Skip to main content

Conference Program

Subpage Hero

Loading

Performance evaluation of a PCR/Nanopore workflow for carrier screening for Cystic Fibrosis, Spinal Muscular Atrophy, and Fragile X Syndrome

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
Cystic Fibrosis (CF), Spinal Muscular Atrophy (SMA), and Fragile X Syndrome (FXS) are among the most common inherited genetic disorders. As such, carrier screening for these conditions is important to identify couples at risk for having a child affected by one of these disorders. Traditionally, carrier screening has required multiple workflows to accurately identify pathogenic variants for each of these disorders and may only target a subset of potential variants. Full gene sequencing provides more robust results to account for the genetic diversity of the population, by increasing the ability to detect unique or rare pathogenic variants. Here, we evaluate a PCR/Nanopore workflow for the detection of pathogenic variants in CFTR, copy number calling and variant detection for SMN1/2, and repeat sizing for FMR1 in a single workflow.

Methods:
Selected samples included DNA obtained from: whole blood from de-identified residual clinical samples (n = 53), DNA from cell lines (n = 17), and CAP proficiency testing samples (n = 3). Using the AmplideX Nanopore Carrier Plus kit reagents, DNA was first amplified via PCR in two separate reactions using Mix A to target CFTR and SMN1/2 and Mix B to target FMR1. Following the gene specific PCR, samples then underwent a subsequent barcoding PCR reaction.  After barcoding, the samples were pooled and then sequenced on MinION flow cells. The sequences were then analyzed and reported using the AmplideX One Reporter software.

Results:
PCR/fragment analysis lab-developed tests were used as reference methods. Of the samples tested, the CFTR results showed >97% concordance with our previous results. Copy number calling for SMN1 and SMN2 was >95% and >82% concordant, respectively. Notably, copy number losses/deletions were called with 100% concordance for both SMN1 and SMN2. Lastly, FMR1 showed 100% concordance within one repeat of our previously tested samples. We also found these results to be reproducible between inter- and intra- run repeats.

Conclusion:
Here, we show that the PCR/Nanopore workflow using the AmplideX Nanopore Carrier Plus reagents is accurate and reproducible for identifying pathogenic variants in CFTR, copy number for SMN1/2, and repeat sizes in FMR1. By testing multiple genes, this assay is capable of screening for carriers for more than one disorder in a single workflow. Additionally, using short- and long-range PCR followed by sequencing, it has the potential to identify more CFTR variants than in traditional genotyping panels, thus facilitating adherence to evolving guidelines regarding the minimum variant set to be tested. Future directions include data re-analysis with an updated version of the software.

Agenda

Sponsors