What are the peaks around 70 – 90 bp in addition to the main library peak on a fragment analyzer trace?
What is peak around 150 -190 bp in addition to the main library peak on the fragment analyzer trace?
They come from incomplete removal of small-molecular-weight materials during magnetic bead purification after digestion. The digestion reagent cuts non-specific amplification products into small pieces, which are then removed during magnetic bead purification. Peaks from 150 – 190 bp are usually caused by the following:
- Using incompatible index primers.
- DNA quantification was inaccurate, especially if using spectrophotometric methods such as nanodrop, OR DNA quality is extremely poor.
- 30% ethanol instead of 70% ethanol OR using TE/H2O instead of 70% ethanol was used in DNA purification.
- Forgetting to add magnetic beads for any of the purification steps.
- Forgetting to add one or both index primers in the 2nd PCR step.
- Over-digestion, forgetting to add Stop Buffer after digestion, or pausing after digestion step.
- Weak or incompatible magnetic rack. Do not use racks designed for 1.5 ml tube.
Please see user guide for examples of other ready-to-use panels.
When using a fragment analyzer such as Agilent Bioanalyzer 2100, the library peak(s) should fall between 200-400bp, depending on the specific panel. A library can exhibit a single peak (such as OncoZoom) or multiple peaks (such as TP53) depending on the panel’s amplicon length distribution. Most importantly, look for sharp and well-defined peaks that span the base pair length range specific for the panel. Each custom panel design will receive an “Amplicon Length Distribution Theoretical Plot” for the user to compare with the completed library.
The fragment analyzer most importantly allows the user to visualize any byproducts such as adapter dimers & digested non-specific products (~150-190bp) and 2nd PCR primer dimers (70-90bp). See following FAQs for details on how to reduce these byproduct peaks.
Concentrations higher or lower than the typical range of yields may lead to lower uniformity due to uneven amplification of target regions. Concentrations much lower in concentration can prove to be difficult to satisfy Illumina’s loading criteria. Please refer to Illumina’s suggestions for minimum loading concentration and volume.
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