=Paper=
{{Paper
|id=Vol-2667/paper45
|storemode=property
|title=The method of generation barcode for DNA certification of plants and other organisms
|pdfUrl=https://ceur-ws.org/Vol-2667/paper45.pdf
|volume=Vol-2667
|authors=Olga Kiryanova,Ilya Kiryanov,Liana Akhmetzianova,Bulat Kuluev,Alexey Chemeris
}}
==The method of generation barcode for DNA certification of plants and other organisms ==
https://ceur-ws.org/Vol-2667/paper45.pdf
The method of generation barcode for DNA
certification of plants and other organisms
Olga Kiryanova Ilya Kiryanov Liana Akhmetzianova
Ufa State Petroleum Technological Corning, Inc. Institute of Petrochemistry and
University Saint Petersburg, Russia Catalisys;
Ufa, Russia ilya.lsc@gmail.com Ufa Federal Research Center, RAS
olga.kiryanova27@gmail.com Ufa, Russia
www.lianab@mail.ru
Bulat Kuluev Alexey Chemeris
Institute of Biochemistry and Genetics; Institute of Biochemistry and Genetics;
Ufa Federal Research Center, RAS Ufa Federal Research Center, RAS
Ufa, Russia Ufa, Russia
kuluev@bk.ru chemeris@anrb.ru
Abstract—In the current paper a new DNA certification Different primers are shown in different colors. Red
method for living organisms was presented. The suggested brackets denote the amplicons sizes.
approach is based on unique barcode that identifies a
particular organism. The studies were conducted using several It is possible to make predictions of amplicons sizes on
species of crops and model plants (Solanum tuberosum, the base of known complete nucleotide sequence of the
Triticum aestivum, Arabidopsis thaliana). The web based analyzed organism. This is a complicated task which could
application was developed on the base of the proposed not be done manually. For example, a genome with 1 billion
technique. pairs of nucleotides has about 103 annealing sites for
decamer primers. To solve this problem a web based
Keywords—polymerase chain reaction, primer design, DNA application was developed. The proposed software allows to
certification, barcode, web application determine the annealing positions of primers in the DNA
chain indicating the length of amplicons. Since the
I. INTRODUCTION probability of obtaining identical results for different
Polymerase chain reaction (PCR) is an experimental genomes is negligible, the obtained data could be represented
method of molecular biology that can significantly increase as unique barcode which, in its turn, represents a digital
the quantity of target DNA fragments with specific DNA passport [5].
nucleotide sequences in a sample [1]. PCR is widely used in
biological and medical practice to isolate new genes, II. PROBLEM DESCRIPTION
diagnose diseases and for other tasks. The global efforts in creation and promotion of new
PCR was invented in the midle of the 1980s. Nowadays it varieties of agricultural crops requires the modernization of
is the leading method in the field of physical and chemical the selection process. Currently existing solutions for DNA
biology. certification of plants do not allow to obtain digitized data.
The proposed barcode system is based on the polymorphism
Primers (short DNA fragments consisting of 10-30 of specific genes (most often the cytochrome oxidase gene).
nucleotides) are important components that affect on success Therefore, the detected degree of polymorphism is quite low
of experiments [2]. Primers in PCR must satisfy the main and allows us to detect only the relationship of individual
requirements: specificity of amplification process and its groups of organisms, as well as their location on the
efficiency. A pair of primers are usually used in PCR. evolutionary tree [6-8]. Some recently dispersed species may
However, in some cases a single primer may be sufficient not be distinguishable based on analysis of several genes.
since it is involved in forward and reverse primers Modern instrumental methods for unambiguous genetic
simultaneously [3]. Such approach with single primer is used identification of biological material do not allow to
for DNA polymorphism elucidation. For multiplex PCR, determine the difference between plant varieties. The
several primers can be used simultaneously, usually up to 12. development of a well-reproducible and relatively
More than one pair of oligonucleotide primers at the same inexpensive method of DNA certification of varieties and
time leads to the coamplification of DNA matrices with their DNA identification is an urgent task. Improved or new
results in multiple PCR products [4]. In this case primers solutions for the abovementioned problem could ensure
could be annealed in pairs in all possible combinations. An significant economic growth in the agricultural sector of
example of primer annealing in the multiplex PCR is shown economy.
on figure 1.
For unambiguous certification and identification we
proposed a new approach: to assign unique genetic barcodes
to plant varieties based on the detected DNA polymorphism
using PCR. It does not require prior knowledge about
genome of any plant species.
There are more than 20 methods for detecting DNA
polymorphism in plants. However, none of them could
Fig. 1. An example of primer annealing in the multiplex PCR. provide true digital data and does not have proper
reproducibility [9-12]. The experimental basis of the DNA
certification method is a modified PCR based on the RAPD -
Copyright © 2020 for this paper by its authors.
Use permitted under Creative Commons License Attribution 4.0 International (CC BY 4.0)
Data Science
Random Amplified Polymorphic DNA amplification algorithm and the Boyer-Moore algorithm [15]. The Boyer-
method. It is preferable to perform computer analysis before Moore algorithm is considered as the fastest among general-
the laboratory experiments conduction. Such computer purpose classical algorithms designed to find a substring in a
modeling could assist to determine the places of possible string. The main advantage of the Boyer-Moore algorithm is
annealing sites and sizes of reaction products (amplicons). that the shift is calculated based on the pattern (but not over
the line where search is conducted). The pattern comparison
In order to determine the amplicon size in silico it is with a fragment of the string occurs from right to left. In
necessary to know positions of direct and reverse primers in addition, the search pattern is not compared with the source
a nucleotide sequence. After that the distance between these text in all positions, most of them are skipped as obviously
primers could be determined. That distance is called the unsuccessful. General evaluation of the computational
amplicon size and must have from 51 to 500 nucleotides
complexity of the linear algorithm – O(nm), where m is the
length inclusive. This range is optimal for most cases of gel
length of the search pattern, n the length of the search string.
electrophoresis and sequencing [13]. A search example is
General evaluation of the computational complexity of the
shown on figure 2.
Boyer-Moore and Knuth-Morris-Pratt algorithms is O(m+n)
[16].
A comparative analysis of algorithms efficiency was
performed using genome with 106 nucleotides. It was shown
that the Boyer-Moore algorithm is more suitable for primers
search [17]. Thus, the Boyer-Moore algorithm was used to
implement the substring (primer) search.
It should be mentioned, that one presented search
technique was implemented using Python language with JIT
Numba-compiler [18].
Fig. 2. An example of searching forward and reverse primers in a
fragment of nucleotide sequence. On the base of data about the length of amplicons a
barcode could be generated. The barcode is represented as a
Following the above-mentioned logic the proposed set of lines which determine the presence of amplicon length
software collects information on all available occurrences of in the range from 51 to 500 nucleotides. We assumed that
primers and amplicons lengths [14]. An example of result is this range includes 450 imaginary DNA cells, which may
shown in table 1. contain DNA (and this will be DNA+-cell) or no DNA
(DNA--cell). The presence of one or more DNA fragments
TABLE I. POSITIONS OF ANNEALING FORWARD AND REVERSE PRIMERS IN with the same size in a specific DNA+-cell is not important
THE SOLANUM TUBEROSUM GENOME (DATA IS PLACED IN
ASCENDING ORDER OF AMPLICON SIZE). EXAMPLE OF OUTPUT since it is a qualitative rather than quantitative analysis.
DATA . Thus, the information about each sample can be presented
GGATCTTT AAAGATCC from alternating zeros and ones in the selected range of
Amplicon size lengths taken in the amplicon analysis. For example,
position position
39883835 39884052 217 consider the range from 101 to 110 nucleotides, where the
finding of DNA fragments has the following form: …101-,
55375264 553775548 284
102+, 103-, 104-, 105-, 106-, 107+, 108-, 109-, 110- …. The
29569657 29569969 312 numbers denote the size of DNA fragment in nucleotides, (+)
38393029 38393375 346
– presence of a DNA fragment, (-) – absence of a DNA
fragment. In binary format the entry for this section will be
49519668 49520023 355 as follows: …0100001000.
41540764 41541163 399 Visually such data could be conveniently represented as
8231987 8232448 461 genetic barcodes in a linear or two-dimensional display. For
example, for the data in table 1 the corresponding barcode is
shown on figure 3.
Information about genome is presented as a single file or
collection of files with text data according FASTA standard.
This is the most common format for digital storage of
nucleotide sequences. Nucleotide sequences are stored as Fig. 3. Barcode example for Solanum tuberosum PCR reaction with forward
strings of characters “A”, “G”, “C”, “T” and sometimes “N”. primer GGATCTTT and reverse primer AAAGATCC.
Each letter means the corresponding nucleobase: adenine,
guanine, cytosine, and thymine respectively. “N” means The main advantage of the proposed approach is easy
unknown nucleotide. FASTA format allows easy data comparison of two independent genetic characteristics. It is
manipulations with sequences using text editors and possible to accurately measure the amplicon length after its
programming languages such as Python, Ruby, Perl, etc. separation in capillary gel electrophoresis under denaturing
That is why FASTA files are widely used for primers conditions.
positions search. According to the FASTA file format The obtained data about the primer(s), the analyzed
specification, above mentioned task could be reduced to the
genome, and the set of selected amplicons are unique. It is
well-known approach: substring search in a string.
completely eliminating the accidental barcode coincidence
There are several well-known algorithms for substring of different samples of strains, races, varieties, breeds, or
search in a string: linear search, Knuth-Morris-Pratt individuals. Since the amplicons can have a huge number of
VI International Conference on "Information Technology and Nanotechnology" (ITNT-2020) 208
Data Science
variants (combinations) of the distribution of these DNA In addition to computer analysis it is possible to compare
fragments on DNA+ cells. wet lab experiments (in vitro found amplicons) and predicted
The total number of occurrences combinations in such PCR outcome by comparing two barcodes.
DNA cells could be calculated as the number of Thus, the generated information is a kind of digital
combinations from m to n using the following formula (1): passport for varieties, breeds, strains of various
organisms [19].
𝑛 𝑚!
𝐶𝑚 =
𝑛!(𝑚−𝑛)! IV. CONCLUSIONS
where C is the total number of probabilistic occurrences We proposed a new approach for cataloging/certifying
combinations in DNA cells, m the number of all DNA-cells diverse groups of plants and other organisms. These
unambiguous certification and identification were carried out
analyzed in the selected range, and n the number of all
by assigning unique genetic barcodes to plant varieties based
DNA+-cells.
on the detected DNA polymorphism. In addition, this method
According to the probability theory, the largest number is applicable for all living organisms besides human. Other
of combinations occurs when half of the cells are occupied methods are used for DNA identification of an individual
with DNA fragments (225 of 450). In this case the number approaches, the most promising for data barcode is
of combinations exceed 10100. This number is more than considered to be single-nucleotide DNA polymorphism.
enough for unambiguous DNA certification of any Currently, many approaches are used for DNA certification
organism. The probability of a random match of two DNA of plant varieties but none of them provides unambiguous
samples with the number of different-sized amplicons equal digital data. Thus, the suggested approach for DNA
to five is about one case per 1012. Thus, the proposed certification (cataloging)/identification of living organisms is
approach is an efficient method for DNA certification of unique. In addition, the web application was developed that
cultivars, lines, breeds, and strains. allows to detect the presence of specific primers in the DNA
(genomes), determine the size of amplicons that are formed
III. ABCDNA_GS (AMPLIFIED BAR-CODED DNA as a result of PCR, and create the corresponding unique
GENOME/SPECIMEN) barcode. In the future, it is planned to translate data into QR
We have developed the web application with database for code and use machine learning methods to classify barcodes
storing information about the amplicons and barcode and compare related varieties. [20].
generation. Web based application allows to catalog wet laboratory
Input data is: domain (Archaea, Prokaryotes, experiments and in silico analysis. The entire genomes of
Eukaryotes), Kingdom (Animals, Plants, Fungus) – only for different organisms including Solanum tuberosum, Triticum
Eukaryotes, genome, primer(s), type of DNA amplification aestivum, Arabidopsis thaliana available from resource
(RAPD, ISSR, AFLP). The entire genomes of different EnsembleGenomes http://ensemblgenomes.org. Thus,
organisms including from resource EnsembleGenomes without conducting a full-scale experiment it is possible to
http://ensemblgenomes.org as FASTA files. test several primers as well as get an idea of the full-scale
experiment success. Due to the uniqueness of the proposed
The output data is: found amplicons sizes and the approach it is possible systematize data for different primers
corresponding barcode. and DNA sequences without taking into account their natural
As a result, found amplicons sizes allow to estimate the affiliation. It was shown that barcoding could enhance the
outcome of any particular PCR experiment. genome comparison by excluding the human factor [21],
allows to get digital data about a certain genome, and leads
In other words, obtained data allows to plan the PCR to the intuitive and clear comparison among other digitized
experiment for any genome. In addition, compare genomes.
experimentally obtained amplicons with those found as a
result of the program. ACKNOWLEDGMENT
User interface example is shown on the figure 4. This research was supported by the Russian Foundation
for Basic Research (project № 17-44-020120).
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