AmpliSeq for Illumina Custom and Community Panels FAQs

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  • Input

  • The assay uses between 1 and 100 ng of DNA per primer pool, with most designs using 10 ng per pool.

    We’ve seen success with low quality inputs using the protocol modifications indicated in the user guides.  Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8–2.0. PicoGreen is recommended for an accurate quantification. 

    Only use FFPE-derived DNA when using short amplicon lengths of 140 or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA. 

    There is a limit of 12-6,144 primer pairs per pool. If generating target region greater than 5Mb, we recommend selecting an enrichment option.

  • Sequencing

  • A 2×150 bp paired-end read is recommended for 140-275 bp amplicon sizes. Up to 2x300 bp paired-end run on the MiSeq is recommended for 375 bp amplicon sizes.

    This kit has integrated sample barcodes that enable pooling of up to 96 samples per sequencing run. However, the actual number of samples that can be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. An online calculator is provided in DesignStudio to help with these calculations.

  • AmpliSeq for Illumina On-Demand – IGV Viewer

  • The “observed coverage” track indicates the number of observed reads for each amplicon of each targeted gene during validation experiments on a NextSeq. Use this track as general guidance for the likely performance when running an experiment. While values can vary among assays, the general coverage trend should remain consistent.

    Gaps occur where there are no amplicons to provide coverage for the intended target. We have made every effort to minimize the occurrence of these regions in our On-Demand designs.

    The Y-axis represents the observed coverage normalized by the mean amplicon coverage for the gene.

    No. The IGV viewer can only focus on your gene of interest. In the Grid View, select a gene, and the IGV viewer updates automatically to center on that gene.

    All amplicons in the design contain reads that are visualized in the “observed coverage” track. If the number of reads covering an amplicon is relatively small in comparison to neighboring amplicons, the “observed coverage” track appears empty. However, if you change the scale to a lower value, you will then be able to visualize the lower number of reads. If the observed coverage track is not present, the designer notifies you why that track is not available.