A method for comprehensive genomic analysis of cell-free DNA.

Parpart-Li, Sonya; Angiuoli, Samuel; Chesnick, Bryan; Galens, Kevin; Jones, Siân; Kadan, Michael J.; Kann, Lisa; Lytle, Karli; Murphy, Derek; Nesselbush, Monica; Phallen, Jillian; Riley, David R.; Shukla, Manish; Zhang, Theresa; Husain, Hatim; Velculescu, Victor E.; Díaz, Luis A.; Sausen, Mark

doi:10.1200/jco.2015.33.15_suppl.e22070

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“…A 58-gene panel ( Supplementary Table S4) interrogating 80,930 nucleotides was employed to detect sequence mutations by next-generation sequencing, using the Agilent SureSelect in-solution targeted enrichment system (Agilent; ref. 21).…”

Section: Cfdna Extraction and Mutant Analysismentioning

confidence: 99%

“…The captured DNA library was amplified and purified as described previously (20). The captured genomic library underwent 100-bp paired-end sequencing on a HiSeq 2500 instrument (Illumina) to obtain an average of 26,574Â total coverage in the targeted region of interest ( Supplementary Table S5), with variant identification performed as previously described (21,22).…”

Section: Cfdna Extraction and Mutant Analysismentioning

confidence: 99%

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The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA

Parpart-Li

Bartlett

Popoli

et al. 2017

Clinical Cancer Research

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Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor, and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied, although these may be a critical consideration for assay performance. To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum with those in plasma. Given the limitations of serum for ctDNA analyses, we evaluated the effects of two plasma processing approaches, KEDTA and Cell-Free DNA BCT (BCT) tubes, on cfDNA and ctDNA recovery. A total of 45 samples from nine patients with cancer were collected in both tube types. Once collected, blood was processed into plasma immediately or kept at room temperature and processed into plasma at 1, 3, 5, or 7 days. As early as 24 hours after collection, plasma isolated from blood collected in KEDTA tubes contained an elevated level of cfDNA that increased over time compared with BCT tubes where no significant increase in cfDNA levels was observed. When samples from an additional six patients with cancer, collected in the same manner, were stored at 4°C in KEDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes. At day 3, there was a trend toward a decrease in ctDNA levels in both tubes that was more pronounced when measuring the mutant allele fraction for cases stored at 4°C in KEDTA tubes. In summary, methods of blood processing have a strong influence on cfDNA and ctDNA levels and should be a consideration when evaluating ctDNA in peripheral circulation. .

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Section: Cfdna Extraction and Mutant Analysismentioning

confidence: 99%