Virtual Phase-Shifting Speckle-Interferometry for Polymorphism Detection in Chlamydia trachomatis

1. Application of virtual phase-shifting speckle-interferometry for detection of polymorphism in the of Chlamydia trachomatis omp1 gene

2. Yury V. Saltykov2, Sergey S. Ulyanov1,2,4, Sergey S. Zaytsev2, Onega V. Ulianova2, Valentina A. Feodorova1,2,3 1Laboratory for Molecular Biology of Chlamydia, Federal Research Center for Virology and Microbiology………………………………………………………………….2Laboratory for Molecular Biology and NanoBiotechnology, Federal Research Center for Virology and Microbiology, Branch in Saratov3Department for Microbiology, Biotechnology and Chemistry, Saratov State Agrarian University………………………………………………………………………….4Department for Medical Physics, Saratov State University

3. 1 Federal Research Center for Virology and Microbiology, 601120, Pokrov, Vladimir region, Russia 2 Federal Research Center for Virology and Microbiology, Branch in Saratov, Ap. 9, Proviantskaya Street, Box 1580, 410028, Saratov, Russia 3 Saratov State Agrarian University, 1, Theatralnaya Square, 410012, Saratov, Russia 4 Saratov State University, 83 Astrakhanskaya Street, 410012, Saratov, Russia 5Saratov State Medical University, 410028, Saratov, Russia

4. Method of phase-shifting speckle-interferometry (PSSI) has been used as a new tool with high potency for modern bioinformatics. Virtual PSSI has been applied for detection polymorphism in the of Chlamydia trachomatis omp1 gene. Fractal dimensions of retrieved phase of gene-based speckles have been analyzed. It has been shown, that suggested method is very sensitive to natural genetic mutations as single nucleotide polymorphism. Effectiveness of proposed method has been compared with effectiveness of classical method of bioinformatics, based on nucleotides sequence alignment

5. Standard optical scheme for phase-shifting speckle-interferometry 1 - incident laser beam, 2 - focusing lens, 3 - beamsplitter, 4- investigated object, 5 - reference surface (may be rough), 6 - piezo, 7 - CCD or CMOS camera.

6. Algorithm of speckle processing

7. Reference paper S. S. Ulyanov, S. S. Zaytsev, O. V. Ulianova, Yu. V. Saltykov, V. A. Feodorova, “Using of methods of speckle optics for Chlamydia trachomatis typing,” Proc SPIE10336, 103360D (2017).

8. Recently it has been demonstrated [*] that transformation of genetic data to computer-generated diffraction optical element (virtual scattering surface) and presenting of sequence of nucleotides as speckle pattern will allow to improve drastically methods of modern bioinformatics.

9. As commonly known, a gene is a discrete sequence of DNA nucleotides in a molecule of nucleic acids. Typically, DNA consists from four types of nucleotides, or “letters”. These nucleotides contain, correspondingly, four nitrogen base, namely, adenine (A), thymine (T), guanine (G) and cytosine (C).

10. So, a nucleic acid sequence is a succession of letters that indicate the order of nucleotides within a DNA or RNA molecule. The nucleotide sequence can be determined using specific procedure of sequencing which allows to present the primary structure of analyzing macromolecule in the form of linear sequence of monomers in a text format

11. To generate gene-based speckles, the initial sequence of letters (taken from initial nucleotide sequence) is transformed in the sequence of numbers. This sequence is re-coded into the sequence of triads, and discrete value h is assigned to each possible triad.

12. Then square matrix Hn,m is packed from obtained sequence of h values. Physical meaning of formed matrix Hn,m is the local height of virtual scattering surface, reflecting the local content of gene structure. 2D speckle pattern, corresponding to initial nucleotide sequence, is generated using diffraction of coherent beam with square profile on the (virtual) rough surface with profile Hn,m. Algorithm of re-coding of nucleotides sequence into speckle pattern is described in details Ref. [*]. In this research algorithm has been optimized.

13. If two virtual gene-based speckle patterns are “placed” in shoulders of interferometer, then virtual phase-shifting speckle-interferometry can be applied for detection of genepolymorphism   Two different variants of omp1 gene from C. trachomatis strains of two different subtypes have been used to generate gene-based speckle pattern

14. Gene-based speckle-pattern#1

15. Gene-based speckle-pattern#2

16. Interference of two speckle patterns

17. Interference of two speckle patterns

18. Interference of two speckle patterns

19. Interference of two speckle patterns

20. Phase retrieved. Appearance of phase structure indicates natural genetic mutations as single nucleotide polymorphism

21. Conclusions • Variation in nucleotide sequence of omp1 gene C. trachomatis is detectable using phase-shifting speckle-interferometry. • Using this technique, it is possible to observe smallest polymorphism even in one nucleotide.

22. Acknowledgments This research has been supported by Russian Scientific Foundation, grant # 17-16-01099