Cell free DNA extraction from plasma and targeted sequencing was performed using the AVENIO ctDNA Expanded Kit (Roche Diagnostics), a panel of 77-gene cancer-related biomarkers. Extracted DNA was quantified using fluorimetry (Qubit, Thermo-Fisher Scientific) and stored at −20°C prior to use. Plasma cfDNA was sequenced using the Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) method for quantifying ctDNA as previously described.12 (link) The method combines optimized library preparations with a multi-phase bioinformatics informatics approach to design cancer specific DNA “selectors,” that target recurrently mutated regions in the cancer of interest. Sequencing was performed on libraries generated from approximately 10 ng total cfDNA. AVENIO ctDNA Analysis Software version 2.0.0 was used to align, call, and filter variants against the hg38 human reference genome. Nonsynonymous, non-germline mutations, known to be mutated in cancer were considered significant. Buffy coat DNA was isolated and sequenced using AVENIO Tumor Tissue Expanded Kit (Roche Diagnostics) that contains the same 77-gene panel, and genes associated with clonal hematopoiesis were removed: ASXL1, PPM1D, DNMT3A, TET2, GNB1, CBL, JAK2, STAT2, MYD88, SF3B1. Both filtered and raw data were obtained.
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Targeted Sequencing of Circulating Tumor DNA
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Corresponding Organization :
Other organizations : University of Melbourne, Royal Melbourne Hospital, Peter MacCallum Cancer Centre, Hebrew University of Jerusalem
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Variable analysis
independent variables
- Cell free DNA extraction from plasma
- Targeted sequencing using the AVENIO ctDNA Expanded Kit (Roche Diagnostics)
- Quantification of extracted DNA using fluorimetry (Qubit, Thermo-Fisher Scientific)
- Sequencing of plasma cfDNA using the Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) method
- Alignment, variant calling, and filtering using AVENIO ctDNA Analysis Software version 2.0.0
- Identification of nonsynonymous, non-germline mutations known to be mutated in cancer
- Buffy coat DNA isolated and sequenced using AVENIO Tumor Tissue Expanded Kit (Roche Diagnostics) with the same 77-gene panel
- Removal of genes associated with clonal hematopoiesis: ASXL1, PPM1D, DNMT3A, TET2, GNB1, CBL, JAK2, STAT2, MYD88, SF3B1
- Buffy coat DNA sequencing using the same 77-gene panel as a control
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