PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

Area of Science:

• Molecular Biology
• Genomics
• Evolutionary Biology

Background:

• High-throughput DNA barcoding is crucial for ecology and evolution research.
• Increasing PCR cycles is common for low-quality DNA but may introduce mutations.

Purpose of the Study:

• To investigate the impact of increased PCR cycles on mutation rates in DNA barcoding.
• To assess the reliability of using elevated PCR cycles with nonproofreading polymerases.

Main Methods:

• Sequencing of 20 COI libraries using Illumina MiSeq.
• Preparation of libraries with varying PCR cycles (40-60) from four cephalopod species.
• Analysis of mutation rates in relation to PCR cycle number.

Main Results:

• No correlation was found between the number of PCR cycles and artificial mutations.
• Elevated PCR cycles (up to 60) resulted in a low number of mutations, not impacting DNA barcoding.
• Mutation rates were low enough not to be an issue for DNA barcoding, but may affect DNA metabarcoding due to chimeras.

Conclusions:

• Increasing PCR cycles in high-throughput DNA barcoding studies does not negatively affect the occurrence of point mutations.
• The practice of using more PCR cycles is acceptable for DNA barcoding, especially with limited DNA.
• Further considerations are needed for DNA metabarcoding applications regarding chimera formation.