Ion AmpliSeq™ On‑Demand Panel design and CNV detection

Use the following guidelines when you design an Ion AmpliSeq™ On‑Demand Panel for CNV calling:

• Generally, CNVs can be robustly detected if at least 10, and ideally 20 amplicons cover a CNV. It can be possible to call CNVs with fewer amplicons (especially if using the algorithm’s low stringency setting), but if the data are somewhat noisy or if the coverage is low, a CNV covered by fewer than 10 amplicons can be missed.

• If the gene on which you want to call CNVs has fewer than 10 amplicons, consider adding genes on the 3ʹ and 5ʹ side of the gene of interest via a spike-in panel (or add catalog genes, if available).

• The algorithm used by Ion Reporter™ Software should be robust if no more than ~20% of amplicons in a panel have a CNV. If you are interested in whether a gene is affected by a CNV, there should typically be at least 5 genes in the panel, only one of which has a CNV in any sample. If more than 20% of the amplicons in the panel have a CNV (for example, in the case of a massively aneuploid tumor sample), the change in copy number can be detected, but the exact copy number value prediction may not be correct. This is because the normal diploid (‘ploidy=2’) state can be hard to identify in such samples, and as a result, CNVs can be called with the wrong copy number.