Unique dual indexing PCR reduces chimeric contamination and improves mutation detection in cell-free DNA of pregnant women.

Allele fraction measurement is an essential component in nucleic acid analysis. The formation of chimeric amplicons during multiplex PCR amplification, however, greatly affects the allele fraction even before downstream analysis. Previous error correction strategy with unique molecular indexing (UMI) targets mainly points mutations rather than chimeras. Since the mutant allele detection in pregnant women cell-free DNA (cfDNA) is limited by chimeric amplicon contamination, a more direct error correction solution is demanded. Here we demonstrate effective reduction of chimeric amplicon contamination by unique dual indexing. With error corrected deep sequencing analysis, we achieved 100% accuracy in 16 tests of the parental mutation inheritance and de novo mutations in cfDNA of pregnant women, whose fetuses were at risk of tuberous sclerosis complex or Marfan syndrome. Our error correction strategy could offer a versatile solution for accurate multiplex PCR amplification.