Figure 2:

Workflow of plasma-Seq for monitoring treatment response in metastatic cancer patients.

After plasma DNA extraction, cfDNA is quality checked on an Agilent Bioanalyzer. An enrichment of fragments with a near mode of 166 bp indicates high quality of cfDNA. In order to select samples with sufficient fractions of tumor DNA (>5%–10%), which are suitable for a comprehensive, genomewide analysis a pre-screening using mFAST-SeqS is performed. Samples with genomewide z-scores above 5 are further analyzed with plasma-Seq. To this end a library preparation is performed using a slightly modified protocol of the Illumina TruSeq Nano Library Prep kit. One part of the library is directly subjected to low-coverage whole genome sequencing on an Illumina MiSeq or a NextSeq. The other part of the library is enriched for the most frequently mutated cancer driver genes and sequenced in a separate run. With this approach we can comprehensively monitor tumor-specific changes on the genomic, the gene and the nucleotide level. For samples with genomewide z-scores below 5, high resolution methods are needed in order to obtain informative results.

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