NOTCH1 mutations are associated with high CD49d expression in Chronic Lymphocytic Leukemia: link between the NOTCH1 and the NF-kB pathways

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1 NOTCH1 mutations are associated with high CD49d expression in Chronic Lymphocytic Leukemia: link between the NOTCH1 and the NF-kB pathways Supplementary information: Supplementary Materials and Methods; Supplementary Figure Legends; Supplementary Figures: o Figure S1. Establishment of an in-vitro model of NICD-transfected CLL-like cells. o Figure S2. EDTA treatment and CLL co-culture with stromal cells induce NOTCH1 activation. o Figure S3. Up-regulation of the NF-kB pathway in NOTCH1 mutated CLL. o Figure S4. Rel A nuclear translocation is higher in NICD MEC-1 than in NICD-null transfectants and is inhibited by treatment with the NF-kB inhibitor BMS o Figure S5. NICD MEC-1 express lower levels of the NF-kB inhibitory molecule IkBα than the NICD-null transfectants. o Figure S6. NOTCH1 expression in NOTCH1-wt and NOTCH1-mut CLL following activation with EDTA. o Figure S7. The NF-kB inhibitor BMS impairs CD49d expression. Supplementary Table: o Table S1. List of CLL cases used in the study. 1

2 Supplementary Materials and Methods: Flow cytometry For CD49d detection in CLL cells, a combination of anti-cd5-fluorescein isothyocyanate (FITC) (catalog ), anti-cd49d-phycoerithrin (PE) (catalog number ), anti-cd19- allophycocyanin (APC) (catalog number ) mabs (BD Biosciences, San Jose, CA, USA) was used. A CLL case was judged CD49d + when expressed in at least 30% of the CD5 + CD19 + CLL cell population. 1 CD49d expression values were also recorded as absolute Mean Fluorescence Intensity (MFI) values in log 10 mode. The reported values were obtained after subtracting from the CD49d specific MFI value the MFI of the background fluorescence as determined by using a isotypematched control mab. Although in the vast majority of cases CLL cells were characterized by a single peak of CD49d expression, 1;2 in a subset of cases (154/1 180, 13.1%) a bimodal pattern of CD49d expression was observed. CD49d expression values, computed either as % of positive cells or absolute MFI values (listed in Table S1) were utilized for class comparisons. In the case of CLL with bimodal CD49d expression pattern, the MFI of the prevalent peak or of the positive peak were used in the various analyses. Other directly conjugated monoclonal antibodies used in this study were: CD20-PE (catalog number ), IkBα-PE (catalog number ) (BD Biosciences), Jagged1-PE (catalog number , ebiosciences, San Diego, CA). Data were acquired on a FACSCanto flow cytometer and analysed by Diva software (BD Biosciences). Data reproducibility was ensured using as instrumental set-up an application setting linked to Cytometer Setup & Tracking Beads (CS&T Beads, BD Biosciences) run according to manufacturer s instructions. Cell sorting of CLL cells was performed using a FACSAriaIII (BD Biosciences) using the above reported antibody combination. A purity >95% was obtained for all samples. NOTCH1 mutational status NOTCH1 mutations were evaluated either by amplification refractory mutation system (ARMS) PCR to detect the most common c delct NOTCH1 mutation, or by Sanger sequencing in the entire NOTCH1 PEST domain, as reported 3, or by next generation sequencing (NGS) using a MiSeq sequencer (Illumina, San Diego, CA), with a ~1000X coverage-fold in the entire exon 34 including the 3 UTR region. All samples that resulted NOTCH1 wild type following ARMS PCR or Sanger sequencing, were evaluated by NGS. In NGS, the cut-off value of 12% of mutated DNA, considered the detection limit of a conventional Sanger sequencing, was used to discriminate between clonal and subclonal mutations, as reported in other reports. 4;5 NOTCH1 mutated cases 2

3 with a mutational burden below the 12% cut-off were either re-evaluated by a second independent NGS or confirmed by allele-specific PCR. 6;7 Multispectral imaging To determine NF-κB nuclear internalization, MEC-1 transfectants ( ) or CD19 FITC (catalog number , BD Biosciences) labelled CLL cells ( ) were fixed with 2% paraformaldehyde, permeabilized using 90% methanol, labelled with anti-nf-κb p65/rela PE NOTCH1 (catalog number 9460S, Cell Signaling Technology, Leiden, The Netherlands), and counterstained with DRAQ5 DNA dye (BioStatus Limited, Loughborough, UK). Cells were run on the ImageStreamX cytometer using the INSPIRE software (Merck Millipore, Billerica, MA, USA) and images were analyzed using the IDEAS software (Merck Millipore). Brightfield, side scatter, fluorescent cell images were acquired at 40 magnification. NF-κB (RelA) nuclear localization was measured using an IDEAS software feature, the Similarity Score (SS), that defines/quantifies the similarity of the nuclear (DRAQ5) and labelled-transcription factor staining patterns (RelA PE). All events showing a positive SS were considered with high similarity between NF-κB and DRAQ5, thus indicating a nuclear localization of the transcription factor. Only viable cells were selected on the basis of morphologic features. In-vitro modulation of NOTCH1 and NF-κB signaling MEC-1 transfectants (2x10 6 cells/ml) or primary CLL cells (10x10 6 cells/ml) were treated with 10µM of the ɤ-secretase inhibitor (GSI) L-685,458 (Sigma-Aldrich, Milan, Italy) or with 5µM of the NF-κB inhibitor BMS (MedChem Express, Monmouth Junction, NJ) in a time point experiment for 1-24 hours. To trigger the NOTCH1 pathway, CLL cells (1x10 6 cells/ml) were treated with 10 mm ethylenediaminetetraacetic acid (EDTA) for 15 minutes, washed and plated in complete medium. At the indicated time points, cells were harvested for subsequent RNA extraction, flow cytometry analysis, and for multispectral imaging. In control experiments, equal volume of the appropriate solvent compound was added. CLL cell co-culture with stromal cells For co-culture experiments M210B4 stromal cells, originally obtained from the ATCC collection (Manassas, VA, USA), were seeded the day before the experiment onto 48-well plates (BD Biosciences) and left to adhere to obtain a layer of JAGGED1-expressing stromal cells. 8 After confirming the confluence of stromal layer by phase contrast microscopy, CLL cells were added onto the M210B4 cells at cell density of cells/ml. For comparison, CLL cells were also 3

4 cultured in suspension at the same cell density. At the indicated time points, CLL cells were collected by washing off the CLL cells, leaving the adherent stromal layer intact, and then used for subsequent RNA extraction and flow cytometry analysis. NICD plasmids and transfection NICD Plasmids were engineered cloning the NICD coding sequence in a pcdna3.1-nt-gfp- TOPO vector (Life Technologies, Monza, Italy), as reported. 3;7 The c.5304g>a (NICD-null) mutation was inserted with the Quikchange II XL Mutagenesis kit (Agilent Technologies, Santa Clara, CA). MEC-1 cells, obtained from the DSMZ collection (Germany) were transfected with the Amaxa Nucleofector (Lonza, Basel, Switzerland), as reported. 3;7 All cell lines were tested for mycoplasma contamination before use. Quantitative real-time PCR (QRT-PCR) RNA for QRT-PCR was extracted using the TRIZOL Reagent (ThermoFisher Scientific, Waltham, MA, USA). Transcript expression levels of genes of interest (HES1, DTX1, ITGA4, and B2M) were evaluated with the TaqMan Gene Expression assay kit (Applied Biosystems, Foster City, CA, USA); the relative amount of each gene was computed using the equation 2- Ct where Ct= (Ct target gene-ct B2M). All qrt-pcr experiments were performed on a CFX96 (BioRad, Hercules, CA, USA). Western blotting Immunoblotting was performed using the following primary antibodies: anti-total NOTCH1 (catalog number 4380, Cell Signaling Technology), anti-nicd (cleaved NOTCH1, Val1744, catalog number 2421, Cell Signaling Technology). β-actin (catalog number A-3854, Sigma- Aldrich) was used as loading control. Detection was performed with HRP-conjugated antibodies (GE Healthcare, Little Chalfont, UK) and ClarityECL (Bio-Rad) on a ChemiDoc Touch Imaging System (Bio-Rad). Image analysis and quantification was performed with ImageLab software (Bio- Rad). Gene expression profiling (GEP) data mining tool GEP was performed using the Whole Human Genome (4x44K) oligo microarray platform (Agilent Technologies), and microarray slides were analyzed with an Agilent Microarray Scanner (Agilent Technologies). The hybridization signal values for the multiple probes were obtained with the use of Agilent Feature Extraction Software (Agilent Technologies). Microarray data are available 4

5 in Gene Expression Omnibus (GEO; under accession number GSE Bioinformatics analyses were performed using GeneSpringGX 11.5 (Agilent Technologies); pre-processing and pre-filtering steps were carried out according to Agilent instruction. Gene Set Enrichment Analysis (GSEA) from the online database available at the GSEA Web site ( was used to identify set of genes showing statistically significant, concordant differences between two CLL groups. Statistical analyses All statistical analyses were performed using MedCalc software and GraphPad Prism 5. The sample size for in-vitro experiments with primary samples was chosen to ensure adequate statistical power. All data were tested for normal distribution. Normally distributed data were compared using t-tests (paired or unpaired), non parametric data using Mann Whitney test (independent) or Wilcoxon high rank test (paired analysis). All statistical tests were two-sided. Categorical data were compared by the χ 2 test. Spearman rank s correlation test was used to assess correlation. Results were considered statistically significant when p<

6 References 1. Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F et al. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 2008;111: Bulian P, Shanafelt TD, Fegan C, Zucchetto A, Cro L, Nuckel H et al. CD49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia. J.Clin.Oncol. 2014;32: Pozzo F, Bittolo T, Arruga F, Bulian P, Macor P, Tissino E et al. NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation. Leukemia 2015;30: Nadeu F, Delgado J, Royo C, Baumann T, Stankovic T, Pinyol M et al. Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia. Blood 2016;127: Rossi D, Khiabanian H, Spina V, Ciardullo C, Bruscaggin A, Fama R et al. Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia. Blood 2014;123: Bittolo T, Pozzo F, Bomben R, D'Agaro T, Bravin V, Bulian P et al. Mutations in the 3' untranslated region (3' UTR) of NOTCH1 are associated with low CD20 expression levels in chronic lymphocytic leukemia. Haematologica Pozzo F, Bittolo T, Vendramini E, Bomben R, Bulian P, Rossi FM et al. NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components. Leukemia Shimizu K, Chiba S, Kumano K, Hosoya N, Takahashi T, Kanda Y et al. Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods. J.Biol.Chem. 1999;274:

7 Supplementary Figure Legends: Figure S1. Establishment of an in-vitro model of NICD-transfected CLL-like cells. (A) NOTCH1 protein expression levels of NICD-null MEC1 and NICD MEC1 cells, as evaluated by western blotting. β-actin was used as loading control. Exogenous transfected NICD is indicated as GFP-NICD, the transmenbrane portion of NOTCH1 is indicated as NOTCH1-TM; (B) Flow cytometry analysis of NICD-null MEC-1 stained with anti-jagged1 (red histogram) or isotype control antibody (grey histogram). Figure S2. EDTA treatment and CLL co-culture with stromal cells induce NOTCH1 activation. (A) HES-1 transcript levels in untreated (UNT, open symbols), and EDTA-treated (EDTA, filled symbols) CLL cells; (B) DTX transcript levels in untreated CLL cells (UNT, open symbols) and CLL cells co-cultured for 24 hours with M2-10B4 stromal cells (filled symbols); HES-1 and DTX transcript levels are expressed as fold change relative to the UNT condition; in both panels square and diamond symbols identify NOTCH1-mut and NOTCH1-wt CLL respectively. P values refer to the Wilcoxon test. Figure S3. Up-regulation of the NF-kB pathway in NOTCH1 mutated CLL. GSEA histograms for the gene set TNFA_NFKB_DEP_UP pathway. The enrichments score (ES; y axis) reflects the degree to which a gene set is overrepresented in NOTCH1 wild type (class A) and NOTCH1 mutated (class B) at the extreme left or right of the entire ranked list. Each solid bar represents 1 gene within the gene set. Heat-map image (below) illustrates gene expression levels of the genes belonging to the pathway. Figure S4. Rel A nuclear translocation is higher in NICD MEC-1 than in NICD-null transfectants and is inhibited by treatment with the NF-kB inhibitor BMS NICD-null MEC-1 and NICD MEC-1 cells were labelled with the nuclear staining DRAQ5 and anti-nf-κb p65/rela PE and analysed through multispectral flow cytometry. (A) Examples of NF-kB cellular localization obtained in NICD MEC-1 cells. Multispectral images of brightfield (left panels), DRAQ5 staining (middle-left panels), NF-kB staining (middle-right panels) and merged images of DRAQ5 and NF-kB (right panels) showing an example of merely cytoplasmic (upper images) and nuclear localized NF-kB (lower images); (B) NF-kB/DRAQ5 similarity histograms showing the extent of NF-kB nuclear translocation in NICD-null MEC-1 (left plots) and NICD MEC-1 (right plots) after two-hour incubation with 5 µm NF-kB inhibitor BMS (lower plots) or DMSO as control (upper plots). 7

8 Figure S5. NICD MEC-1 cells express lower levels of the NF-kB inhibitory molecule IkBα than the NICD-null transfectants. Flow cytometry analysis of NICD-null MEC-1 (blue histogram) and NICD MEC-1 (pink histogram) stained with anti-ikbα-pe. Figure S6. NOTCH1 expression in NOTCH1-wt and NOTCH1-mut CLL following activation with EDTA. (A) Analysis of the NICD protein expression by western blot in CLL cell lysates from 5 NOTCH1-mut CLL. Cells were cultured for 24 hours after treatment (filled squares) or not (open squares) with 10 mm EDTA. NICD protein expression levels were calculated by densitometric analysis, using the β-actin as loading control; the p value refers to the paired Student s t test; (B) Western blot analysis of NICD and total NOTCH1 expression in CLL cells from 3 NOTCH1-wt and 3 NOTCH1-mut CLL treated as in (A). Figure S7. The NF-kB inhibitor BMS impairs CD49d expression. (A) ITGA4 transcript levels in NOTCH1-wt (diamonds) and NOTCH1-mut (squares) CLL cells cultured for 3 hours in the absence (open symbols) or presence of 5 µm BMS (filled symbols). ITGA4 levels are expressed as fold change relative to the untreated condition; (B) CD49d expression in CLL cells treated as in (A). CD49d MFI values are expressed as fold change relative to the untreated condition; p values refer to the Wilcoxon test. 8

9 Figure S1 A NICD-null MEC1 NICD MEC1 150 kda > 100 kda > < GFP-NICD < NOTCH1-TM IB: total NOTCH1 42 kda > IB: β-actin B NICD-null MEC-1 JAGGED1

10 Figure S2 A 15 p<0.001 B 2.5 p<0.005 HES-1 fold change (relative to UNT) 10 5 DTX1 fold change (relative to UNT) UNT EDTA 0.5 UNT SC

11 Figure S3 Ranked list metric Enrichment Score (ES) TNFA_NFKB_DEP_UP Class A positevely correlated Zero cross at 7001 Class B positevely correlated Rank in ordered dataset NOTCH1-wt (class A) NOTCH1-mut (class B) GCH1 TNFAIP2 NFKB2 TRAF1 CXCL2 TNFRSF9 CXCL3 NFKBIA TNIP1 TNFAIP3 NFKBIE PLK2 CD83 NFKB1 RELB

12 Figure S4 A brightfield DRAQ5 RelA merged cytoplasmic localized RelA 10 µm nuclear localized RelA 10 µm B NICD-null MEC-1 NICD MEC % % UNT Normalized frequency % % BMS similarity RelA/DRAQ5

13 Figure S5 NICD MEC-1 NICD-null MEC-1 IkBα

14 Figure S6 A NICD normalized expression (relative to UNT) NOTCH1-mut CLL p<0.01 UNT EDTA B NOTCH1-wt NOTCH1-mut EDTA kda > 120 kda > NICD Total NOTCH1 42 kda > β-actin

15 Figure S7 A 1.0 ITGA4 fold change (relative to UNT) BMS p< p< NOTCH1 wt mut 3-hour CLL culture B 1.0 CD49d MFI fold change (relative to UNT) p<0.05 BMS p< NOTCH1 wt mut 3-hour CLL culture

16 Table S1. List of CLL cases used in the study. case %CD49d a MFI CD49d b mutational NOTCH1 status NOTCH1 mutation mutation type Assay IGHV FISH d trisomy 12 burden c wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS ND del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del17 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del17 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no

17 wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA Sanger, NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS ND del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM normal no wt 0 NA NGS ND normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA Sanger, NGS UM normal no wt 0 NA ARMS-PCR, NGS M del17 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del11 no

18 wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA Sanger, NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA NGS UM del11 no

19 wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS ND del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del17 no wt 0 NA Sanger, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no

20 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS ND normal no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA Sanger, NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no 175 e 2 7 wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no

21 wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS ND normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del11 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del11 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del11 no wt 0 NA NGS ND del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del17 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del17 no wt 0 NA NGS UM normal no

22 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M tri12 yes wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS ND normal no wt 0 NA NGS M del17 no wt 0 NA NGS M del17 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M normal no wt 0 NA Sanger, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del17 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no

23 wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA NGS M del17 no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS ND del17 no wt 0 NA NGS M del17 no wt 0 NA NGS UM del17 yes wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS ND del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no

24 wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS ND del17 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS ND del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del17 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no

25 wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA NGS M del17 yes wt 0 NA NGS UM del13 no wt 0 NA NGS UM del17 no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS ND del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no 372 e 5 22 wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no

26 wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA Sanger, NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del17 no wt 0 NA NGS M tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del11 no

27 wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS ND normal no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS UM normal no wt 0 NA NGS M del17 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA Sanger, NGS M del13 no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del17 no wt 0 NA NGS ND normal no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA Sanger, NGS M del13 no wt 0 NA NGS M normal no

28 wt 0 NA NGS UM del11 no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA Sanger, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del11 no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del11 no

29 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS UM del17 no /1084 wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no /1820 wt 0 NA ARMS-PCR, NGS M del13 no /1341 wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no /1373 wt 0 NA NGS M del13 no /1883 wt 0 NA ARMS-PCR, NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no

30 /1462 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M del13 no /816 wt 0 NA NGS M del13 no wt 0 NA NGS M del17 no /1273 wt 0 NA NGS UM del17 no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no /1245 wt 0 NA NGS M normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS ND normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M del17 no wt 0 NA NGS UM del17 no /2711 wt 0 NA NGS M del13 no /1230 wt 0 NA NGS ND normal no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no /841 wt 0 NA NGS M del11 no /1154 wt 0 NA NGS M tri12 yes wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA NGS M del13 no /925 wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del11 no /1398 wt 0 NA NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA ARMS-PCR, NGS M del17 yes wt 0 NA NGS M normal no wt 0 NA NGS M normal no /1529 wt 0 NA ARMS-PCR, NGS ND del13 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del11 no

31 wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS ND del13 no /1758 wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA Sanger, NGS M del13 no /1041 wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS UM del13 no /1004 wt 0 NA NGS M normal no /1278 wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M del11 no /1586 wt 0 NA NGS M del13 no /1114 wt 0 NA NGS M normal no /1366 wt 0 NA NGS M del17 no /1808 wt 0 NA NGS ND tri12 yes /1248 wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA Sanger, NGS UM del11 no wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA NGS M del17 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no /1663 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA NGS M del13 no /888 wt 0 NA ARMS-PCR, NGS M normal no /1254 wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M del13 no /1008 wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no /1082 wt 0 NA NGS UM del13 no /1708 wt 0 NA ARMS-PCR, NGS M normal no /890 wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA Sanger, NGS UM del17 yes wt 0 NA ARMS-PCR, NGS M normal no /990 wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no

32 wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS ND normal no /888 wt 0 NA NGS M del13 no wt 0 NA NGS M normal no /865 wt 0 NA NGS M del13 no /1152 wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M del13 no /1740 wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no /846 wt 0 NA NGS M del13 no /1437 wt 0 NA NGS M normal no wt 0 NA NGS M del13 no /1771 wt 0 NA NGS M del13 no wt 0 NA NGS ND del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no /1258 wt 0 NA NGS M tri12 yes /1798 wt 0 NA NGS M del17 no /1155 wt 0 NA NGS UM del11 no wt 0 NA NGS M del11 no /1149 wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del17 no wt 0 NA ARMS-PCR, NGS ND del17 no wt 0 NA Sanger, NGS M del11 no wt 0 NA NGS M normal no /535 wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no /818 wt 0 NA NGS UM del17 yes /1152 wt 0 NA NGS ND normal no /1228 wt 0 NA NGS M normal no /1177 wt 0 NA NGS UM del11 no /1290 wt 0 NA NGS UM del17 no /1410 wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS M normal no

33 wt 0 NA NGS M del13 no /1259 wt 0 NA NGS UM normal no wt 0 NA NGS ND normal no /3847 wt 0 NA NGS UM tri12 yes /1476 wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no /1032 wt 0 NA NGS M normal no wt 0 NA NGS UM del17 no /2561 wt 0 NA NGS ND tri12 yes /805 wt 0 NA NGS M del13 no /1569 wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS M normal no /1448 wt 0 NA NGS UM del13 no /1230 wt 0 NA NGS M tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM del13 no wt 0 NA NGS UM normal no /1256 wt 0 NA ARMS-PCR, NGS M normal no /1049 wt 0 NA NGS M del13 no /1558 wt 0 NA ARMS-PCR, NGS M normal no /1095 wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del11 no wt 0 NA NGS UM del17 no wt 0 NA NGS UM del13 no /1705 wt 0 NA NGS M tri12 yes /1708 wt 0 NA NGS M normal no wt 0 NA NGS ND del11 no wt 0 NA NGS UM del13 no /1198 wt 0 NA NGS M del13 no wt 0 NA NGS M normal no /1798 wt 0 NA NGS M normal no wt 0 NA NGS M tri12 yes /967 wt 0 NA NGS M del13 no /878 wt 0 NA NGS M del13 no /1487 wt 0 NA NGS M del13 no

34 /2423 wt 0 NA NGS M tri12 yes /1556 wt 0 NA NGS UM tri12 yes /1069 wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no /2364 wt 0 NA NGS M del13 no /1157 wt 0 NA NGS M del17 no wt 0 NA NGS M normal no wt 0 NA NGS UM del13 no wt 0 NA NGS UM del11 yes wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no /1784 wt 0 NA NGS M tri12 yes /1766 wt 0 NA ARMS-PCR, NGS UM del13 no 694 e wt 0 NA NGS UM del17 no /1851 wt 0 NA NGS M del17 no wt 0 NA NGS UM normal no wt 0 NA NGS M normal no /752 wt 0 NA NGS M normal no /1764 wt 0 NA NGS UM normal no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M del13 no /1042 wt 0 NA NGS M tri12 yes /1610 wt 0 NA ARMS-PCR, NGS M normal no /2808 wt 0 NA NGS UM normal no wt 0 NA NGS M del17 no wt 0 NA NGS UM tri12 yes wt 0 NA ARMS-PCR, NGS M del13 no /2211 wt 0 NA ARMS-PCR, NGS M normal no /1132 wt 0 NA NGS M normal no /1238 wt 0 NA NGS M del11 no /1666 wt 0 NA NGS M normal no /1865 wt 0 NA NGS M del13 no wt 0 NA NGS M normal no /1229 wt 0 NA NGS M del13 no /1282 wt 0 NA NGS M normal no /2537 wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no wt 0 NA NGS UM del13 no

35 wt 0 NA NGS UM del13 no /1573 wt 0 NA NGS M del13 no wt 0 NA NGS UM del17 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del13 no /855 wt 0 NA NGS UM normal no wt 0 NA NGS M del17 no /1465 wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no /2107 wt 0 NA ARMS-PCR, NGS ND tri12 yes wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del13 no /1230 wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no 738 f wt 0 NA NGS UM normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del11 no /2115 wt 0 NA NGS UM del13 no wt 0 NA NGS UM del11 yes wt 0 NA NGS UM del11 yes wt 0 NA NGS M del13 no /1771 wt 0 NA NGS M del13 no /966 wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no /1224 wt 0 NA NGS M del17 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no

36 wt 0 NA NGS M del11 no wt 0 NA NGS UM tri12 yes /1264 wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS UM del11 no wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS UM del17 yes wt 0 NA NGS UM del11 no wt 0 NA NGS UM del13 no wt 0 NA NGS UM normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del11 no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del17 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM del17 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA Sanger, NGS UM del11 no wt 0 NA NGS UM del11 no wt 0 NA NGS ND normal no wt 0 NA NGS UM del17 no wt 0 NA ARMS-PCR, NGS UM normal no 787 f wt 0 NA NGS UM normal no wt 0 NA NGS M tri12 yes wt 0 NA ARMS-PCR, NGS UM del11 no wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS UM del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M tri12 yes

37 wt 0 NA NGS UM tri12 yes wt 0 NA ARMS-PCR, NGS UM tri12 yes wt 0 NA ARMS-PCR, NGS UM tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM del11 no wt 0 NA NGS M del11 no wt 0 NA NGS M normal no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no 804 e wt 0 NA NGS UM normal no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS ND normal no 808 f wt 0 NA NGS M tri12 yes wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del11 no 813 f wt 0 NA NGS UM normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM del11 no wt 0 NA NGS UM tri12 yes wt 0 NA ARMS-PCR, NGS M del13 no wt 0 NA NGS M del11 no wt 0 NA NGS M normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS UM del17 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA NGS M normal no 827 e wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM del13 no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M del13 no

38 832 f wt 0 NA NGS M del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del17 no wt 0 NA NGS M normal no 836 f wt 0 NA NGS M normal no wt 0 NA NGS UM tri12 yes 838 f wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del11 yes wt 0 NA NGS M del13 no 847 f wt 0 NA ARMS-PCR, NGS UM del11 yes wt 0 NA NGS UM normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM normal no wt 0 NA NGS M del17 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS UM del13 no wt 0 NA NGS M normal no 857 f wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M normal no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M del17 yes wt 0 NA ARMS-PCR, NGS M tri12 yes wt 0 NA Sanger, NGS UM del11 no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS M normal no 868 f wt 0 NA NGS UM tri12 yes

39 wt 0 NA NGS UM normal no wt 0 NA NGS UM del11 no 871 f wt 0 NA NGS UM normal no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS UM normal no wt 0 NA NGS M del13 no wt 0 NA NGS UM normal no wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M del11 no wt 0 NA NGS M del13 no wt 0 NA NGS M del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS M normal no wt 0 NA ARMS-PCR, NGS ND normal no wt 0 NA ARMS-PCR, NGS UM tri12 yes wt 0 NA ARMS-PCR, NGS M tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS UM tri12 yes wt 0 NA NGS UM del13 no wt 0 NA ARMS-PCR, NGS M tri12 yes wt 0 NA NGS UM normal no wt 0 NA ARMS-PCR, NGS M del17 no 897 f wt 0 NA NGS M del13 no wt 0 NA ARMS-PCR, NGS UM tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS UM tri12 yes wt 0 NA NGS M del13 no wt 0 NA NGS M normal no wt 0 NA NGS UM normal no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM del17 no

40 wt 0 NA NGS M tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS M normal no wt 0 NA NGS M del13 no wt 0 NA NGS M tri12 yes wt 0 NA NGS UM tri12 yes wt 0 NA NGS M normal no mut 1 c.7541_7542delct NGS M normal no mut ND c.7541_7542delct ARMS-PCR M normal no mut ND c.7541_7542delct ARMS-PCR UM del11 no mut 1 c.7541_7542delct NGS UM del13 no mut 2 other NGS UM del11 no mut 2 c.7541_7542delct NGS M del13 no mut 3 other NGS M normal no mut 3 c.7541_7542delct NGS UM del11 no mut 5 c.7541_7542delct NGS UM del13 no mut 8 c.7541_7542delct NGS M del13 no mut 23 c.7541_7542delct NGS UM del13 no mut 39 c.7541_7542delct NGS UM normal no mut ND c.7541_7542delct ARMS-PCR M normal no mut 1 c.7541_7542delct NGS M normal no mut 2 c.7541_7542delct NGS M normal no mut 2 c.7541_7542delct NGS M del17 no mut 1 c.7541_7542delct NGS UM normal no mut ND c.7541_7542delct ARMS-PCR UM del13 no mut 2 c.7541_7542delct NGS UM normal no mut ND c.7541_7542delct ARMS-PCR M normal no mut 3 c.7541_7542delct NGS M normal no mut 2 other NGS UM del11 no mut 4 3'UTR NGS UM del11 no mut ND c.7541_7542delct ARMS-PCR M del13 no mut 21 c.7541_7542delct, other NGS UM del11 no mut ND c.7541_7542delct ARMS-PCR M del13 no mut ND c.7541_7542delct ARMS-PCR M del13 no mut 8 c.7541_7542delct NGS M normal no mut 31 3'UTR NGS M normal no mut ND c.7541_7542delct ARMS-PCR M normal no