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FAQ

Does the digestion step work at other temperatures than 37°C?

Digestion may work at room temperature (25°C) for certain panels with ≤ 220 primer pairs. However, other similar or larger panels may experience incomplete digestion of non-specific amplification products at room temperature. This may cause under-amplification of the library during the 2nd PCR step.

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

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 the right). Therefore, we recommend using higher amounts of DNA for somatic variant detection. This is especially true when DNA quality is uncertain (such as DNA from FFPE tissues and liquid biopsy). Theoretically, 10 ng human DNA (~1500 cells and ~3000 copies of each locus) will allow detection of 30 somatic alternative alleles at 1% frequency. With CleanPlex® technology, lower amount (even less than 1 ng) of high-quality genomic DNA may be used for detection of germline alterations.
      

There are peaks from 70 – 90 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart?

Peaks from 70 – 90 bp (see the graph 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 making a large number of libraries in a short period of time OR insufficient removal of ethanol during washing steps. To remove any possible small-molecular-weight materials, it is wise to pool all indexed libraries (that will be sequenced in the same lane) and do one additional round of 1.2X magnetic bead purification, before the quantification step prior to sequencing.

There is a peak around 150 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart.

Peaks from 150 – 160 bp (see the graph below on the left) are residues of digested non-specific amplification products. 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 from150 – 160 bp are usually caused by inaccurate pipetting of magnetic beads when making a large number of libraries, OR insufficient removal of ethanol during washing steps. To remove any possible small-molecular-weight materials, it is wise to pool all indexed libraries (that will be sequenced in the same lane) and do one additional round of 1.2X magnetic bead purification, before the quantification step prior to sequencing.

Significant peaks from 150 – 160 bp may be caused by under-amplification of the library (see the graph below on the right). Certain panels may tend to produce more non-specific amplification products. In this case, two rounds of magnetic bead purification after the digestion step solves the problem.

There is very little or no library peak when assayed with Agilent™ 2100 Bioanalyzer.

The possible causes are:

(1) 30% ethanol instead of 70% ethanol is used in DNA purification with magnetic beads; or

(2) forgetting to add magnetic beads after digestion. The workflow does not require users to change tubes, nor to remove magnetic beads from previous steps. One can easily tell if magnetic beads in each purification steps are added or not by checking the volume; or

(3) forgetting to add Stop Buffer after digestion, especially when handling a large number of samples.

(4) over-drying beads can decrease library yield, whereas under-drying beads can leave ethanol that will inhibit PCR.

(5) inaccurate volume of magnetic beads added. Ensure magnetic bead aliquot is well mixed prior to each dispense.

(6) insufficient mixing. Ensure all reactions and solutions are thoroughly mixed and homogeneous prior to proceeding to thermocycling and incubation steps.

What are the AT and GC biases of CleanPlex® target enrichment libraries?

The observed AT and GC dropouts are below 3% in CleanPlex® target enrichment libraries. Below is a typical graph of Coverage Depth vs. GC Content of a library made with CleanPlex® OncoZoom Panel.

What does a typical CleanPlex® target enrichment library look like?

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 the library.

What is the CleanPlex® amplicon sequencing workflow for a multi-pool panel?

For a panel with two or more primer pools, follow the procedure shown below.

What is the CleanPlex® amplicon sequencing workflow for a single-pool panel?

For a single-pool panel, follow the procedure shown below.

What is the optimal range of library yields for sequencing?

CleanPlex® workflow generates a single peak of library at 20,000 – 100,000 pM when measured with Agilent™ 2100 Bioanalyzer instrument and Agilent™ high sensitivity DNA reagents, or 3.5 – 20 ng/µl when measured by Qubit™ dsDNA HS Assay Kit, depending on each specific panel. Concentrations higher or lower than this range of yields may lead to lower uniformity due to uneven amplification of target regions.

What is the uniformity of a typical CleanPlex® target enrichment library?

The uniformity ( ≥ 0.2X mean depth) of most CleanPlex® libraries are larger than 95%. For example, the observed uniformities (at ≥ 0.2X mean depth) of CleanPlex® OncoZoom Panel are greater than 99% for both NA12878 (a genomic DNA standard) and Horizon Discovery’s HD780 (a cfDNA standard) (see the graph below).

What should I do when the library yield is lower or higher than expected?

Increase or decrease the number of cycles for the 2nd PCR by 2 to 3.

Why are the long amplicons under-represented in my target enrichment library?

CleanPlex® supports the amplification of up to 300 bp insert length. Please make sure that the correct read length is chosen during sequencing.

Why are the short amplicons under-represented in my target enrichment library?

Short amplicons in your target enrichment library may be removed during magnetic bead purification. Please add the  correct amount of magnetic bead suspension in each purification step.

How to 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)

Does Paragon offer wetlab optimization services for custom panels?

Yes, please contact [email protected] for a quote.