SRA

Quantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich for the sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification has the potential to introduce significant quantitative error to these measurements. Here we describe a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We demonstrate that this PCR-free quantification barcode sequencing approach is amenable to multiplexing through use of orthogonal restriction enzymes. Finally, we use this technique to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.