Fig 1.
Hybridization experiment scheme with tiling microarray.
The DNA target region is represented by (A) two gene copies, (B) one copy or (C) no gene—one copy (X chromosome) combined with deletion of DMD gene fragment in a male patient.
Fig 2.
Schematic representation of probes’ hybridization characteristics.
Fig 3.
G-effects influence on hybridization specificity.
Categorization of oligoprobes according to numbers of G nucleotides (A) or GGG-blocks (B) is presented. Averaged hybridization signals were calculated for each bin and their values are shown along primary Y axes as columns of assorted colors. Overall (purple) hybridization and cross-hybridization (dark blue) are shown on the upper panels; specific hybridization (pink) is presented on the middle panels. Hybridization specificity (black), defined as ratio between specific and cross-hybridization, is presented on the lower panels. Numbers of the probes in each bin are shown along the secondary Y axes as light blue columns on the upper panels. A. Relationship between probes’ hybridization signals and the numbers of G nucleotides. B. Relationship between probes’ hybridization signals and the numbers of GGG-blocks in the positions of the probe. Probes were categorized into bins according to GGG-block counts in the probe’s positions from 1 to 23 (numeration from probe’s 5’ end). Location of GGG-blocks was assigned for three groups: for position 1, positions from 2nd to 4th, and positions from 5 to 23rd. The left histograms show averaged signals for probes with GGG-blocks located at the first position of the probe, middle histograms show results for probes with GGG-blocks located in the 2nd to 4th positions. The numbers of GGG-blocks in any position from the 5th to 23rd is presented on the right histogram.
Fig 4.
Averaged cross-hybridization signals of probes with various locations of GGG(G)-blocks.
The upper panel shows results of probes’ binning and averaging for GGG-blocks and the lower panel for GGGG-blocks along the probe positions from the 1st to 23rd (5’→ 3’). Averaged absolute cross-hybridization signals are shown along the primary Y axes as dark blue columns and the numbers of probes in each bin are shown along the secondary Y axes as light blue columns.
Fig 5.
Nucleotide bias and folding potential affect hybridization specificity.
Oligoprobes were categorized into bins according to nucleotide bias (SAS-score, left panel) and self-folding vulnerability (right panel). Averaged hybridization signals are shown along the primary Y axis and numbers of probes in each bin are shown along secondary Y axes as light blue columns on the top histograms. Averaged values of overall (dark blue) and cross-hybridization (grey) signals are shown on the top histograms, specific hybridization (pink) is shown on the middle histograms and hybridization specificity (black) is present on the bottom histograms. Numbers of probes in each bin are shown along the secondary Y axis as light blue columns on the top histograms.
Fig 6.
Categorization of probes by binding energy after filtration using all analyzed parameters.
Oligoprobes were categorized into bins according to their binding energy. Averaged hybridization signals and other hybridization characteristics calculated for each bin are shown as columns of assorted colors. The columns with variable shades of a particular color illustrate the filtration process that gradually removes from the database all probes with a defined sequence characteristic, which negatively affect hybridization specificity because of involvement in parallel hybridization reactions. Colors of the columns becomes lighter after each filtration step. The darkest columns represent all probes from the database, while lightest columns represent probes after all filtration steps. The filtration process entails the following sequential probe removal steps: 8 or more of G in a sequence, at least one GGG block, SAS score above 0.05, -ΔG folding above 1.5 kcal/mol and minimum genomic occurrence among all 11-mers in oligo above 250. A. Overall hybridization (derived from the dataset of probes with specific targets). B. Cross-hybridization (absolute) (derived from the dataset of probes without specific targets). C. Specific hybridization (represented by subtraction values between overall and cross-hybridizations). D. Hybridization specificity (represented by ratio between specific and cross-hybridization). E. The percentage of probes (from complete dataset) in each bin.
Fig 7.
Categorization of probes by theoretically estimated S function after filtration using all analyzed parameters.
Oligoprobes were categorized into bins according to the estimated S function, where S is calculated as a combination of genomic occurrence of all k-mers (7 ≤ k ≤ 11) in an oligoprobe and their binding energy (see Material and methods for details). Averaged hybridization signals and other hybridization characteristics calculated for each bin are shown as columns of assorted colors depending on the bin type. The columns with variable shades of a particular color illustrate the filtration process that gradually removes from the database all probes with a defined sequence characteristic, which negatively affect hybridization specificity because of involvement in parallel hybridization reactions. Colors of the columns become lighter after each filtration step. The darkest columns represent all probes from the database, while lightest columns represent probes after all filtration steps. The filtration process entails the following sequential probe removal steps: 8 or more of G in a sequence, at least one GGG block, SAS score above 0.05, -ΔG folding above 1.5 kcal/mol. A. Overall hybridization (derived from the probe’s dataset with specific targets). B. Cross-hybridization (absolute) (derived from the dataset of probes without specific targets). C. Specific hybridization (represented by subtraction values between overall and cross-hybridizations). D. Hybridization specificity (represented by ratio between specific and cross-hybridization). E. The percentage of probes (from complete dataset) in each bin.
Table 1.
Relationships between hybridization values and probes’ sequence characteristics.