Duplex Sequencing/CRISPR/Cas9 Duplex Sequencing: Challenges and Solutions for Early Cancer Detection

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method,has limited efficiency for small genomic regions, contributing to low recovery. Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among > 1 × 107wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. To overcome the high error rate of next-generation sequencing and thereby facilitate the study of subclonal and random mutations DS was developed. Mutations introduced during PCR by DNA polymerase misincorporations or arising from DNA damage will appear in only one of the two DNA strands and thus are not counted as real mutations. The other approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input.