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Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors  Jennifer L. Winters, Jaime I. Davila, Amber.

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Presentation on theme: "Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors  Jennifer L. Winters, Jaime I. Davila, Amber."— Presentation transcript:

1 Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors  Jennifer L. Winters, Jaime I. Davila, Amber M. McDonald, Asha A. Nair, Numrah Fadra, Rebecca N. Wehrs, Brittany C. Thomas, Jessica R. Balcom, Long Jin, Xianglin Wu, Jesse S. Voss, Eric W. Klee, Gavin R. Oliver, Rondell P. Graham, Jadee L. Neff, Kandelaria M. Rumilla, Umut Aypar, Benjamin R. Kipp, Robert B. Jenkins, Jin Jen, Kevin C. Halling  The Journal of Molecular Diagnostics  Volume 20, Issue 4, Pages (July 2018) DOI: /j.jmoldx Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Representation of the spanning reads supporting a fusion (in this case the novel STXBP6-BRAF fusion observed in case V7-96). Top panel: The scale in base pairs of the region. Middle panel: The reads supporting the fusion. Bottom panel: The transcripts involved (STXB6 exon 4 and BRAF exon 10) in the fusion. In this panel, the translations for the three different reading frames are shown on top of the transcript, with green squares representing start codons and red squares representing stop codons. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Postanalytical assessment of RNA integrity number (RIN). The x axis represents the distance of the fusion from the 3′ end of the mRNA with known distances for several common gene fusions being shown on the x axis (eg, the BCR-ABL fusion at 5 kb), and the y axis represents the probability of detecting a fusion at that distance. Both plots show the degree of degradation for universal human reference RNA that has been degraded to RINs of 8.6, 7.6, 5.9, 4.9, and 3.9 (red lines). The degradation plots for samples V1-35 and V (blue lines) are shown. Both samples had a RIN of 7.5 by the Agilent Bioanalyzer, but the RNA sequencing data reveal that the RNA from sample V1-35 is significantly more degraded than the RNA from sample V The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 Prevalence of fusions detected by distance from 3′ end of mRNA. The x axis shows the distance of the fusion from the 3′ of the mRNA, and the y axis shows the number of fusions identified at that distance. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 Figure 4 Gene expression heat map showing a representative example of the expression of a subset of the 571 genes assessed in tumor case V1-35 (lung adenocarcinoma). Each row shows a gene, and each column shows the number of reads at the beginning and end of each exon for that gene. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 Figure 5 Limit of quantitation. Measured coverage was plotted against the mean coverage for supporting read intervals of 0 to 4, 5 to 9, 10 to 14, and 15 to 20. The red line indicates the fusion detection threshold, which is a minimum of five supporting reads to detect a fusion. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 Supplemental Figure S1 Representative example of RT-PCR confirmation of a gene fusion detected by RNA sequencing. Two PCR primer pairs were designed to detect a JAZF1-SUZ12 gene fusion detected in an endometrial stromal sarcoma (sample V1-6). Top panel: Lane 1, no template reaction; lane 2, primer pair designed to detect a 380-bp JAZF1-SUZ12 product; lane 3, primer pair designed to detect a 499-bp JAZF1-SUZ12 product; and lane 4, 1-kb size ladder. The RT-PCR products gave the expected sizes with the two primer pairs. Bottom panel: Sanger sequence of isolated RT-PCR product. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8 Supplemental Figure S2 Diagram of the bioinformatics workflow for calling fusions in clinically relevant genes. IGV, Integrated Genome View; RSEQC, RNA sequencing quality control. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9 Supplemental Figure S3 Supporting reads consist of spanning reads for which both paired-end reads span the fusion, split reads that have paired-end reads in the different gene fusion partners, and hybrid reads that have one paired-end read spanning the fusion and the other read entirely in just one of the fusion partner genes. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

10 Supplemental Figure S4 A: Inner distance histogram. This is an example of a histogram for a case that shows the distances between the ends of two reads on the x axis and the fraction of fragments showing that size on the y axis. The red line indicates the cumulative distribution function curve for the histogram that represents mRNA fragment size. This plot is helpful for determining if there is overfragmentation or underfragmentation of the RNA used for library preparation. B: The gene body coverage plot shows the mean level of coverage across all genes normalizing the length of each gene to 100 and provides information on the degree of RNA degradation. Increasing levels of degradation as shown by RNA integrity numbers (RINs) lead to a skewing of the gene body coverage toward the 3′ end of mRNAs that are poly(A) captured. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

11 Supplemental Figure S5 Gene body coverage for intrarun (three replicates on run 1) and between-run replicates (1 replicate on run 2 compared with the three replicates on run 1). The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

12 Supplemental Figure S6 RNA degradation as assessed from RNA sequencing data. The x axis represents the distance of the fusion from the 3′ end of the mRNA (eg, the BRD4-NUT fusion at 3.5 kb), and the y axis represents the probability of detecting a fusion at that distance. The degree of degradation for universal human reference RNA that has been degraded to RNA integrity numbers (RINs) of 8.6, 7.6, 5.9, 4.9, and 3.9 is shown (red lines). The degradation plot for samples V3-65 is shown as a blue line. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

13 Supplemental Figure S7 Gene expression heat maps showing a representative example of the expression of a subset of the 571 genes (detailed view) and all 571 genes (high-level view) for a sample with an RNA integrity number (RIN) of 10 and a sample with an RIN of 3.4. Each row shows a gene and each column shows the number of reads at the beginning and end of each exon for that gene. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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