Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• PCR amplicon sequencing is a key tool for DNA/RNA analysis.
• High multiplex PCR allows enrichment of hundreds of amplicons simultaneously.
• Challenges include duplicate reads, polymerase artifacts, and amplification bias.

Purpose of the Study:

• To develop a protocol for incorporating molecular barcodes into high multiplex PCR.
• To address limitations of existing PCR amplicon sequencing methods.

Main Methods:

• Integration of molecular barcodes into PCR primer design for high multiplex reactions.
• Application of the protocol to reference materials for validation.

Main Results:

• Successful implementation of molecular barcodes in high multiplex PCR with hundreds of amplicons.
• Demonstrated accurate variant calling at very low fractions over large regions.
• Showcased utility in targeted RNA quantification and FFPE sample profiling.

Conclusions:

• The new protocol combines benefits of high multiplex PCR and molecular barcodes.
• Enables analysis of large regions with low DNA input and high reproducibility.
• Accurately detects low-frequency mutations (1%) with minimal false positives.