Fig. 1

NGS technology as the backbone of ctDNA analysis. A Starting with whole blood collected in specialized cfDNA collection tubes, the plasma layer containing cfDNA is separated via centrifugation, followed by extraction of cfDNA from plasma. Typically, two vials of blood corresponding to ~17-20ml are submitted for analysis for both research studies or analysis by commercial vendors to ensure that sufficient amounts of plasma are available for extraction and harvesting of the ctDNA signal. B Simplified theoretical (Illumina) library fragment as a result of NGS library preparation. The dark green and dark blue bars represent the Illumina adapters P5 and P7, respectively, which enable hybridization to the sequencing flow cell and subsequent bridge amplification after ligation to the cfDNA fragment (gray bars). Sample-specific indexes, which are used to identify the patient sample, are typically in dual format and are shown here as i5 and i7. Additionally, unique molecular identifiers (UMI) serve as molecule-specific barcodes that enable the bioinformatics filtration of amplification or sequencing errors to ensure high-quality variant calling. C Sequencing-by-synthesis (SBS) on an Illumina instrument allows for one fluorescently-tagged nucleotide to be added to the growing read per cycle. Here, G’s, A’s, C’s and T’s are tagged with pink, blue, green and yellow fluorochromes, respectively. After the instrument has converted the captured images to base calls, the data is converted into a FASTQ file containing reads and quality scores. D Since the reads in the FASTQ file do not describe genomic location of the read, the data must first be aligned to a reference genome. This alignment is referred to as a SAM file, which has a binary counterpart called a BAM file. The BAM file contains all information in the original FASTQ file along with the mapping information of the read, i.e. the genomic coordinates to which it aligned. The BAM (alignment) file serves as the core data for diverse downstream analyses, e.g. calling of SCNAs or variants, estimation of tumor fraction from plasma, calculation of fragment size distributions, or nucleosome mapping. E Example of a clinical report summarizing the interpretation of genomic alterations detected from cfDNA. Such a clinical report describes the detected genomic alterations alongside their variant allele frequencies (VAF) and pathogenicity with potential clinical implications. Such findings should be discussed at a molecular tumor board and aligned to the patient’s clinical status