Single Sperm Cell RNA Sequencing for Rare Mutation Detection

The invention segments the bulk sperm sample into individual single sperm cells for separate analysis. By isolating and sequencing individual sperm cells rather than analyzing bulk samples together, the method can detect mutations present in less than 5% of the sample. Each single cell is processed independently through reverse transcription of RNA to cDNA, allowing rare mutations to be identified without being diluted or obscured by the high error rates that plague bulk sequencing of millions of cells.

The invention introduces reverse transcription as an intermediary step between single sperm cell isolation and sequencing. By converting RNA to cDNA through reverse transcription, the method creates a stable DNA intermediate that can be more easily sequenced and analyzed. This intermediary process enables high-precision detection of mutations in individual sperm cells while managing the technical complexity through a well-established molecular biology technique.

The invention inverts the conventional approach by analyzing single sperm cells instead of bulk samples. Rather than pooling millions of sperm cells and accepting that only common mutations will be detected, the method examines individual cells separately. This inversion allows rare mutations present in less than 5% of the sample to be identified, thereby improving the reliability of detecting disease-causing mutations while maintaining productivity through automated single-cell processing.