Abstract
Next-generation sequencing (NGS) is an incredibly useful tool for genetic disease diagnosis. However, the most commonly used bioinformatics methods for analyzing sequence reads insufficiently discriminate genomic regions with extensive sequence identity, such as gene families and pseudogenes, complicating diagnostics. This problem has been recognized for specific genes, including many involved in human disease, and diagnostic labs must perform additional costly steps to guarantee accurate diagnosis in these cases. Here we report a new data analysis method based on the comparison of read depth between highly homologous regions to identify misalignment. Analyzing six clinically important genes—CYP21A2, GBA, HBA1/2, PMS2, and SMN1—each exhibiting misalignment issues related to homology, we show that our technique can correctly identify potential misalignment events and be used to make appropriate calls. Combined with long-range PCR and/or MLPA orthogonal testing, our clinical laboratory can improve variant calling with minimal additional cost. We propose an accurate and cost-efficient NGS testing procedure that will benefit disease diagnostics, carrier screening, and research-based population studies.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
We would like to show our gratitude to Dr. Yiping Shen and Dr. Samuel Strom, who provided expertise and insightful comments that improved the manuscript. We thank Dr. Kyle Proffitt for editing and improving the manuscript and references. We also thank Dr. Becky Tsai for organizing the review process. This study was supported by Fulgent Genetics, Inc.
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C-YY and H-YY proposed the model and reviewed the clinical data. AZ conducted the experiments. HG provided suggestions on research goals and supervised the project.
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Lee, Cy., Yen, HY., Zhong, A.W. et al. Resolving misalignment interference for NGS-based clinical diagnostics. Hum Genet 140, 477–492 (2021). https://doi.org/10.1007/s00439-020-02216-5
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DOI: https://doi.org/10.1007/s00439-020-02216-5