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Hum Mutat. 2019 Sep;40(9):1225-1234. doi: 10.1002/humu.23866. Epub 2019 Aug 17.

Future directions for high-throughput splicing assays in precision medicine.

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Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island.
Center for Computational Molecular Biology, Brown University, Providence, Rhode Island.
Genomic Medicine Institute, Geisinger, Danville, Pennsylvania.
Hassenfeld Child Health Innovation Institute of Brown University, Providence, Rhode Island.


Classification of variants of unknown significance is a challenging technical problem in clinical genetics. As up to one-third of disease-causing mutations are thought to affect pre-mRNA splicing, it is important to accurately classify splicing mutations in patient sequencing data. Several consortia and healthcare systems have conducted large-scale patient sequencing studies, which discover novel variants faster than they can be classified. Here, we compare the advantages and limitations of several high-throughput splicing assays aimed at mitigating this bottleneck, and describe a data set of ~5,000 variants that we analyzed using our Massively Parallel Splicing Assay (MaPSy). The Critical Assessment of Genome Interpretation group (CAGI) organized a challenge, in which participants submitted machine learning models to predict the splicing effects of variants in this data set. We discuss the winning submission of the challenge (MMSplice) which outperformed existing software. Finally, we highlight methods to overcome the limitations of MaPSy and similar assays, such as tissue-specific splicing, the effect of surrounding sequence context, classifying intronic variants, synthesizing large exons, and amplifying complex libraries of minigene species. Further development of these assays will greatly benefit the field of clinical genetics, which lack high-throughput methods for variant interpretation.


assay; disease; high-throughput; precision medicine; splicing; variant

[Available on 2020-09-01]

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