Adapter dimers causes, effects, and how to remove them

**What are adapter dimers?**During quality checks of sequencing libraries with chip-based capillary electrophoresis instruments, eg, the BioAnalyzer or Fragment Analyzer, or on agarose gel, an unexpected small peak at 120-170 bp indicates the presence of adapter dimers (Figure 1).

Adapter dimers contain full-length adapter sequences that are able to bind and cluster on the flow cell and generate sequencing data. In contrast, primer dimers do not contain complete adapter sequences, and are not able to bind or cluster on the flow cell, so are not sequenced.

What causes adapter dimers?

Insufficient starting material

• Using too little input material can lead to an increase in adapter dimer formation. Quantification with a fluorometric-based method is recommended to ensure accuracy of input amount. Using an input amount within the range recommended for the workflow minimizes the chance that adapter dimers will be present in the final libraries.

Poor quality of starting material

• Using fragmented or degraded input nucleic acid is not recommended for certain workflows. Using degraded input material for library preparation methods that are not compatible with degraded input can lead to adapter dimers.

Inefficient bead clean-up

• It is important to follow best practices when handling beads to ensure proper size selection and removal of adapter dimers that may have formed during library preparation.

Why is it important to remove adapter dimers?

Adapter dimers have complete adapter sequences, so they can cluster and sequence. Because of their small size, they cluster more efficiently than the intended library fragments. Depending on the quantitative proportion relative to the specific library, they can subtract a significant portion of sequencing reads from the desired library fragments. Moreover, they can negatively impact sequencing data quality, and may even cause a run to stop prematurely (Figure 2).

How to remove adapter dimers?

If adapter dimers are present in the library, perform an additional clean-up step with beads (AMPure XP, SPRI, or Sample Purification beads, “SPB”) or gel purification. A second round of purification may reduce the library yields. A bead ratio of 0.8x to 1x is usually recommended and sufficient to remove the unwanted adapter dimers.

What do adapter dimers look like?

Figure 1. BioAnalyzer electropherogram showing a library with an adapter dimer peak at 126 bp.

Figure 2. If adapter dimers are present during sequencing, this is evident in the percent base (%base) plot in Sequence Analysis Viewer, or in BaseSpace. Adapter dimers give the following signature:

1. A region of low diversity

2. The index region

3. Another region of low diversity

4. An increase in "A" when the adapter sequence ends and the sequencing read runs into the flow cell. This A overcall may be a G overcall, depending on the sequencing platform used

The overcall pattern may differ depending on the proportion of adapter dimers compared to the intended library fragments, ie, it can be less recognizable with a lower proportion of adapter dimers present in the run.

Note: Patterned flow cells are more susceptible to reduced run performance when adapter dimers are present. Illumina recommends limiting adapter dimers to 0.5% or lower when sequencing on patterned flow cells and 5% or lower when sequencing on non-patterned flow cells. (Percentages refer to the percent of adapter dimers relative to the full library; this can be determined by regional analysis with trace/electropherogram instruments.) Any level of adapter dimers will take away reads from the proper library fragments.

For any feedback or questions regarding this article (Illumina Knowledge Article #1911), contact Illumina Technical Support [e-mail].