Rapid identification of variants associated with cystic fibrosis using a long-read multiplexed amplicon panel

Cystic fibrosis is a recessive genetic condition caused by mutations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein (Fig. 1a), which controls movement of salt and water into and out of cells (Fig. 1b). Patients are prone to breathing difficulties and infections (Fig. 1c) and have reduced life expectancy. There are many disease- related CFTR mutations (Fig. 1d). Detecting these variants provides a way to screen populations, confirm clinical diagnosis, determine treatment options and to perform prenatal diagnosis. Sequencing long amplicons allows unambiguous identification of paralogous genes (Fig. 1e).

We constructed a multiplexed PCR panel consisting of twenty four ~1.5 kb amplicons covering all major CFTR variants. We called single nucleotide variants and small indels from human cell line NA11472 using Clair and correctly identified all confirmed heterozygous SNPs as well as an unreported SNP (Fig. 2a). The intron 8 poly-T tract represents a more challenging variant to detect, consisting of 5, 7 or 9 consecutive Ts followed by a dinucleotide repeat. Our new nanopore, R10 enables resolution of longer homopolymers, such as the (T)9 heterozygous homopolymer, which can be seen in approximately half of the aligned sequences (Figs. 2b and 2c).

The MUTCF-2 panel consists of 23 samples, each with a unique CF-related variant. We used the panel to determine variant-level specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) for the multiplexed PCR assay (Fig. 3a). The multiplexed PCR was performed on each sample individually along with a negative sample (NA12878) and libraries were barcoded, pooled and run on a MinION flow cell. Variant calling was performed with Clair after down-sampling to a maximum read depth of 150 per amplicon. We achieved a sensitivity of 94.44% and specificity of 100%, with a PPV of 100% and NPV of 99.63%. Figure 3b shows the most common pathogenic variant (delta F508) in homozygous and heterozygous form.

Because sequencing on nanopore devices takes place in real time, sufficient coverage for variant-calling can be obtained from all amplicons in around five minutes of sequencing (Fig. 4a). VolTRAX is a portable, USB-powered library preparation device which can perform a wide range of molecular biological manipulations without the need for human intervention (Fig. 4b). VolTRAX utilises an array of pixels and by applying a charge, reagent and sample droplets are moved in a path programmed by software, which allows library preparation processes to be performed sequentially. The device is also capable of heating and cooling droplets, enabling PCR (Fig. 4c). We successfully performed the 24-plex CFTR PCR on VolTRAX in ~3 hours in a volume of 10 ml.