Abstract

NGS Libraries From Cell-free DNA Containing Molecular Tags Prepared with ThruPLEX® Technology Improve Ability to Detect Rare Alleles

Karl Hecker, Vice President, Rubicon Genomics Inc

Next Generation Sequencing (NGS) of cell-free DNA (cfDNA) enables non-invasive access to genetic information from liquid biopsies. Key to identifying rare genetic mutations is improved sequencing accuracy and the ability to distinguish between biological and PCR duplicates. Rubicon Genomics’ ThruPLEX Plasma-seq was developed specifically for cell-free DNA. The addition of molecular tags improves sequencing accuracy by accounting for polymerase and sequencing errors and increases confidence in rare allele identification.
ThruPLEX libraries with and without molecular tags were prepared using Horizon Discovery’s Multiplex I cfDNA Reference Standard Set and enriched with Agilent ClearSeq Comprehensive Cancer Panel. Enriched libraries were sequenced on Illumina’s NextSeq 500 System using a high-output flow cell. Six single nucleotide variants (SNV) for 4 different genes (EGFR, KRAS, NRAS, PIK3CA) were sequenced at average total read coverage of approximately 20,000 X (SD = 3125). Removal of PCR duplicates reduced read coverage to 295 X (SD = 18). The use of molecular tags allowed identification of biological duplicates, which when included in the analysis, increased read coverage by a factor of 7.1 (SD = 0.8), increasing coverage by unique reads to 2110 X (SD = 348). The number of false positives was significantly reduced by the use of molecular tags, eliminating 73% (SD = 6.1%) of background noise.

The use of molecular tags in the preparation of NGS libraries from cfDNA enhances sequencing accuracy and enables distinction between biological duplicates and PCR duplicates. The confident identification of biological duplicates significantly increases read coverage and concomitantly leads to a significant decrease in background noise, reducing false positives resulting in more confident SNV calls.