Comparison of different semi-automated cfDNA extraction methods in combination with UMI-based targeted sequencing

Abstract

The analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood can serve as a minimally invasive alternative to tumor tissue biopsies in cases with impaired access to tissue. Its clinical utility has been well demonstrated for EGFR T790M testing in lung cancer patients suffering progress after tyrosine kinase inhibitor treatment. At present, highly sensitive unique molecular identifiers (UMI)-based NGS for liquid biopsy testing is less established compared to single gene assays. However, the critical bottleneck are sufficient cfDNA yields, which are essentially required to obtain meaningful test results.We compared four different cfDNA extraction methods (Qiagen, Promega, Thermo and Stratec) using the same plasma samples in order to evaluate their suitability for further NGS analysis. We managed to draw 60 ml blood from 12 patients each and equally collected 30ml in PAXgene and EDTA tubes at the same time point, sufficient for total of 96 cfDNA extractions. CfDNA concentrations and total amounts were highest for Qiagen and Promega protocols, showing the best read length profiles after sequencing.Known oncogenic driver mutations were identified in 9 out of 12 patients with at least one of the cfDNA extraction methods, again favoring the extraction protocols from Qiagen and Promega. We also uncovered putative sequencing artefacts including known driver genes pointing to a careful consideration for the limit of detection of this methodology. Our study shows that pre-analytical optimization is necessary to achieve the maximum sensitivity of UMI-based sequencing but also highlights the low abundance of tumor-derived cfDNA in lung cancer samples.

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

“…In-house analysis of numerous cfDNA samples using TapeStation has shown that the majority of cfDNA fragments are indeed within the 50–200 bp range (with the median being ~167 bp, associating cfDNA with mononucleosomes [ 41 ]), showing, again, that contamination with germline DNA is minimalized (expected range for germline DNA is >10,000 bp). This has also been shown by others [ 42 , 43 , 44 ], confirming that robust pre-analytical handling of blood samples is a critical factor in cfDNA analysis [ 19 , 36 , 45 ]. Germline DNA was extracted from 200 μL of buffy coat using the QIAamp DNA Blood Mini kit (Qiagen), according to manufacturer’s instructions.…”

A novel method for extracting circulating cell‐free DNA from whole blood samples and its utility in the non‐invasive prenatal test

“…For laboratorydeveloped tests using NGS, automation in library construction, sequencing, bioinformatics, and report management has been gradually realised. [19][20][21] However, there has been a continuous demand for automation in sample processing. Automated sample processing systems are complicated, expensive, and not suitable for application in small and medium-throughput laboratories.…”

“…There have been continual demands for automation of experimental operations in molecular laboratories, especially in clinical testing centres that use mature testing technology. For laboratory‐developed tests using NGS, automation in library construction, sequencing, bioinformatics, and report management has been gradually realised 19‐21 . However, there has been a continuous demand for automation in sample processing.…”

Circulating Tumor DNA Testing for Homology Recombination Repair Genes in Prostate Cancer: From the Lab to the Clinic

“…Besides high throughput and low hands-on time, the use of automated methods reduces variability and the risk of sample-to-sample contamination, though they generate somewhat lower cfDNA yields than manual extraction [ 71 , 72 ]. Recently, results from a comparison study on four commercially available (semi-)automated cfDNA extraction protocols have been published [ 73 ]. The same plasma samples were processed with Qiagen, Promega, Thermo, and Stratec extraction methods in order to evaluate their suitability for further NGS analysis.…”