Rapid Molecular Profiling of Myeloproliferative Neoplasms Using Targeted Exon Resequencing of 86 Genes Involved in JAK-STAT Signaling and Epigenetic Regulation  Graham W. Magor, Michael R. Tallack, Nathan M. Klose, Debra Taylor, Darren Korbie, Peter Mollee, Matt Trau, Andrew C. Perkins  The Journal of Molecular Diagnostics  Volume 18, Issue 5, Pages 707-718 (September 2016) DOI: 10.1016/j.jmoldx.2016.05.006 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Read coverage metrics for the custom AmpliSeq MPN Panel. A: A box and whisker plot showing the distribution of read coverage for each amplicon. Each of the 1647 individual amplicons were placed in bins according to their normalized coverage value for each of the samples analyzed, and the number of amplicons in each bin plotted for all of the samples. Approximately nine amplicons were absent in every sample (ie, a normalized coverage value of zero), and approximately 52 amplicons were represented at <10% of their expected read coverage. Whiskers show the 25th to 75th percentiles; median value is represented by the horizontal line. B: A box and whisker plot showing the distribution of read coverage for each amplicon, as a function of GC content. Each of the 1647 individual amplicons were placed in bins according to their GC% for each of the samples analyzed, and the distribution across all of the samples plotted. Whiskers show the 25th to 75th percentiles; median value is represented by the horizontal line; black circles are the fifth and 95th percentiles. A red line is shown to indicate a median amplicon expression value of 100 (Materials and Methods). C: The normalized coverage value for each of the 1647 amplicons was calculated for each of 16 samples analyzed, and the distribution within each sample was plotted. Whiskers show the 25th to 75th percentiles; median value is represented by the black horizontal line; the red line represents the expected mean value of 100; black circles are the fifth and 95th percentiles. D: Performance of MPN Ampliseq panel (x axis) compared with a commercially available real-time quantitative PCR–based assay (y axis) for the evaluation of JAK2 V617F allele burden. Linear regression was used to determine the line of best fit as shown by the red line on the graph. Slope = 1.15, R2 = 0.996. MPN, myeloproliferative neoplasm. The Journal of Molecular Diagnostics 2016 18, 707-718DOI: (10.1016/j.jmoldx.2016.05.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Mutations identified in MPN and related disorders by targeted exon resequencing. A: The number of mutations found in each gene is shown with mutations separated into either those previously reported in the COSMIC database (blue) or those not reported in the COSMIC database (red). B: Mutations are grouped according to diagnosis. The number of mutations in genes that were mutated in more than one patient are shown and subdivided into the different diagnostic categories (ET, PV, PMF, ET-MF, PV-MF, or other). The category other incorporates patients participating in the screen with a diagnosis of CNL (three patients), CMML (two patients), MDS/MPN overlap (four patients), or additional diagnoses only represented by a single patient (Table 1). C: The histogram displays the number of patients from the study according to mutation number. All patients were found to have between 0 and 4 mutations by our method. D: Frequency of mutations according to mutational type. Mutations were classified according to the presumed effect on the protein encoded. Missense mutations are by far the most common mutations found in this screen. CMML, chronic myelomonocytic leukemia; CNL, chronic neutrophilic leukemia; COSMIC, Catalog of Somatic Mutations in Cancer; ET, essential thrombocytosis; MDS, myelodysplastic syndrome; MF, myelofibrosis; MPN, myeloproliferative neoplasm; PMF, primary myelofibrosis; PV, polycythemia. The Journal of Molecular Diagnostics 2016 18, 707-718DOI: (10.1016/j.jmoldx.2016.05.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Targeted exon resequencing reveals a complex array of patient-specific gene mutations. A: A screen capture from the IGV for a patient found to contain two different missense mutations in the MPL gene (S505N and W515L as indicated by boxes). The genomic coordinates, amino acid sequence, and genomic sequence are found at the top of the image. A subset of individual DNA sequencing reads is found beneath with reads from either strand of the DNA shown by either red or blue bars. Reads are shown for both a patient blood sample and a sample of matched germline DNA (buccal). Segments of reads differing from the normal human genome reference are indicated by either showing the new DNA base or a gap. Note that contiguous reads for this patient only show one of the individual mutations and not both (ie, G→A or G→T), suggesting that this patient is a compound heterozygote. There are no abnormal sequencing reads in this patient's buccal sample, suggesting these are also somatic mutations. B: A screen capture from IGV as shown for panel A. Blood and buccal sample reads for the gene SH2B3 are shown. There is an 11-bp frameshift deletion (L310 fs*12) found on both strands of the DNA. This same mutation is also found at very low frequency in the patient buccal sample. IGV, integrative genomics viewer; PGM, Personal Genome Machine. The Journal of Molecular Diagnostics 2016 18, 707-718DOI: (10.1016/j.jmoldx.2016.05.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions