Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA...

Area of Science:

• Genomics
• Molecular Biology
• Aging Research

Background:

• Somatic mutations accumulate with age in healthy tissues, contributing to cancer and aging.
• Studying these mutations in non-neoplastic tissues is crucial for understanding aging and disease.
• Existing methods may not be suitable for analyzing small cell populations from tissue sections.

Purpose of the Study:

• To develop and describe a comprehensive workflow for sequencing genomes from small cell populations.
• To enable the study of somatic mutations in non-neoplastic human tissues.
• To provide a WGA-free (whole-genome amplification-free) protocol for low-input DNA sequencing.

Main Methods:

• The workflow integrates laser-capture microdissection (LCM) with low-input genome sequencing.
• It avoids whole-genome amplification (WGA) by using enzymatic fragmentation for library generation.
• The protocol includes tissue processing, LCM, low-input library preparation, and mutation calling/filtering.

Main Results:

• The workflow successfully sequences genomes from small cell populations (100-1,000 cells) in microbiopsies.
• It has been applied to thousands of samples across diverse human tissues.
• The protocol is compatible with standard sequencing facility equipment and liquid handling platforms.

Conclusions:

• This WGA-free, low-input DNA sequencing protocol facilitates the study of somatic mutations in aging and disease.
• The method provides a robust approach for analyzing genomic alterations in non-neoplastic tissues.
• The workflow can be completed in 1-3 weeks, making it accessible for research.