Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits

Abstract

Ultrasensitive methods for rare allele detection are critical to leverage the full potential offered by liquid biopsies. Here, we describe a novel molecular barcoding method for the precise detection and quantification of circulating tumor DNA (ctDNA). The major benefits of our design include straightforward and cost-effective production of barcoded adapters to tag individual DNA molecules before PCR and sequencing, and better control over cross-contamination between experiments. We validated our approach in a cohort of 24 patients with a broad spectrum of cancer diagnoses by targeting and quantifying single-nucleotide variants (SNVs), indels and genomic rearrangements in plasma samples. By using personalized panels targeting a priori known mutations, we demonstrate comprehensive error-suppression capabilities for SNVs and detection thresholds for ctDNA below 0.1%. We also show that our semi-degenerate barcoded adapters hold promise for noninvasive genotyping in the absence of tumor biopsies and monitoring of minimal residual disease in longitudinal plasma samples. The benefits demonstrated here include broad applicability, flexibility, affordability and reproducibility in the research and clinical settings.

A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

“…Using results from the ProDuSe-aligned reads [14] we compared the allelic fractions of 19 genome-sequence derived somatic single-nucleotide variants (SNVs) across four cases with matched blood-direct and plasma ctDNA data. Of the 19 SNVs, 15 were found in the plasma ctDNA and 16 were found in the blood-direct using a cutoff of 0.5 allelic ratio.…”

“…[14]. The ligation products were purified using Aline magnetic beads with a 0.8:1 ratio of beads to sample, and amplified in an eight-cycle PCR reaction with Phusion Hot Start High Fidelity DNA Polymerase (Fisher Scientific Ltd, Canada) and dual index primers [14]. Library PCR products were purified using beads with a 0.8:1 ratio and analyzed using Quant-iT and caliper high sensitivity DNA assays.…”

“…The repaired DNA fragments were subsequently adenylated, then ligated with barcoded adapters in an overnight reaction at 16C as described by Alcaide etal . [14]. The ligation products were purified using Aline magnetic beads with a 0.8:1 ratio of beads to sample, and amplified in an eight-cycle PCR reaction with Phusion Hot Start High Fidelity DNA Polymerase (Fisher Scientific Ltd, Canada) and dual index primers [14].…”

“…The hybridization capture reaction was carried out according to the protocol of Alcaide etal . using xGen Universal Blocker-TS Mix (Integrated DNA Technologies, USA) as the blocking reagent [14]. The captured products were amplified with 13 cycles of PCR, and bead purified with a 1:1 volume ratio of capture DNA to beads.…”

“…Sequenced reads were aligned and analyzed with ProDuSe 0.2.2 to create BAM files and putative variant calls [14]. …”