Peak Fronting in NGS Library Smears on Agilent TapeStation

Answer

This Information Applies To: 2200/4200/4250 TapeStation systems, TapeStation D1000 ScreenTape assay, TapeStation High Sensitivity D1000 ScreenTape assay, TapeStation analysis software

Issue:

A sharp peak appears at the lower end of the NGS library smear in Agilent TapeStation D1000 ScreenTape and High Sensitivity D1000 ScreenTape assays (see Figure 1).

Figure 1. Region analysis (blue borders) of an NGS library smear. The red arrow indicates the peak fronting.

Resolution:

Peak fronting is an electrophoretic artifact. The cause is a concentration-dependent stacking effect on D1000 and High Sensitivity D1000 ScreenTape assays. In certain circumstances, some fragments might show a similar mobility and stack together, forming a distinct peak within the smear. It is more obvious in low-input libraries with medium to low concentrations. It does not represent a contamination in the library. Potential causes include factors such as the sample resuspension buffer, sample smear size, and concentration range. Therefore, rerunning the samples on the same ScreenTape kit will not help as the effect is inherent of the sample.

Peak fronting does not affect the overall quantification as long as the smear is correctly integrated (see region included in Figure 1). However, the sizing might be slightly shifted and therefore the molarity might be marginally altered as well. Nevertheless, quantification and sizing results are usually meeting the kit accuracy and precision specifications. Peak fronting typically does not alter results significantly, as long as the samples are meeting kit sizing and concentration specifications (see also Quick Guides and for smear specifications technical overview):

Kit	Sizing range	Quantification range (DNA fragments)	Quantification range (DNA smears)
D1000	35–1000 bp	0.1–50 ng/µl	5–100 ng/µl
High Sensitivity D1000	35–1000 bp	10–1000 pg/µl	0.5–15 ng/µl

Please use the following search terms to find more information about this and other NGS library phenomena in the Agilent Information Center (AIC) on your PC: TapeStation Analysis in the SureSelect and HaloPlex Workflow.

Root cause:
Concentration-dependent stacking effect