The next example of detection for the Mycoplasma pneumoniae pathogen demonstrated a case where there was only a single ProSeq for the target pathogen. A total of 48 test samples were performed using multiplex PCR (via method in Lin et al., submitted for publication) where for 46 of the samples M.pneumoniae organism was spiked into nasal wash with several other pathogens from 100 to 100 000 colony forming units per ml, the remaining 2 samples were purified with nucleic acid from culture stock at a concentration of 1000 genome copies per reaction volume. This ProSeq was also not optimal for fine discrimination because it was selected from a highly conserved region (345 bp) of the cytadhesin P1 gene. In every case taxonomic database entries for M.pneumoniae or its one recognized distinct strain tied for MaxScore (Supplementary Table 3 ). To better understand these returns, the database sequences were examined and subdivided into three groups of sequences, A, B and C, based on how well they matched the reference sequence used to make the ProSeq. The placement of the database entries into the three groups was determined from a CLUSTAL alignment of the sequences of this gene. This alignment confirmed that the database entries differed significantly more from each other in regions not represented by the ProSeq and contained sufficient variability that would have allowed finer discrimination. Members of Group A exactly matched the ProSeq and could not be distinguished between on the microarray. Similarly, members of group B matched the Pro-Seq except at the 199th position where the base called was C rather than T. Group C sequences contained a few database entries that were more variable and might be distinguished from other entries within the ProSeq. For the 48 experimental tests of M.pneumoniae, as much as 80% of the ProSeq hybridized for 19 samples, yet only 5 of these samples had an unambiguous base call at the 199th position. When it was unambiguous, it always matched group B sequences. In the cases where an N base call was made at the 199th location, both groups A and B sequences were returned with the same score. Regardless of this, the target pathogen positively identified was M.pneumoniae for every sample tested.