RPM Technology – How it Works

 

The TessArae RPM technology is unique in that direct nucleotide sequence is generated from pathogen-specific targets or loci present in the sample. Reference sequences or tiles, complementary to the pathogen-specific loci in the sample, are represented on the TessArray RPM microarray, with each basepair of the pathogen-specific gene sequence interrogated by eight related 25-base probes on the array. Figure 1 demonstrates how the first base of a particular pathogen-specific gene sequence in the sample is derived.

 

Four of these eight related probes are complementary to, and thus may hybridize (bind) to the sequence of one strand of the pathogen-specific gene sequence present in the sample (probes 1-4 in Figure 1). These four probes differ only by the nucleotide, either A, G, C, or T, at the center position (#13 of 25 bases) of their otherwise identical sequences. Generally only one of these four probes will be a perfect match to the pathogen-specific sequence and will have the greatest fluorescence signal of the four probes. The other three probes will have lower fluorescence signals because of poor hybridization caused by their center position mismatches. The other four probes are complementary to the other strand of the pathogen-specific sequence in the sample (probes 5-8), again differing at only the center position, to derive the sequence of the complementary strand of the pathogen-specific sequence.

 

The perfectly matching pair of oligonucleotide probes are probe 1 (bold font) to the target positive ( + ) strand and probe 7 (bold font) to the target negative ( – ) strand, because the center nucleotide of probe 1 is T, complementary to the corresponding A in the target plus ( + ) strand sequence, and the center nucleotide of probe 7 is A, complementary to the corresponding T in the target negative ( - ) strand sequence. Probes 1 and 7 will exhibit greater fluorescence signal on the array compared with the other three in each set of four probes.

 

In completely similar fashion, there are another eight related probes on the microarray that represent the pathogen-specific gene sequence from position 2 through 26, to interrogate the identity of the target basepair at position 14. These are followed by another eight probes that represent the pathogen-specific gene sequence from position 3 through 27, to interrogate the identity of the target basepair at position 15, and so forth, demonstrating the iterative strategy of microarray-based resequencing by hybridization (rSBH).

 

Fluorescent signal is read from successive sets of four related probes on the microarray, arranged as a square of four features, to determine the nucleotide identity of each base position in the pathogen-specific gene sequence hybridized on the microarray, as illustrated in Figure 2.

 

The translation key indicates the feature at each corner of the square that corresponds to the center base, either A, G, C, or T of each probe in the set. A basecalling algorithm converts the signal intensity data from the image of the hybridized microarray into nucleotide sequence by comparing the intensities at each of the four features.

 

The succession of such probe sets as squares between the blue lines is readily translated to a portion of the target gene sequence aligned below the image. On another part of the array a similar succession of probe sets interrogates the corresponding complementary strand of the same pathogen-specific gene sequence.

 

Typically, a base is called if the signal from the most intense probe/feature is greater than the sum of the other three. Empirical studies (Zwick et al. 2005) demonstrate that the accuracy of the RPM basecalling algorithm is routinely two orders of magnitude greater than conventional DNA sequencing methodology or greater than 1 x 10^-6 (less than one error per million bases called).

 

Next: RPM Protocol