Validation of Next Generation Sequencing.

<p>A) The absolute number of samples with a mutation in various genes as denoted on the x-axis that were used for the validation of NGS by means of the Ion Torrent platform. All samples are colour coded: dark blue are the concordant samples with the same mutation in standard versus NGS, the intermediate blue are the concordant samples showing no mutation, the light blue bar represents the discordant samples B) The same data as depicted in Fig 5A, however represented as percentages of all tested samples for a given gene.</p

Next Generation Sequencing workflow for routine diagnostics.

<p>A) Number of samples included in this study. B) Ion Torrent NGS workflow analysis by which routine diagnostics from tissue arrival to reporting results to the clinician is performed within 5 working days. First, tumour percentage is determined in control sections, and subsequently tumour tissue is macro dissected, DNA is isolated and NGS is performed. If insufficient DNA is isolated and no further tissue is available, conventional techniques like Sanger sequencing is performed. Somatic variants are identified by the Torrent Variant Caller supplemented with variant annotation and filtering. Variants are manually checked in IGV and discussed in a multidisciplinary meeting with necessary clinicians. Finally, results are reported to the responsible clinician.</p

Run and library statistics.

<p>A) Boxplot of run statistics of FFPE (green) and FF (orange) samples for 4 variables: 1. the percentage of ISP (Ion Sphere Particle) density (the addressable wells on the chip which have detectable loading); 2. usable reads of the total number of reads (percentage of ISPs that pass the polyclonal, low quality, and primer dimer filters); 3. polyclonals, ISPs that contain more than one template sequence per ISP and 4. low quality, ISPs with a low or unrecognizable signal. The upper and lower “hinges” of the boxplots correspond to the first and third quartiles (the 25^th and 75^th percentiles). The upper “whisker” extends from the hinge to the highest value that is within 1.5*IQR of the line, where IQR is the inter-quartile range (the distance between the first and third quartiles). The lower “whisker” extends from the hinge to the lowest value within 1.5*IQR of the hinge. Data beyond the end of the vertical lines are outliers and plotted as points. B) Library statistics of FFPE (green) and FF (orange) samples; the mean target base read depth (including non-covered target bases); the number of reads mapped to the full reference genome; and the percentage of mapped reads which are aligned to the target region. Significant differences calculated by means of an independent t-test between FFPE and FF samples are depicted with ** p = 0.002 or ***p = 0.0009).</p

Variant distribution of the complete dataset.

<p>A) Heatmap of number of variants per tumour group. On the y-axis the different primary tumour site is depicted and on the x-axis all genes with mutational data are depicted. The relative number of mutations is defined as the number of mutations normalized per number of samples in the tumour group. B) co-occurrence of different variants in colorectal tumours. The size of the circle around a gene is indicative of the number of times a variant is identified in the gene. The lines represent co-occurrences between genes where the line thickness indicates the number of co-occurrences. The colour of the circles indicates the function of the gene: green–tumour suppressor genes and oncogenes, purple–receptor tyrosine kinases, pink–PI3K pathway, yellow–KRAS/BRAF pathway.</p

Correlation between tumour percentage and allele frequency.

<p>The observed allele frequency for all variants detected using NGS is plotted against the tumour cell percentage as determined by a pathologist. The green line depicts the theoretical line of expected allele frequency of a heterozygous (somatic) mutation versus tumour cell percentage. A forced linear regression line (black line) was plotted to determine whether increased tumour percentage affects the mean allele frequency detected with a correlation coefficient of 0.041.</p

Theoretical model of the detection sensitivity for different variant detection limits.

<p>Lines depict the coverage needed for a certain tumour percentage. In this study a detection limit of 20 variants was used, which, combined with a tumour percentage of at least 10%, leads to a needed coverage of 800x.</p

Concordance between conventional techniques and Ion Torrent based NGS analysis.

<p>Concordance between conventional techniques and Ion Torrent based NGS analysis.</p