Fig 1.
The integrated workflow of FoundationOne®CDx and FoundationOne®RNA assay.
Both DNA and RNA were simultaneously extracted from FFPE samples or slides. RNA underwent sequencing and analysis using the FoundationOne®RNA pipeline, and it is highly recommended to include DNA sequencing by FoundationOne®CDx to generate a comprehensive clinical report.
Table 1.
The concordance analysis result of genes with actionable fusion and diagnostic fusion pairs.
Table 2.
The reproducibility and repeatability results of precision study.
Fig 2.
Precision study design and results of FoundationOne®RNA assay.
(A) The experiment design of precision study. Ten Fusions from 10 valid commercial FFPEs were processed on 3 different days, with 3 replicates per day, for a total of 9 replicates per source sample. Equal cDNA input (500 ng) was used for each replicate. All replicates (9 replicates X 10 source sample) passed quality control steps and therefore were valid for reproducibility and repeatability analysis; (B) The distribution of supporting reads of 10 fusions. The supporting reads of 9 replicates were plotted for each fusion. Dots with the same shape were processed from the same day. First five fusions have low supporting reads and last 5 have high supporting reads. The prefix’FM_RNASeq_’ was omitted in sample names for brevity.
Table 3.
The results of LoD study for five known fusions.
Fig 3.
LoD study design and results of FoundationOne®RNA assay.
(A) The experiment design of LoD study. Five known fusions from 5 fusion-positive cell lines were selected. Extracted RNA from fusion-positive cell lines were titrated to five dilution levels. The RNA from each cell line was pooled and diluted as needed. Fusion Percent input was based on 300ng cDNA synthesis reaction; (B) The boxplots of supporting reads per input RNA level for each of the five known fusion. The hit rate of each dilution level was annotated along x-axis and the hit rate of LoD was highlighted.
Fig 4.
Gene expression analysis on FoundationOne®RNA.
(A)Ten samples with 9 replicates each (x-axis and y-axis were in same identifier order) from precision study were used in this sample-to-sample distance plot. Darkest shade of blue corresponds to the minimum value (sample-sample distance calculated from Euclidean distance using log2(TPM) of all 1521 gene expression reporting genes) in the data, while the lightest shade of blue will represent the maximum value. The gradient used to represent the range of values in the distance matrix. All replicates were clustered in its sample-wide clade, which suggested remarkable gene expression reporting reproducibility that arrived the experimental design. The prefix ’FM_RNASeq_’ was omitted in row and column names for brevity; (B) The FGFR3 signature was determined by calculating the sum of expression (measured in TPM – Transcripts Per Million) of three genes: FGFR3, TP63, and WNT7B. FGFR2 and NTRK1 expression level was determined by calculating the expression (measured in TPM – Transcripts Per Million) of FGFR2 and NTRK1 gene, respectively. FGFR3 fusion positive samples (2 samples in red color with 9 replicates for each) indicated up-regulated differential gene expression levels (Wilcoxon rank sum test, fold change > 17, p < 0.001) compared to samples with other fusions (8 samples with 9 replicates for each). FGFR2 fusion positive sample (1 sample in green color with 9 replicates for each) indicated up-regulated differential gene expression level (Wilcoxon rank sum test, fold change > 16, p < 0.001) compared to samples with other fusions (9 samples with 9 replicates for each). NTRK1 fusion positive sample (1 sample in blue color with 9 replicates for each) indicated up-regulated differential gene expression level (Wilcoxon rank sum test, fold change > 83, p < 0.001) compared to the samples with other fusions (9 samples with 9 replicates for each). The prefix ’FM_RNASeq_’ was omitted in sample names for brevity.