Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays

Abstract

We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers. It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence. Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software. Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list. Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements. Using a TimeLogic hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy. Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes.

Figure 1

Workflow diagram of oligo selection in Osprey. Sequences, assembly information and default parameters are read from disk. Many parameters and modes can be overridden on the command line. Note that the probe selection part of the processing is common to all oligo design modes. Also, the probe selection test cascade can be run concurrently for many sites.

Figure 2

Range of candidate sequencing primers for elucidation including position T1 (e.g. an assembly ambiguity to resolve), with user-specified parameters. Candidates are checked from 3′ to 5′ within the range to minimize the number of primers required. An early candidate could also resolve T2 in the same reaction. U = number of unreadable bases, L = primer length, R = expected sequence read length, M = minimum number of bases to elucidate after T1.

Figure 3

Every segment of the gene that is either unique (noted by the .uniq suffix) or repeated by a distinct subset of the gene set is isolated for oligo design. To qualify for analysis, sections must be at least as long as the oligo length minus the maximum allowable repeat (default 20); hence some overlapping regions are not represented in the final oligos.

Figure 4

Profile scoring used to encode caloric values from the NN thermodynamic model at 37°C with 1 M NaCl. The base and its 5′ NN determine the score for a match; C scores 1300 in positions 1, 10 and 17 of the top sequence (the oligo) because T preceeds it and it scores 1830 in position 18, because C proceeds it. The score position 8 is the sum of the two 5′–3′ mismatch NN values, minus the NN value overcounted in the following match. The AG/TA mismatch data is inverted to demonstrate that the actual 3′–5′ mismatch is equivalent to this standard 5′–3′ representation. The profile score summation, minus the standard initiation penalty of 1.0 kcal/mol is the duplex's free energy summation, 20.91 kcal/mol.

Figure 5

A dot plot of HyTher predicted free energy versus predicted melting temperature in 0.1 M NaCl for random exact matching oligos of lengths 10, 15, 20, 25 and 50 (forming clear groups from lower right to upper left).

Figure 6

Prediction correlation between HyTher server (x) and Osprey (y) results for ΔG in kcal/mol using the 250 oligomers from Figure 5. R² = 0.9996, with linear regression y = 1.0029x + 0.055, indicating Osprey's close conformity to the reference Unified Model predictions.