FIG 1.

Oligonucleotide selection for 16S rRNA. (A) Alignment of 16S sequences from 8 bacterial species (Ec, E. coli; Pa, P. aeruginosa; Rp, R. parkeri; Cc, C. crescentus; Bs, B. subtilis; Ms, M. smegmatis; Mtb, M. tuberculosis; Sa, S. aureus). Alignment gaps are shown as red lines in the particular species of the gap. Regions with a gap in any species are highlighted in pink; these regions were not considered when designing oligonucleotides. (B) The position, length, and minimum T_m of all oligonucleotides plotted against the 16S alignment after the indicated number of optimization cycles (top). The information content at each nucleotide position of aligned regions is also shown (bottom, points). To highlight conserved regions, a sliding average information content is also plotted (bottom, line). (C) Oligonucleotide T_m statistics after multiple cycles of the T_m optimization algorithm. For each oligonucleotide (n = 250), we calculated the minimum T_m across the 8 species considered and then plotted the mean of this value across all oligonucleotides (black). The T_m cannot be accurately estimated for oligonucleotides with multiple sequential mismatches; the number of oligonucleotides with an undefined T_m is also plotted (blue). (D) Histograms of minimum T_m for oligonucleotides at the indicated number of optimization cycles. Data were generated as in panel C, but oligonucleotide T_m minima were used to generate histograms rather than taking the mean across all oligonucleotides. Oligonucleotides with an undefined T_m were not included in the histograms. (E) Distribution of T_m values for each 16S-targeting oligonucleotide (n = 8) for each individual species indicated. The mean T_m of oligonucleotides for each species is also shown (red lines). Note that the same oligonucleotides are used for each species, but because of 16S sequence variability, the T_m can vary, as illustrated for one particular oligonucleotide (blue).