What should I expect my NGS library to look like on a fragment analyzer trace?

Please see user guide for examples of each 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. …

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What are the peaks around 70 – 90 bp in addition to the main library peak on a fragment analyzer trace?

Peaks from 70 – 90 bp (see the trace below) are primer dimers from the 2nd PCR that result from incomplete removal of small molecular materials during the final magnetic bead purification. These are usually caused by inaccurate pipetting of magnetic beads volume, poor mixing prior after adding magnetic beads, and/or incomplete removal of supernatant …

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What is peak around 150 -190 bp in addition to the main library peak on the fragment analyzer trace?

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 …

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What is the typical yield of a CleanPlex or CleanPlex UMI library?

CleanPlex and CleanPlex UMI workflow generates a single peak of library of 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 …

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There is little or no library peak when assayed with a fragment analyzer.

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 for DNA purification. Forgetting to add magnetic beads for any of the purification steps. Forgetting to add one …

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

If the library yield is low, but other wise clean and free from by-product peak(s), the library may require optimization in 2nd PCR cycles.  Increase or decrease the 2nd PCR cycles by 2 to 3. If significant by-product peak is present, please refer to FAQ’s above for detailed troubleshooting suggestions.

What is the recommended minimum read coverage for Cleanplex NGS panels?

For Genotyping applications, minimum read coverage per amplicon is 20X. However, we suggest starting with 1000 PE reads/amplicon/sample for the first sequencing run to account for any potential off target, non-mapping, or poor quality sequencing runs. For subsequent sequencing runs, the allocated PE reads/amplicons can be significantly reduced based on the panel’s performance and sequencing …

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What is the recommended minimum read coverage for Cleanplex UMI NGS panels?

The suggested minimum read coverage is 7,500 paired-end reads/ amplicon/ nanogram of DNA input for detection of 0.1% allele frequency, when the minimum DNA input amount is also met. 7,500 paired-end reads equates to 3,750 clusters or single-end reads on the sequencing chip. Please see FAQ for DNA input requirement for detecting 0.1% somatic variant …

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Does Paragon offer bioinformatics and data analysis support for ready-to-use panels and custom panels?

We’re actively developing a cloud-based data analysis package and customer facing software using our analysis algorithms. In the near term, our bioinformatics team can provide plug-ins specific to Cleanplex libraries for common data analysis pipelines. The team can also offer support, assistance, and guidance to customers who may need additional help in data analysis for our …

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