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.
      

Peaks from 70 – 90 bp (see the trace below) are primer dimers from the 2^nd 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 of supernatant  and ethanol during washing steps. If these peaks are significant, one can pool all indexed libraries (that will be sequenced in the same lane) and perform one additional round of 1.2X magnetic bead purification, before the quantification step prior to sequencing.

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 usually caused by the following:

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 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.

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.

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.

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.

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

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

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 to QC completed library with a fragment analyzer to confirm the quality of the library prior to sequencing. Qubit measurements only give the total yield, and does not indicate if the library was poorly prepared and had resulted in large amount of byproducts.

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.

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).

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

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

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.

 

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