Specifications CGD Ampliseq Panel

The CGD AmpliSeq Panel v1 (IAD63499_185) exists of 137 amplicons and is covering 26 Kbase. 98.1% of desired areas (exons with a 10 base exon padding and the promotor region of CYBB) are covered in the design that includes the following seven genes: CYBB, CYBA, NCF1, NCF2, NCF4, G6PD and RAC2.

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Coverage of the CGD Ampliseq Panel

The percentage of target bases that is covered at least 20 times is at least 97% for the recommended Mapped Reads of 250,000. The regions that are missed, either in the design or due to practical coverage, are listed Table 1 below.

Three small regions of G6PD are missed in the design. However, when there are specific G6PD requests, our RBC Enzym AmpliSeq Panel will be used and those three regions are covered well in this panel. There are some CGD patients known to have a pathogenic variant in G6PD (NM_000402:c.653C>T) and this position is covered well with the CGD panel.

NCF1 has two pseudogenes and is therefore a difficult region to sequence. We decided to include both pseudogenes in the panel, to get as many positions covered as possible with as many specific and distinguishable positions as possible. However, in the regions in NCF1B or NCF1C that are not covered, there won’t be any pathogenic variants. Therefore, those regions are not listed in Table 1.

There are two frequent occurring pathogenic variants known in NCF1. One is a cross-over between the gene and one of its pseudogenes, resulting in a two base pair deletion at the start of exon two. This variant occurs in 80% of the CGD patients with a defect in NCF1. Reads with this deletion won’t be aligned on exon 2 of the gene, but will be aligned to the pseudogenes. This variant will be seen in the failure of coverage in exon 2. With this technique we won’t be able to detect carriers for this variant. Therefore it is needed to perform a NCF1 genescan to exclude or confirm whether a person has only one normal NCF1 gene. This genescan will be performed by us when an X026 is requested and for familiar NCF1 requests.

The second frequent occurring pathogenic variant is a nonsense variant located in exon 7 and occurs in 8 to 10% of the CGD patients with a defect in NCF1. This location is covered well and gene specifically with this Ampliseq CGD-panel.

Other variants in NCF1 are less common and difficult to find. We try to analyse the other regions of NCF1 on a research base (possibly with another Ampliseq panel), but cannot guarantee any findings.

Table 1: Missed regions in design (Part 1) and possibly by practical coverage (part 2).

Read more about the technique and reporting in the background information.