How much DNA should I use to detect 1% somatic variant frequency?


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:
There is little or no library peak when assayed with a fragment analyzer.
- 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.
What are the AT and GC biases of CleanPlex® target enrichment libraries?
What does a typical CleanPlex® target enrichment library look like?
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.
What is the CleanPlex® amplicon sequencing workflow for a multi-pool panel?
What is the CleanPlex® amplicon sequencing workflow for a single-pool panel?
What is the typical yield of a Cleanplex® library?
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.
What is the uniformity of a typical CleanPlex® target enrichment library?
What should I do when the library yield is lower or higher than expected?
Why are the long amplicons under-represented in my target enrichment library?
Why are the short amplicons under-represented in my target enrichment library?
How do I place an order using a Purchase Order?
Register an account and place an order online (US customers only)
- You can pay with credit card at the last step of Checkout, or
- You can use a Purchase Order number at the last step of Checkout (as shown below)