[A method for reliable detection of genomic point mutations based on single-cell target-sequencing]

Area of Science:

• Genomics
• Oncology
• Molecular Biology

Background:

• Tumor clonal evolution is typically studied using bulk sequencing, which can obscure low-frequency mutations and specific cell population dynamics.
• Current bulk sequencing methods may not accurately represent the heterogeneity of tumor cell populations.

Purpose of the Study:

• To develop and validate a single-cell resolution strategy for analyzing genomic point mutations in prostate basal cell carcinoma (BCC).
• To overcome limitations of bulk sequencing in capturing tumor heterogeneity and low-frequency mutations.

Main Methods:

• Optimized single-cell whole genome amplification (SC-WGA) using HepG2 cells.
• Captured single cells from BCC tissue using microfluidics for SC-WGA.
• Performed whole exome sequencing to identify mutations in SCUBE3 and MST1L.
• Validated mutations using single-cell targeted amplification and Sanger sequencing.

Main Results:

• Successfully established and optimized a single-cell genomic mutation analysis strategy.
• Identified and confirmed SCUBE3 and MST1L mutations in BCC at the single-cell level.
• Demonstrated the method's capability to reconfirm known mutations with high accuracy.

Conclusions:

• The developed single-cell strategy provides a powerful tool for precise analysis of tumor clonal evolution.
• This approach enhances the detection of genomic mutations in specific tumor cell populations.
• Offers a valuable method for studying cancer at single-cell resolution.