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Possibilities include: 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 …
There is little or no library peak when assayed with a fragment analyzer. Read More »
Lower DNA input will generally increase the possibility of inaccurate calling of variant frequencies. As shown in the following figure on the left, when less and less DNA was amplified with CleanPlex™ OncoZoom Panel, increasingly larger variations were observed for calling alternative alleles at 50% frequency, even though the uniformity may not deteriorate significantly (figure below on …
How much DNA should I use to detect 1% somatic variant frequency? Read More »
For a panel with two or more primer pools, follow the procedure shown below.
For a single-pool panel, follow the procedure shown below.
Below is a typical library made with CleanPlex® OncoZoom Panel and assayed with Agilent™ 2100 Bioanalyzer instrument and Agilent™ high sensitivity DNA reagents. Please note that the “Regions” in “Smear Analysis” function in Agilent™ 2100 Bioanalyzer software (under tab “Global/Advanced”) is set from 200 – 330 bp in order to obtain an accurate concentration of …
What does a typical CleanPlex® target enrichment library look like? Read More »
CleanPlex® workflow generates a single peak of library at approximately 8,000 – 20,000 pM when measured with a fragment analyzer such as Agilent™ 2100 Bioanalyzer instrument and Agilent™ high sensitivity DNA reagents, or approximately 1.5 – 4 ng/µl when measured by Qubit™ dsDNA HS Assay Kit, depending on each specific panel. It is good practice …
What is the typical yield of a Cleanplex® library? Read More »
Increase or decrease the number of cycles for the 2nd PCR by 2 to 3.
Peaks from 70 – 90 bp (see the trace below) are primer dimers from the 2nd PCR and result from incomplete removal of small molecular materials during the final magnetic bead purification. These are usually caused by inaccurate pipetting of magnetic beads when working with a large number of samples at once OR insufficient removal …
Peaks from 150 – 190 bp are residues of digested non-specific amplification products (see examples below). 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 …