Fig. 1

Schematic representation of the WGS laboratory and bioinformatics flow. Short-read WGS protocols can in general be divided into four separate steps: 1. Sample preparation, 2. Library preparation, 3. Cluster generation, and 4. Sequencing. Panel 1, WGS is routinely performed with DNA from EDTA or citrate stabilized whole blood or surgically removed or biopsy tissue. DNA is isolated by conventional methods, but to facilitate CNV detection high molecular DNA is preferred. Historically, WGS required a DNA amplification step, but with newer protocols this step is no longer needed. Omission of the amplification step eliminates the PCR-bias and provides a more uniform coverage and quality [12]. The library is generated by fragmenting the high molecular DNA followed by ligation of adapters that will bind to the linker DNA on the chip surface. Moreover, barcodes allowing pooling of samples from different patients on the same chip may be attached. Panel 2, The libraries are subsequently loaded onto a flow cell and placed on the sequencer, after which the individual DNA fragments are clonally amplified by a polymerase, generating small single-stranded clusters of the particular fragments. The sequencing is in principle a conventional Sanger sequencing [5], where elongation is initiated by the addition of a sequence primer and polymerase and the nucleotide sequence is determined by the incorporation of complementary fluorescent-tagged nucleotide terminators. The fluorescent signal from the incorporated terminators is detected by scanning the chip and the individual clusters with a high-resolution confocal fluorescence laser detector after every round of nucleotide incorporation. Panel 3, Data are compiled in a fastq file that is being transferred to the high performance computer (HPC). In the HPC the reads are mapped and compiled in a .BAM file before variants are called listed in a .VCF file. Panel 4, The VCF is finally uploaded to the interpreters in the genomic laboratory for filtration, annotation and prioritization