PCRinduced

PCRinduced refers to artifacts and biases introduced during polymerase chain reaction (PCR) amplification that can skew molecular analyses. It is of concern in cloning, sequencing, metagenomics, diagnostics, and gene expression studies.

Common PCRinduced artefacts include sequence errors from polymerase misincorporations, PCR chimera formation via template switching, and preferential amplification leading to certain sequences being overrepresented or underrepresented. In sequencing libraries, PCR can also generate duplicates and introduce copy number biases.

Causes and mechanisms: standard DNA polymerases have a nonzero error rate; errors accumulate with cycle number. Slippage in repetitive regions can cause insertions/deletions. Template switching between similar sequences can create chimeric molecules, especially in mixed templates. GC-rich regions can be underamplified, AT-rich overamplified, leading to GC bias.

Consequences: inaccurate variant calls, distorted representation of alleles, biased estimation of gene expression or microbial diversity, inflated duplicate counts, and potential misassemblies in metagenomes or amplicon sequencing data.

Mitigation: minimize PCR cycles, use high-fidelity DNA polymerases, optimize primer design, balance annealing temperatures, use UMIs to identify original molecules, plan replicates, and include control experiments. Alternative approaches like linear amplification or PCR-free library preparation can reduce PCRinduced artefacts, but may not always be practical.