Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Detecting extremely rare DNA variants is crucial for research areas like circulating tumor DNA analysis.
• Existing DNA barcoding methods for next-generation sequencing (NGS) often involve lengthy and complex protocols.
• Polymerase errors in NGS can hinder the accurate detection of low-frequency variants.

Purpose of the Study:

• To introduce Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq).
• To provide a streamlined and efficient method for preparing barcoded DNA libraries for sensitive mutation detection.
• To enable the detection of rare variant alleles with high accuracy and minimal DNA input.

Main Methods:

• SiMSen-seq utilizes a three-cycle barcoding PCR followed by adaptor PCR.
• The protocol requires minimal DNA input and allows flexible target selection (1 to 40+ amplicons).
• Library preparation is short (approximately 4 hours) and includes bead purification before sequencing.

Main Results:

• SiMSen-seq successfully detects variant alleles at frequencies below 0.1%.
• The method simplifies the identification and removal of polymerase errors through DNA barcoding.
• The protocol is adaptable for various molecular biology laboratories and target selections.

Conclusions:

• SiMSen-seq provides a robust and accessible method for sensitive mutation detection using NGS.
• The simplified protocol facilitates the study of rare variants in clinical and basic research.
• This technique enhances the reliability of detecting low-frequency DNA variants, such as those found in cancer diagnostics.