An Empirical Study of Choosing Efficient Discriminative Seeds for Oligonucleotide Design

1. An Empirical Study of Choosing Efficient Discriminative Seeds for Oligonucleotide Design Won-Hyong Chung and Seong-Bae Park Dept. of Computer Engineering Kyungpook National University, South Korea

2. Motivation • Issues for designing oligonucleotides • To minimize the cross-hybridizations • To minimize the computing time • Seeding (or indexing) have been widely used for concurring those issues by means of pre-screening unreliable sequence regions before calculating cross-hybridizations. • Although many types of seeding methods have been proposed, measure of evaluating the seeds regarding how adequate and efficient they are in the oligonucleotide design is not yet proposed.

3. Difference between alignment and oligonucleotide design • Alignment • To find all possible alignments which have enough scores. • Sensitivity is important, while specificity is usually guaranteed by seed’s own specificity. • Oligoncleotide design • To find optimal oligonucleotides to differentiate target sequences from the others. • Specificity should be considered as well as sensitivity for checking cross-hybridization.

4. Objectives • We propose novel measures of evaluating the seeds based on the discriminability and the efficiency. • We examine five seeding methods in oligonucleotide design. • continuous, spaced, transition-constrained, BLAT, and Vector seed • We provide a software package SeedChooser which enables users to get the adequate seeds under their own experimental conditions.

5. What is Seed? • Seeding process • Filtering step: short fixed-length common words which are found at both query and target sequences are selected. • Extension step: the selected words are extended to the size of oligonucleotide and be checked the cross-hybridization. Seed = the filtering template of the fixed-length words

6. Seeding methods (1/2) • Continuous seed: a seed to find k-length exact matches • BLAST employs 11-bp length seed 11111111111 • Spaced seed: allowing don’t care letter labeled ‘0’ in the seed • 18-bp-length seed containing 11-bp matches 101101100111001011 is used at PatternHunter. • Transition-constrained seed: adopting transition (A <-> G, C <-> T) letter ‘@’ in the seed • YASS used such seed 1110@10010@1010111, it consists of 18-bp length, 10-bp matches and 2 transitions.

7. Seeding methods (2/2) • Blat seed: a continuous seed allowing one or two mismatches at any positions of the seed. • Vector seed: a generalized seed by combining the idea of BLAT seed and spaced seed. • BLAT seed and Vector seed allow some mismatches in any positions. • They greatly increase the sensitivity but spends much more computing time than the previous seeds.

8. The Issues of seeds for oligo design • An ideal seed should filter all regions as fast as possible that have no possibility of being chosen as an oligo. a seed should find as many oligos as possible a seed should avoid to find non-oligo region a seed should minimize the cost of indexing to find oligos Discriminability Efficiency Efficient Discriminability

9. Discriminability The discriminability is a balance between precision and recall to minimize both false positives and false negatives. alpha jump

10. Efficiency The efficiency is the proportion of usefulregions filtered by a seed. • the duplication ratio ofgenerated indices • the average number of indices in each oligo jump beta, gamma

11. Efficient discriminability The efficient discriminative seed is the seed that has the maximum efficient discriminability value for the given

12. Experiments • Empirically chosen seeds were evaluated by three measures, discriminability, efficiency, and efficient discriminability, respectively. • We tested the seeds for designing the 50mer oligos. • The parameters are set to 1 for evaluation. • Simulated data set • A set of random sequences which are generated by OligoGenerator in SeedChooser. • Biological data set • Ecologically important genes involved in the nitrogen and carbon cycles. • nirS: nitrite reductase gene set • pmoA: methane monooxygenase gene set

13. Discriminability of the five seeding methods

14. Efficiency of the five seeding methods

15. Efficient Discriminability the five seeding methods

16. Evaluation results of pmoA data set

17. Evaluation results of nirS data set

18. SeedChooser: Seed Evaluation and Recommendation Tools • SeedChooser : To recommend best seeds by the evaluation parameters. It uses genetic algorithm to find best seeds. • SeedEvaluator : To evaluate a set of the seeds by the parameters. • OligoGenerator : To generate a set of oligos for the desired experimental conditions. • SeedChooser homepage http://ml.knu.ac.kr/~whchung/seedchooser.html

19. CONCLUSION • The novel measure for evaluating the seeds in the oligo design based on the discriminability and the efficiency. • The spaced seed was generally preferred to the other seeding methods. • Our study can be applied to the oligo design programs in order to improve the performance by suggesting the experiment-specific seeds. • We expect that our study will be helpful to the other genomic tasks.

20. Supplementary materials

21. P0 T0 P1 T1 P2 T2 • T1, T2, T3: the target sequences. • P1 and P2 are the matched oligos for an oligo P0 • S1, S2 and S3 are the seed indices for S0 by a seed. S0 T3 S1 S2 S3 back

22. Relations of precision, recall and discriminability

23. Discriminability according to values of α back

24. Efficiency according to values of β and γ back

25. Efficient Discriminability for 70mer Oligos