Research Article: Sequence characteristics define trade-offs between on-target and genome-wide off-target hybridization of oligoprobes

Date Published: June 21, 2018

Publisher: Public Library of Science

Author(s): Olga V. Matveeva, Aleksey Y. Ogurtsov, Nafisa N. Nazipova, Svetlana A. Shabalina, Ruslan Kalendar.


Off-target oligoprobe’s interaction with partially complementary nucleotide sequences represents a problem for many bio-techniques. The goal of the study was to identify oligoprobe sequence characteristics that control the ratio between on-target and off-target hybridization. To understand the complex interplay between specific and genome-wide off-target (cross-hybridization) signals, we analyzed a database derived from genomic comparison hybridization experiments performed with an Affymetrix tiling array. The database included two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization and (ii) genomic cross-hybridization only. All probes from the database were grouped into bins according to their sequence characteristics, where both hybridization signals were averaged separately. For selection of specific probes, we analyzed the following sequence characteristics: vulnerability to self-folding, nucleotide composition bias, numbers of G nucleotides and GGG-blocks, and occurrence of probe’s k-mers in the human genome. Increases in bin ranges for these characteristics are simultaneously accompanied by a decrease in hybridization specificity—the ratio between specific and cross-hybridization signals. However, both averaged hybridization signals exhibit growing trends along with an increase of probes’ binding energy, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The same trend is evident for the S function, which serves as a combined evaluation of probe binding energy and occurrence of probe’s k-mers in the genome. Application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, we showed that high values of specific and cross-hybridization signals are not mutually exclusive for probes with high values of binding energy and S. In this study, the application of a new set of sequence characteristics allows detection of probes that are highly specific to their targets for array design and other bio-techniques that require selection of specific probes.

Partial Text

Many biotechnology applications involve oligoprobe hybridization with complementary targets in DNA or RNA as a basic procedural step. One such application is microarray technology. High throughput sequencing is gradually replacing microarrays as the preferred method for studying cellular transcript expression levels. However, microarrays are still dominating certain applications, such as identification of transcription binding sites [1], and gene copy number evaluations and genotyping [2–3]. It is possible to envision a powerful symbiosis between microarrays and new generation sequencing technologies [4].

In this study, we discriminate between absolute and relative cross-hybridization values using two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization or (ii) genomic cross-hybridization only. Absolute cross-hybridization applies to cross-hybridization signal that derives from contributions of all off-target interactions of each probe. The relative cross-hybridization term applies to a proportion of absolute cross-hybridization in an overall probe’s signal, which includes two components: the target specific signal and cross-hybridization. Therefore, while the absolute cross-hybridization represents a signal, the relative one represents a calculated signals’ ratio.

In this study, we demonstrated that elevated levels of hybridization specificity and absolute cross-hybridization are not mutually exclusive and may be attributed to the same probe sets. Moreover, trends in which hybridization specificity and absolute cross-hybridization are changing along with a sequence characteristic can differ significantly. They might be either both positive, either both negative, or opposite of each other, depending on the sequence probe characteristic.

Hybridization specificity and absolute cross-hybridization values of oligoprobes both increase with increasing binding energy of probes in the analyzed bins. We showed that high specific hybridization and high cross-hybridization are not mutually exclusive, and may be attributed to the same probe sets. In other words, the level of non-specific interactions for some molecules may be high, but the ratio between off-target and total hybridization signals may be low. This also means that the specific signal is sufficient in magnitude for a high on-target/off-target ratio, which defines interaction specificity.




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