Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer

Abstract

Summary:The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

“…Since interaction effects between the method for cfDNA extraction and the method applied for mutation detection may lead to increased artifacts, careful comparison during establishing liquid biopsy procedures is warranted . Different abilities to recover longer or shorter fragments have been described in the literature . Besides biological and pathological parameters reviewed above in chapter 1, the concentration of isolated cfDNA is influenced by the efficiency of the DNA extraction method and the final elution volume of the respective method.…”

“…78,83 Different abilities to recover longer or shorter fragments have been described in the literature. 84,85 Besides biological and pathological parameters reviewed above in chapter 1, the concentration of isolated cfDNA is influenced by the efficiency of the DNA extraction method and the final elution volume of the respective method. In a comparative study, cfDNA concentrations from identical artificially spiked plasma samples extracted at 56 different laboratories revealed wide variations in cfDNA yields ranging from 2.87 to 224.02 ng/mL with a median of 23.15 ng/mL of plasma.…”

Targeted Sequencing of Circulating Tumor DNA to Monitor Genetic Variants and Therapeutic Response in Metastatic Colorectal Cancer

“…CtDNA can be detected by PCR-based methods that are highly sensitive, such as droplet digital PCR (ddPCR). However, the number of detected mutations is limited for this method, and therefore, it may not always be able to detect mutations of interest (10,(12)(13)(14). Using broader next-generation sequencing (NGS) sequencing panels may overcome these limitations.…”

Optimization of DNA extraction from human urinary samples for mycobiome community profiling

“…Because naturally occurring carriers, such as salmon sperm DNA, contain rDNA sequences with partial homology to other eukaryotic DNA, we chose a synthetic carrier, polyadenylic acid, which has shown efficacy in enhancing recovery of low abundance DNA from human biological samples. [43] Supplementation of carrier DNA increased both overall (data not shown)and fungal-specific DNA yields 2-4 fold across all samples (Figure 1C). The best combination of all techniques tested resulted in an almost 14-fold increase in fungal DNA yields, comprising an optimal protocol utilizing low-speed centrifugation, enzymatic, mechanical, and thermal cell wall disruption, inclusion of carrier DNA, and proteinase K digestion in combination.…”