ABSTRACT
Current next-generation sequencing techniques suffer from inefficient target enrichment and frequent errors. To address these issues, we have developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion. By designing all fragments to similar lengths, regions of interest can be size-selected prior to library preparation, increasing hybridization capture efficiency. Additionally, homogenous length fragments reduce PCR bias and maximize read usability. We combine this novel target enrichment approach with ultra-accurate Duplex Sequencing. The result, termed CRISPR-DS, is a robust targeted sequencing technique that overcomes the inherent challenges of small target enrichment and enables the detection of ultra-low frequency mutations with small DNA inputs.
ABBREVIATIONS
- DS
- Duplex Sequencing
- DCS
- Double-stranded consensus sequence
- SSCS
- Single-stranded consensus sequence
- gRNA
- Guide RNA
- crRNA
- CRISPR RNA
- tracrRNA
- Trans-activating crRNA
- NGS
- Next-generation Sequencing
- ng
- Nanogram
- bp
- Basepair
- ssDNA
- Single-stranded DNA
- dsDNA
- Double-stranded DNA
- DIN
- DNA integrity number