CleanPlex™ OncoZoom Panel

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CleanPlex™ OncoZoom Panel

$936.00$28,537.00

CleanPlex™ OncoZoom Panel contains a single pool of 601 primer pairs, multiplex PCR reagent and other reagent components necessary for amplifying target regions of human DNA and constructing amplicon libraries for Next-Generation Sequencing on Illumina Sequencers. The panel targets more than 2900 commonly observed mutational positions (“hot spots”) from 65 oncogenes and tumor suppressor genes.

Product Description

CleanPlex™ OncoZoom Panel contains a single pool of 601 primer pairs, multiplex PCR reagent and other reagent components necessary for amplifying target regions of human DNA and constructing amplicon libraries for Next-Generation Sequencing on Illumina Sequencers. The panel targets more than 2900 commonly observed mutational positions (“hot spots”) from 65 oncogenes and tumor suppressor genes.

Product Features

  • Extremely uniform amplification of target regions. 100% observed uniformity at ≥ 0.2x mean depth and 97% observed uniformity at ≥ 0.5x mean depth.

  • Compatibility with a variety of sample types. The panel  is compatible with genomic DNA, FFPE, frozen tissue, fine needle aspirate, blood and cfDNA.
  • Low DNA input and high sensitivity. only 100 pg of input DNA is needed for germline genotype calling and 10 ng of DNA for somatic mutation detection (down to 1% low frequency allele).

Gene List

 

Sequencing Platform Illumina Sequencers (MiniSeq, MiSeq, NextSeq, Hiseq)
Enrichment Method Multiplex PCR
# of Primer Pools 1
# of Primer Pairs 601
# of Target Genes 65
Target Region Size (bp) 55199
Amplicon Size Average 146 bp (from 125-175 bp)
Species Human
Recommended DNA Input (Amount) For germline genotype calling: minimum 100 pg;
For somatic mutation calling with an LOD of 1%: minimum 10 ng
Sample Type Genomic DNA, FFPE DNA, cfDNA, and DNA from Blood, Tissue, Cell Culture, and Fine Needle Aspirate (FNA)
Sample Multiplexing
(at ~2000x mean coverage)
Miseq 2×150 Read Length: ~25 samples
NextSeq Series Mid Output 2×150 Read Length: ~200
NextSeq Series High Output @ 2×150 Read Length: ~600
Coverage Uniformity (at ≥ 0.2x mean coverage) >95%
On-target Reads % (% reads aligned to target regions out of total aligned reads) >95%

Additional Information

Weight 4 kg
Dimensions 30 x 50 x 20 cm
Pack Size (Reactions)

8, 96, 384

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.

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. 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 in one day. 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.

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 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.

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