Image extraction software

Cy5 fluorescence intensity of the arrays was measured with an MS200 scanner (Roche NimbleGen, Madison, WI) at resolution 2 µm, wavelength 635 nm, gain 25%, and laser intensity 100%. Cy5 signal intensities were extracted using Image Extraction Software (Roche NimbleGen). Data pre-processing, normalization, and statistical tests were performed using the language R. Data visualization and analysis was performed with the Spotfire 6.5.0 (Tibco, Boston, MA) software platform. Distance analysis and principle component analysis of distance matrices were performed with the R package PEPLIB^{19 (link)}.

The relative fluorescent unit (RFU) signals for all the probes were extracted from the images using Roche Sequencing Solutions image extraction software. The RFU signals were converted into intensity plots after quantile normalization, background and spatial correction, and deconvolution for redundant peptides. To determine background noise, we obtained signal intensity data for each antigen from 26 control serum and 20 cerebrospinal fluids (CSF) samples found to be negative for all agents tested. A reactive epitope was identified by a a continuous set of overlapping 12-mer peptides with signal intensities above threshold in samples with known TBDs. The signal threshold was defined for each reactive epitope by calculating the mean plus 3 standard deviations of the signal intensity for the same epitope in the 26 negative control samples. An epitope was considered reactive when the signal intensity for at least two contiguous 12-mer peptides was above threshold. An individual sample was called positive when reactive with at least one specific epitope.