Although PCR is a cost-effective way to enrich for genomic regions of interest for DNA sequencing, amplifying regions with extreme GC-content and long stretches of short tandem repeat (STR) sequences is often problematic and prone to sequence artifacts. This is especially true when developing multiplexed PCR assays for clinical applications such as carrier screening for multiple genes. The additional challenge is that all PCR primer pairs must be carefully selected to be compatible based on amplicon size and PCR conditions. Due to these experimental design constraints, a single tube with a high number of multiplexed PCR amplicons is difficult to achieve. We have developed an amplification-free enrichment method harnessing the CRISPR-Cas9 system to target hard-to-amplify genomic regions, circumventing potential PCR artifacts for PacBio highly accurate long-read (HiFi) sequencing using this No-Amp Targeted Sequencing approach. The method utilizes a pair of guide RNAs to target and excise each region of interest. This allows simple experimental design for multiplexing targets with variable GC-content and length. In addition, since the method is amplification-free, the DNA base modifications are also preserved. In this study, we show successful enrichment and sequencing of a Thalassemia-a/ß panel for HBA/HBB gene variation detection as well as an Ataxia panel targeting the disease-causing repeat expansion regions in 15 Ataxia genes. In addition to multiplexing several targets, we have also multiplexed at least 20 samples in one experiment making the No-Amp Targeted Sequencing method a cost-effective option. By removing the need for employing multiple assays, our results show the versatility of the method and the potential for using it for various genetic disease carrier screening applications in the future.
February 5, 2021 | Video Poster