## Results: 10

1.

2.

3.

4.

EBV miRNAs are derived from two transcripts: BHRF1 and BART (white bars). Precursor miRNAs are listed in the order of genomic location. The location of latent proteins (EBNAs and LMPs –grey boxes), other transcripts found in latency states (EBER – red box) and their promoters (black flags) are also shown. The V-snoRNA1[57] is also indicated at the end of the BART transcript.

5.

The data sets used in Figure 3 and 4 were normalized based on the fraction that each miRNA comprised of the total EBV miRNA measured in each sample. A complete description is given in materials and methods. The relative up and down regulation of miRNAs is indicated by red and green respectively. Dendrograms of clustering analysis for samples and miRNAs are displayed on the top and left respectively. Tumors, forming a distinct branch on both the sample and miRNA dendrograms, are boxed in blue.

6.

Note that with the exception of HD less than half of the cells are non-tumor infiltrating lymphocytes. A. Nasopharyngeal carcinoma stained for the EBV nuclear antigen EBNA1. B. Hodgkin's disease stained for EBNA1 Note the sparse appearance of the tumor cells compared to the other tumor types. C. Gastric carcinoma stained for EBNA1 D. Burkitt's lymphoma stained for the EBV encoded small RNAs EBER.

7.

PCA was performed on the same data set used in Figure 3A. A. The first two principal components resolve the four tumor types. For a movie demonstrating the first three principal components see Video S2. B. Subsets containing as few as 3 miRNAs resolve the tumor types. This figure plots the failure rate of different sized sets of miRNAs. For each set size, 10,000 randomly chosen subsets were tested. N.B. this represents over sampling for the subset n = 3

8.

The average and standard deviation of miRNA expression after normalization to the level of expression of the ubiquitous human small RNA RNU6b is shown. The stars indicate miRNAs that were consistently absent from HD (blue) and LCL (grey) only. A. LCL versus the B cell tumors. Blue – Hodgkin's disease; yellow – Burkitt's lymphoma; grey LCL. B. LCL versus the epithelial tumors. Green - gastric carcinoma; red - nasopharyngeal carcinoma; grey - LCL. This is the same data set for the tumor biopsies as shown in Figure 3. N.B. The miRNAs are presented on the x-axis from left to right in the order that they appear in the viral genome (see Figure 1).

9.

The data is expressed as average and standard deviation of the fraction that each miRNA constitutes of the total of all EBV miRNAs. A. The four tumor types tested. Blue – Hodgkin's disease (n = 3): yellow – Burkitt's lymphoma (n = 6): green - gastric carcinoma (n = 6): red - nasopharyngeal carcinoma (n = 6). B. Normal infected tissue. Grey – GCB (n = 5), black – MemB (n = 4). The y intercept is a conservative approximation to the sensitivity of the assay (this will vary according to the input number of infected cells). It represents the fraction for a miRNA present at 10 copies, the lower limit of sensitivity for detection of all the miRNAs [16]. N.B. The miRNAs are presented on the x-axis from left to right in the order that they appear in the viral genome (see Figure 1).

10.

The data is expressed as copy number of each miRNA per infected cell. A. Spontaneous lymphoblastoid cell lines LCL (n = 6). miRNAs that are significantly expressed in LCL but not GCB and MemB are highlighted in red. B. Normal infected tissue. Grey – GCB (n = 5), black – MemB (n = 4). This is the same data set as Figure 3A except now expressed as copy number /cell instead of fraction. The y intercept is an approximation to the sensitivity of detection based on the following. The maximum number of infected cells tested for GCB and MemB was 4–6×10

^{3}(Table 1). Approximately 0.1% of the RNA from these samples was used for each PCR reaction i.e. equivalent to ∼5 infected cells. Since we can detect as few as 10 copies of each miRNA [16], we conclude that failure to detect a miRNA in any sample indicates it is present at a copy number ≤1/cell. The red arrow indicates miRNA 17-5p which was the only miRNA to show a striking differential expression between GCB and MemB cells. N.B. The miRNAs are presented on the x-axis from left to right in the order that they appear in the viral genome (see Figure 1).