attaches to another library’s fragment, the resulting mismatched index pair will not match any valid sample and can be discarded during demultiplexing.
Example
Two libraries, A and B. With UDI primer sets, A gets index tags A1 (for i7) and A2 (for i5), and B gets B1 and B2. If during sequencing some of A’s index hops onto B’s DNA, B’s read might end up with index sequence A1 on one end and B2 on the other. The demultiplexing software will flag this read as undetermined because “A1–B2” is not an assigned index pair, and thus it won’t be incorrectly attributed to either sample. Therefore, unique dual indexing allow for the detection (and removable) of index hopping.
In addition to preventing false assignments, UDIs also allow for greater error tolerance in index reads. UDI sets include indices that are sufficiently different from each other. Even if the sequencing platform makes an error during base call in one or two bases of an index read, it’s unlikely to accidentally turn one index into another one and misassign a read. The result is highly accurate demultiplexing.
Eurofins Genomics’ NGS UDI Primer Sets: Features and advantages
Our NGS UDI Primer Sets are specialized index primer plates for Illumina platforms.
Each UDI in the set is 12 bases long
Longer index sequences offer more unique combinations and reduce the chance of index collision or misreading. All index pairs are unique and no index sequence is repeated across the 96 primers.
High edit distance
The index sequences have a minimum edit distance of ≥4 bases between any two indexes in the set. This ensures exceptionally low index cross-talk.
Low cross-contamination manufacturing
The cross-contamination levels of our NGS UDI Primers is around 0.0014% in real-world tests of the UDI sets. In a NovaSeq run with 96 libraries using these UDIs, 99.9986% of reads were correctly assigned to their proper index pair.
Verified performance and quality standards
The production of our UDI Primer Sets adheres to ISO 13485 quality management standards. All UDI Primer Sets are quality-controlled in our NGS facility and shipped to our customers only after passing strict QC criteria.
Ideal for high-throughput
We supply UDI Primer Sets in ready-to-use 96-well plates, with each well containing a unique primer pair at 10 µM concentration. The 96-well plates are ideal for robotic pipetting or multichannel use.
When are UDI Primer Sets used – Use cases
Not every sequencing run will demand unique dual indices, but certain scenarios greatly benefit from them. UDI primer sets is highly recommended for:
- Large multiplexed cohorts
For sequencing of tens to hundreds of samples together (e.g., a 96 or 384-sample pool), UDIs are crucial. The more samples are multiplexed, the more pairs of indices are used and the greater the impact of even tiny index cross-talk percentages. UDIs ensure that even in a huge pool, each sample’s reads remain isolated. - Variant detection and rare event sequencing
In cancer genomics (liquid biopsies, low-frequency somatic mutations), infectious disease detection, or ancient DNA analysis, even 0.1% false assignment can create a phantom variant. If an experiment’s conclusions depend on distinguishing a 0.5% allele frequency from the background noise, UDIs are pivotal. - Low-complexity or PCR-free libraries
Certain library types are more prone to index hopping, notably PCR-free libraries (which lack an enrichment step) have exhibited higher hopping rates around 1–2%. Also, libraries with low diversity (e.g. 16S rRNA gene pools) can suffer more from index misassignment because subtle cross-talk stands out amid uniform reads. Unique dual indices ensure that any misindexed reads are invalidated rather than incorrectly included into another sample’s data. - Clinical and regulated sequencing workflows
When sequencing is performed in a regulated environment (clinical diagnostics, accredited labs), UDI Primer Sets provide an extra level of assurance for auditors and quality reports. They also reduce the risk of having to repeat tests due to sample swaps, which in clinical settings could delay critical results.
Quality matters – From adapters to indexes
Unique Dual Index Primer Sets offer a powerful tool for multiplexed sequencing. By using UDIs, laboratories can substantially reduce index misassignment, ensuring that each sample’s data is trustworthy and actionable. Eurofins Genomics’ UDI primers are a dependable choice for any lab looking to upgrade its indexing strategy.
It’s worth noting that UDIs are one piece of the puzzle. Even with perfect index assignment, the overall success of an NGS experiment relies on the quality of oligonucleotides at every step. From the moment adapters are ligated to DNA, to the PCR primers that amplify a library, using high-purity NGS Oligos ensures that no unwanted variables are introduced.
Read more about NGSgrade Oligos in our blog post and learn how ultra-pure primers and adapters improve library prep efficiency and reduce sequencing noise across all NGS workflows. By combining NGSgrade oligo quality with Unique Dual Indexing, a robust sequencing pipeline is created, where both accuracy and purity are maximized at every step.
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