A Multiplexed Amplicon Sequencing Technology for FFPE and Circulating, Cell-free DNA

Detection of somatic mutations is challenging since percent tumor content in clinical samples is variable and is compounded by tumor heterogeneity. Additionally, circulating cfDNA and FFPE samples are typically limited in quantity, and FFPE samples can also be highly damaged. To address these challenges, we developed a single-tube, multiplexed amplicon sequencing method that employs hundreds of primer pairs for amplification of target loci, producing ready-to-run libraries for Illumina® sequencing. The two-step method- multiplexed PCR followed by a 10 minute adapter ligation- results in amplicons 120-160 bp in length, enabling amplification and variant calling from cfDNA-sized DNA fragments or damaged FFPE DNA. Using this technology, an oncology panel was developed to target known, clinically relevant mutations in 56 genes. For example, EGFR resistance was targeted to enable liquid biopsy monitoring in cfDNA samples. The panel design encompasses single exons (e.g. BRAF) as well as comprehensive coding exon coverage of entire genes (e.g. TP53), depending on the allele distribution across each target gene. To validate this panel, a cohort of control and clinical samples with pre-validated genotypes was tested using 10 ng of input DNA. Amplicon libraries were quantified by qPCR and sequenced on a MiSeq. Alignment and variant calling were performed using validated, publicly available tools and confirmed manually. Robust detection of 5% mutant frequency was observed for samples, and in performing spike-in experiments, the limit of detection was as low as 1% mutant frequency. The percent on-target bases and coverage uniformity were both >95%, where uniformity is defined as the percent bases covered at >20% of the mean coverage. These results indicate that this multiplexed amplicon panel is an excellent tool to assess multiple oncogenes in limiting clinical samples, enabling high throughput, cost effective NGS analysis.