2023 ACMG Annual Clinical Genetics Meeting Digital Edition: Poster Gallery-Long-Range PCR and Nanopore Sequencing Method Resolves F8, GBA, CYP21A2, SMN1, and TNXB Variants Using a Single Streamlined Workflow

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Researchers at Asuragen have developed a prototype single-platform, multi-gene AmplideX PCR-based nanopore sequencing assay with custom informatics pipelines that can detect key genetic variants associated with disorders such as Gaucher Disease and Hemophilia A. The assay utilizes sequence deconvolution, amplicon read depth, Paralog Specific Variants (PSVs)-based copy number, and machine learning models to automate the identification of variants specific to each disease. In reference cell-line samples, more than 90% of known variants across the five genes were detected. The assay also identified 27 carriers in a presumed normal whole blood testing cohort, consistent with known carrier rates.<br /><br />The study focused on the development of a single-tube workflow using long-range PCR amplification and long-read nanopore sequencing to detect various classes of genetic variation in challenging genes such as F8, GBA, CYP21A2, TNXB, and SMN1. These genes are difficult to sequence due to the presence of complex structural variants and pseudogenes. The workflow allows for the amplification of large, highly GC-rich regions and the detection of copy number and structural variants in highly homologous sequences.<br /><br />The assay was tested on cell-line samples containing all major classes of variation and whole blood samples in four sequencing runs, demonstrating its performance. The researchers used custom software for sequence deconvolution and variant identification. The assay showed accurate resolution of copy number and pseudogene fusions in the CYP21A2 gene cluster and accurate detection of F8 intron 1 and 22 inversions.<br /><br />The prototype assay offers a single-tube, streamlined workflow that can multiplex up to 95 samples in a sequencing run, replacing four independent assays that target fewer variants. The assay provides comprehensive sequencing and allows for variant phasing and the identification of complex fusions, resulting in the detection of over 90% of known variants. The study highlights the importance of a unified carrier screening approach for identifying carriers of multiple genetic disorders.

Asset Subtitle

Presenting Author - Cody Edwards, BS; Co-Author - Bradley Martin, BS, MS, PhD; Co-Author - Monica P. Roberts, MS; Co-Author - Jon N. Kemppainen, BS; Co-Author - Ryan Routsong, MS; Co-Author - Bryan Killinger, PhD; Co-Author - Adrian N. Adrian, BSc; Co-Author - Christopher J. Fraher, BS; Co-Author - Gary J. Latham, PhD; Co-Author - Bradley Hall, PhD;

Meta Tag

Genetic Testing

Population Genetics

Sequencing

Variant Detection

X-Inactivation/X-Linked Disease

Co-Author Bradley Martin, BS, MS, PhD

Co-Author Monica P. Roberts, MS

Co-Author Jon N. Kemppainen, BS

Co-Author Ryan Routsong, MS

Co-Author Bryan Killinger, PhD

Co-Author Adrian N. Adrian, BSc

Co-Author Christopher J. Fraher, BS

Co-Author Gary J. Latham, PhD

Co-Author Bradley Hall, PhD

Presenting Author Cody Edwards, BS

Keywords

Asuragen

PCR-based

nanopore sequencing assay

genetic variants

Gaucher Disease

Hemophilia A

structural variants

pseudogenes

copy number

carrier screening