Reliability of liquid biopsy analysis: an inter-laboratory comparison of circulating tumor DNA extraction and sequencing with different platforms.
Author(s): Koessler T, Paradiso V, Piscuoglio S, Nienhold R, Ho L, Christinat Y, Terracciano LM, Cathomas G, Wicki A, McKee TA, Nouspikel T
Publication: Lab Invest, 2020, Vol. , Page
PubMed ID: 32616816 PubMed Review Paper? No
Purpose of Paper
The purpose of this study was to compare the yield, fragment size, and suitability for next-generation sequencing (NGS) of cell-free DNA (cfDNA) extracted from the blood of one healthy donor at different laboratories using different methods.
Conclusion of Paper
The cfDNA yield was mostly comparable between the kits tested but the resultant cfDNA concentrations differed among the kits as the input and elution volumes varied. Electropherograms showed a clear cfDNA peak at 170 bp and no detectable high molecular weight DNA, regardless of extraction method. Further, all cfDNA extracts demonstrated ratio of the 305 bp to 41 bp amplicons in the Kappa assay of 0.10-0.25, indicating high quality cfDNA and the absence of cellular contamination. The authors report all specimens were suitable for sequencing with good coverage and alignment quality.
Studies
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Study Purpose
The purpose of this study was to compare the yield, fragment size, and suitability for NGS of cfDNA extracted from the blood of one healthy donor at different laboratories using different methods. Blood from one healthy donor was collected into multiple Streck BCT tubes, stored at room temperature, and then mailed next-day to four different laboratories. After arrival (approximately 24 h after collection), plasma was obtained by centrifugation at 1600 x g for 10 min followed by centrifugation at 16,000 x g for 10 min. DNA was extracted from plasma using the MagMAX Cell-Free DNA Isolation Kit, QIAamp Circulating Nucleic Acid Kit with QIAvac 24 Plus, Avenio cfDNA Isolation Kit, MinElute, Cobas cfDNA SP Kit, and QiaSymphony robot with DSP Circulating DNA Kit. DNA yield was evaluated by Qubit High-Sensitivity Kit and the size profile was investigated using a TapeStation or bioanalyzer. DNA quality and integrity were further evaluated using the 305 bp/41 bp ratio in Kapa hgDNA Quantification and QC Kit. NGS libraries were constructed using the Oncomine Lung cfDNA assay and sequenced on an IonS5XL and using the Avenio ctDNA expanded and QIAseq human lung cancer panel kits and sequenced on a NextSeq 500 sequencer.
Summary of Findings:
The cfDNA yield was mostly comparable between the kits tested but the concentrations differed with much more concentrated cfDNA obtained using the MagMax Kit than any of the others (1283 ng/µL versus 93-333 ng/µL). The difference in cfDNA concentration was attributed to the higher plasma volume (8 mL versus 2.2-4 mL) and lower elution volume (18 µL versus 30-90 µL) used for the MagMax Kit. Electropherograms showed a clear cfDNA peak at 170 bp and no detectable high molecular weight DNA, regardless of extraction method. Further, all cfDNA extracts demonstrated ratio of the 305 bp to 41 bp amplicons in the Kappa assay of 0.10-0.25, indicating high quality cfDNA and the absence of cellular contamination. The authors report all specimens were suitable for sequencing with good coverage and alignment quality.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Real-time qPCR DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp Circulating Nucleic Acid Kit with QIAvac 24 Plus
Avenio cfDNA Isolation Kit
QiaSymphony robot with DSP Circulating DNA Kit
MagMAX Cell-Free DNA Isolation Kit
MinElute
Cobas cfDNA SP kit