KR101447593B1 - Method for determining whole genome sequence of chloroplast, mitochondria or nuclear ribosomal DNA of organism using next generation sequencing - Google Patents

Abstract

The present invention relates to a method for separately or simultaneously determining a whole genome sequence of a chloroplast, a mitochondria or a nuclear ribosomal DNA of an organism using next generation sequencing (NGS) and a computer readable medium to perform the same. The method includes: (a) determining a sequence of a whole genome of the organism using the NGS; (b) generating an NGS data set based on a genome coverage amount of the chloroplast using reads (sequence pieces) generated by determining the sequence in step (a); (c) assembling the reads of the NGS data set generated in step (b) using assembly software; (d) separating contigs including at least one sequence selected from the group consisting of sequences of a chloroplast, a mitochondria or a nuclear ribosomal DNA from contigs generated after assembling of step (c); and (e) connecting the contigs separated in step (d) using a sequence comparison program to correct an error in the assembly.

Description

Description: TECHNICAL FIELD The present invention relates to a method of decrypting a complete genome sequence of a chloroplast, mitochondrial or nuclear ribosomal DNA of an organism using a next generation sequencing method,

The present invention relates to a method for decoding a complete genome sequence of a chloroplast, mitochondrial or nuclear ribosomal DNA of an organism using a next generation sequencing method, and more particularly, to a method for sequencing a whole genome of an organism (NGS, next decoding the nucleotide sequence by a genomic sequencing method; (b) generating an NGS data set based on the amount of chloroplast genome coverage using the leads (sequence fragments) generated through the nucleotide sequence decoding in the step (a); (c) assembling leads of the generated NGS data set of step (b) using assembly software; (d) isolating contigs comprising at least one sequence selected from the group consisting of chloroplasts, mitochondria, and nuclear ribosomal DNA (nrDNA) sequences in the contig produced after assembly in step (c); And (e) connecting the isolated contigs of step (d) using a nucleotide sequence comparison program and correcting errors occurring in the assembly, wherein the complete genome of the chloroplast, mitochondrial or nuclear ribosomal DNA of the organism A method for decoding the sequence alone or simultaneously, and a recording medium on which a computer-readable program for performing the above method is recorded.

Plant cells have a genome in the nucleus, chloroplast, and mitochondria. Chloroplasts are the main organ responsible for photosynthesis, and generally produce maternal heredity. The size of the chloroplast genome is 120 ~ 217kb, which is conserved and maintained as a mutation with about 130 genes. However, a relatively large number of single nucleotide polymorphism (SNP), insertional deletion (InDel), inversion, and translocation. The chloroplasts are circular in all plant cells and there are hundreds of copies in one cell.

Nuclear ribosomal DNA (nrDNA, nuclear ribosomal DNA) is present in the nucleus of plant cells and is concentrated in the tandem repeats of one or two chromosomes and repeats from thousands to tens of thousands of copies, nucleolar organizer region, and it is known that homogenization occurs very quickly even when the genomes of both parents are recombined. Plant nrDNA has a high conservation level in the plant nucleotide sequence because it conserves gene regulation of ribosome assembly and nucleoplasmogenesis. In higher plants, four rRNA components are located in two chromosomal regions, 5S nrDNA and 45S nrDNA, but in some ancient plants, ginkgo, moss and algae, 45S nrDNA and 5S nrDNA are in the same tandem unit .

The 45S nrDNA consists of one 45S cistron unit containing 18S, 5.8S and 25S / 26S / 28S gene clusters and internal transcribed spacers 1 (ITS1) and ITS2, which are relatively mutated between each gene in all seed plants . Each 45S cistron unit is divided into IGS of various sizes and arranged in a columnar arrangement.

The chloroplast genome and nrDNA sequences are very well preserved as essential genomic components and represent important cytoplasmic and nuclear genomes, respectively, providing important clues to the diversity and evolution of the entire plant genome. To date, some 360 chloroplast genome sequences (GenBank Organelle Genome Resources, July 2013) and only one nearly complete 45S nrDNA sequence (May 2013) in plants have been published in GenBank (www.ncbi.nlm.nih.gov/genbank/) . Although some chloroplast genome sequences have been achieved by plant genome sequencing projects, most chloroplast genome sequences have been created by several independent researchers. In other words, most chloroplast genome sequences were obtained by identifying the nucleotide sequences of chloroplast genomic DNA fragments inserted into BAC clones or by PCR working and sequencing methods using reference genomic sequences. On the other hand, in many plants that have been genetically deciphered, the region of the nucleolar organizer region (NOR) in which 45S nrDNA units are clustered remains as yet incomplete information. The currently reported complete 45S nr DNA unit sequence has been reported by the BAC clone sequencing method (GenBank No.OSJNBb0013K10; AP008245.2) in a complete sequence of 45s rDNA sequence of 7,928 bp at the terminal region of chromosome 9 of rice. In addition, a complete nrDNA unit of about 9 kb was assembled on chromosome 2 (Genbank No. AC215459.2) and No. 3 (Genbank No. AC246968.1) of Solanum lycopersicum . Up to now, the 45s rDNA unit has been reported in over 20 species including the Arabidopsis chromosome 2 and 3 (based on ncbi blastn).

Recently, there have been some reports on the completion of the chloroplast genome using the next generation sequencing method (NGS) using 454 GS-FLX, SOLiD and Illumina's nucleotide sequencing equipment. However, most of the chloroplast DNAs are purely isolated, Which is then completed with additional PCR and sequencing to perform the de novo sequence assembly and fill many gaps, which still requires much effort and time. Recently, we have introduced a method to simultaneously produce nrDNA units and partial organelle genome sequences using the entire genome sequence based on the GS-FLX platform in two species of moss (Liu et al., 2013, Mol Phylogenet Evol, 66: 1089 -1094). However, most of the introduced NGS approach is difficult to apply high throughput and requires a lot of time and effort.

NGS technology is able to analyze nucleotide sequences by greatly reducing time and cost, but obtaining meaningful complete data from large-scale data is a critical challenge. Therefore, we have developed a highly efficient method to produce paired end sequences from whole genomic DNA that is generally prepared and to obtain a complete chloroplast genome sequence and complete nrDNA units simultaneously using a small amount less than 1 Gbp Respectively. In addition, it suggests a method to analyze and resolve all types of errors that may occur in the assembly process, so that it is possible to complete the complete sequence without any additional PCR or ABI sequencing process, and it is possible to analyze more than 50 species by analyzing a lane . This method can be applied to onions and lilies of very large genomes, such as lichens and lichens, and can be used as a breakthrough in exploring species diversity and the origins of evolution in all plant kingdoms I expect to be. In addition, various species of chloroplasts and nrDNAs can be identified to identify differences between strains, thus suggesting practical applications such as breed identification markers, protection of biological sovereignty, and protection of breeder's rights.

Korean Patent Laid-Open Publication No. 2013-0134269 discloses an 'ultra high density gene mapping technique using a next generation nucleotide sequence-based SNP genotype analysis', Korean Patent No. 1313087 discloses 'a method and apparatus for recombination of sequences for NGS' There has been no description of a method for decoding a complete genome sequence of chloroplast, mitochondrial or nuclear ribosomal DNA of an organism using the next generation sequencing method of the present invention.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have discovered that by using a small amount of genomic DNA of a photosynthetic organism to decode a nucleotide sequence with next generation sequencing (NGS) The present invention has been accomplished by carrying out an efficient assembly and developing a method for quickly and accurately completing the complete sequence of an organism's chloroplast, mitochondrial or nuclear ribosomal DNA simultaneously or independently.

In order to solve the above problems,

(a) decoding a nucleotide sequence of a whole genome of an organism by a next generation sequencing (NGS) method;

(b) generating an NGS data set based on the amount of chloroplast genome coverage using the leads (sequence fragments) generated through the nucleotide sequence decoding in the step (a);

(c) assembling leads of the generated NGS data set of step (b) using assembly software;

(d) isolating contigs comprising at least one sequence selected from the group consisting of chloroplasts, mitochondria, and nuclear ribosomal DNA (nrDNA) sequences in the contig produced after assembly in step (c); And

(e) linking the separated contigs of step (d) using a nucleotide sequence comparison program and correcting errors occurring in the assembly, wherein the complete genomic sequence of the chloroplast, mitochondrial or nuclear ribosomal DNA of the organism Either alone or at the same time.

The present invention also provides a recording medium on which a computer-readable program for performing the above method is recorded.

In the present invention, a new method and an error elimination method for de novo assembling a complete chloroplast genome sequence, a mitochondrial sequence and a 45s nrDNA major unit sequence simultaneously with a small amount of whole genome sequence information and a highly efficient method have been developed. The base sequence decoding method of the present invention is based on the study of high copy essential genome region and major repetition site, development of DNA bar coding markers for interspecific or interspecific identification, origin discrimination, seed purity assay, investigation of evolutionary mechanism of photosynthetic organisms, And the protection of the rights of the breeder to a specific breed, it is considered to be useful industrially.

1 is a rice (Oryza sativa ) and ginseng ( Panax ginseng ), a is a bar graph showing the number of contigs corresponding to chloroplasts, nuclear ribosomal DNA (nrDNA), mitochondria, and others. Means the ratio of the length of each reference sequence to the total length of the reference sequence extracted. Also, b is the result of cotigmassing and chloroplast sequence mapping using rice Os2 data set.
FIG. 2 is a diagram showing contigs and errors participating in each data set of rice. FIG.
FIG. 3 shows the result of showing the assembly error position and the error base pair information of the data sets of rice and ginseng.
FIG. 4 is a diagram showing errors and correcting processes of the insertion-deletion assembly. FIG.
FIG. 5 is a diagram showing a column redundancy region that may appear as an assembly error. FIG. a represents the type of erroneous assembly appearing in the columnar redundant portion of ginseng, b is a schematic diagram showing the case of correct assembly in the region where there is 4 copies of the 18-bp column redundancy generated from ginseng, and c indicates that the 18- And the depth of the lead when assembled into 2 copies and 4 copies, respectively.
Figure 6 is a picture showing various types of thymine (T) homopolymer sites.
Figure 7 is an illustration showing an example of a false assembly site caused by a mitochondrial lead (sequence fragment).
FIG. 8 is a graph showing the effect of the 100- sativa ), ginseng ( Panax As a result of the chloroplast base sequence of ginseng and Panax quinquefolius , the Y axis represents the mapping depth of each primitive data, and the colored boxes represent the individual contigs.
Fig. 9 shows the results of comparison between chloroplast sequences and reference sequences completed by the method of the present invention.
10 shows the distribution tendency of ribosomal DNA (rDNA) of rice and ginseng, where a is the result of blastz of 7,928 bp of the rDNA unit of the finished rice variety (Nipponbare) on Nipponbare chromosome 9, and b and the structure of the unit nrDNA 1, c is the American ginseng (Panax quinquefolius) ginseng (Panax ginseng ) and rice ( Oryza sativa ), d is the primer made in the conserved region of 45s to confirm the IGS length of the nrDNA completed with de novo assembly, and e and f are the numbers of ginseng IGS length and interspecific variation.
Figure 11 is a chloroplast genome map of various plant species completed by the method of the present invention.
FIG. 12 shows the results of comparing the chloroplast genome of ginseng with that of American ginseng using the mVISTA program.
FIG. 13 shows the results of comparing 12 chlorophyll genomes with 12 ginseng cultivars using the mVISTA program.
14 shows the results of comparing the chloroplast genomes among 17 rice varieties using the mVISTA program.
15 shows the results of analysis of phylogeny among 17 rice varieties based on the chloroplast genome.
Fig. 16 shows the result of comparing the structure of nrDNA of 16 species of rice with the sequence of nrDNA of each species.
FIG. 17 shows the results of analysis of phylogeny of 13 ginseng based on the sequence of 45s rDNA.
18 shows the results of analysis of the lineage of 16 species of rice based on the sequence of 45s rDNA.
Figure 19 shows examples of species specific chloroplast genome based bar coding markers using the method of the present invention.
20 is a diagram showing the diversity of the nucleotide sequences in the chloroplast genome between the two kinds of ginseng cultivars 'Chunpoong' and 'Yeonpoong'.
FIG. 21 shows the result of indicating a species-specific marker of the ginseng cultivar 'Chunhoong'.
22 is a diagram showing a unique marker that can be utilized for species classification and breed identification through a ribosomal DNA sequence.
23 is a diagram illustrating the flow of the de novo assembly method of the chloroplast and ribosomal DNA of the present invention.
24 is a flowchart of a method for decoding complete sequence information of chloroplast and nrDNA from whole genome sequence (WGS) of the present invention.

In order to achieve the above object,

(a) decoding a nucleotide sequence of a whole genome of an organism by a next generation sequencing (NGS) method;

(b) generating an NGS data set based on the amount of chloroplast genome coverage using the leads (sequence fragments) generated through the nucleotide sequence decoding in the step (a);

(c) assembling leads of the generated NGS data set of step (b) using assembly software;

(d) isolating contigs comprising at least one sequence selected from the group consisting of chloroplasts, mitochondria, and nuclear ribosomal DNA (nrDNA) sequences in the contig produced after assembly in step (c); And

(e) linking the separated contigs of step (d) using a nucleotide sequence comparison program and correcting errors occurring in the assembly, wherein the complete genomic sequence of the chloroplast, mitochondrial or nuclear ribosomal DNA of the organism Either alone or at the same time.

In the method according to an embodiment of the present invention, the step (e) of decrypting the complete nucleotide sequence of the chloroplast or mitochondria comprises:

Sorting and joining the contigs comprising the chloroplast sequences among the separated contigs of step (d) into a complete circular sequence, mapping the generated raw data sequence and eliminating assembly errors ,

The step (e) of decrypting the complete nucleotide sequence of the nuclear ribosomal DNA comprises:

Among the separated contigs of step (d), contigs containing the 45s rDNA sequence are artificially arranged in two, an artificial gap is given therebetween, and a gene gap gene program But is not limited to, filling the physical gaps in the intergenic spacer (IGS) region, completing the complete 45s rDNA unit, mapping the raw data sequence of the completed complete 45s rRNA unit, and eliminating assembly errors .

In the method according to an embodiment of the present invention, the NGS data set of step (b) may be an amount capable of covering 50 to 500 times of the chloroplast genome, but is not limited thereto.

Also, in the method according to an embodiment of the present invention, the assembly software of step (c) may be SOAP de novo, CLC de novo, Bowtie, Velvet or BWA, and preferably SOAP de novo or CLC de novo Software, but is not limited thereto.

In the method according to one embodiment of the present invention, the nucleotide sequence comparison program of step (e) may be a program such as Blast, Clusatal X, Bioedit or Phydit, preferably Blast or Bioedit, , But is not limited thereto.

In the method according to an embodiment of the present invention, the organism may be selected from the group consisting of microorganisms such as algae such as microbes, mosses, mosses, algae such as algae, algae or brown algae, fungi including mushrooms, , An insect, a fish or an animal, and the like.

In the method according to an embodiment of the present invention, the assembly error may be a sequence error, a false gap, a tandem repeat error, a monopolymer error, or a single nucleotide polymorphism (SNP) error But is not limited thereto.

The present invention also provides a recording medium on which a computer-readable program for performing the above method is recorded. Specifically, a recording medium on which a computer-readable program for performing a method for analyzing complete nucleotide sequences of chloroplasts, mitochondria, or nuclear ribosomal DNA of an organism using NGS experimental data is provided.

A computer-readable recording medium is any recording medium that can be directly read and accessed by a computer. Examples of the recording medium include magnetic recording media such as a floppy disk, a hard disk and a magnetic tape; optical recording media such as CD-ROM, CD-R, CD, RW, DVD-ROM, DVD- , And mixtures of these categories (e.g., magnetic / optical recording media such as MO), but are not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Chloroplast genome using whole genome sequence nrDNA of de novo  assembly

To perform whole genome assembly, about 3 to 10 times for Sanger sequencing, about 13 to 22 times for 454 pyrosequencing, and about 60 to 100 times for Illumina sequencing Duplicate genome data is required. Recently, the Illumina platform, which is the most widely used platform, has been assembled using about 100 times more whole genome sequence (WGS) sequences, and more than 30 plant genome decodings have been completed. However, it is difficult to obtain a complete chloroplast genome and nrDNA sequence even with such a large amount of WGS. Most of the contigs, including the chloroplast sequences generated at this time, were found to be in the form of chimeric contigs fused with genomic DNA sequences in the nucleus. On the other hand, as a result of assembly with the CLC de novo assembler using small quantities of WGS data approximately one-and-a-half times the size of the genome, most of the long-produced con- tents are present in very high copies in the cell, such as chloroplasts, mitochondria and ribosomal DNA Genome sequence. In the case of rice ( Oryza sativa ), five of the longest assembled congig sequences were found to cover the entire chloroplast genome with an overlap of about 20 bp. In addition, the nucleotides containing the nucleotide ribosomal DNA (nrDNA) One contig of 6,889 bp was identified. While 15 contigs covered about 50% of the mitochondrial genome (Table 1 and Figure 1).

Panax ginseng ) showed three chloroplasts, thirteen mitochondria and one nrDNA sequence of 9,422 bp in 30 consecutive contig sequences (Table 2 and Fig. 1).

Based on these results, it is suggested that rice germ with a relatively small genome size of 430 Mbp and a comparatively large genome size of ginseng, which have not yet been studied, can complete a chloroplast and nrDNA sequence by providing appropriate assembly conditions using WGS. Therefore, the present invention has been carried out to find the optimum assembly condition.

Example  2. De novo  Genome Assembler  Selection

Some popular genomic assemblers are SOAP de novo 2.04 (http://soap.genomics.org.cn/) and CLC-NGS-CELL 4.06 beta (www.clcbio.com/products/clc- assembly-cell) was used to compare the ability of constructs containing the chloroplast genome. Both genome assemblers formed contigs containing chloroplast sequences, but for the SOAP de novo 2.04 version, a somewhat larger number of short contigs in the 50x and 250x data sets than in the CLC-NGS-CELL 4.06 beta version And the coverage was also low and the conditions to complete the chloroplast genome were very sensitive. However, in the case of the CLC assembler, the increase in the data set was able to cover the entire chloroplast dielectric with a relatively long congigence of less than five. In particular, even in the relatively large genome size of plants (about 3.2 Gbp), the CLC assembler covered the entire chloroplast with a small contiguous number (Table 3).

Therefore, additional studies were conducted to establish optimal conditions to complete the chloroplast genome and nrDNA sequences using the CLC assembler.

Example  3. Need for chloroplast genome assembly NGS  Amount of data

WGS 44,425,734,760bp of Nipponbare rice variety and WGS 220,948,250,844bp of Ginseng varieties 'Chunpoong' were used to find the appropriate amount of data to assemble chloroplast and nrDNA sequences using WGS data. These amounts corresponded to about 100 times and 70 times the coverage of rice and ginseng dielectrics, respectively, and each WGS contained 1.69% and 6% of chloroplast genomes, respectively. Based on this, 10 WGS data sets containing 50 to 5,000 times based on the chloroplast genome were generated as shown in Table 4 below.

In rice and ginseng, the genomic coverage of chloroplast genome coverage was 50 times and 1,050 times, respectively, and rDNA coverage was 324 times and 3,560 times, respectively. After assembly for each data set, we checked the number of contigs and assembly errors including chloroplast sequences (NCBI accession No. GU592207.1).

Rice ( Oryza sativa ) dataset from OS3-OS6, ginseng ( Panax ginseng ) data set showed a small amount of sequence errors as shown in Table 5 below, with a small number of contigs in PG3-PG7.

Data sets with a chloroplast genome reference depth of less than 50 times and 1,000 times greater did not cover the entire chloroplast genome or increased the number of chloroplast contigs and increased assembly errors (FIGS. 2 and 3).

Using the NGS data set of rice and ginseng, the appropriate amount of WGS data showed that there was a small gap and mismatch in the data set of about 100 to 500 times based on chloroplast, which ranged from 0.86 to 4.3 Gbp The amount of WGS is equivalent to 0.3 ~ 1.5Gbp of ginseng. Therefore, it is confirmed that even a plant having a large genome size such as ginseng can be assembled with a certain amount of WGS sequence according to the degree of chloroplast genome incorporation there was.

Example  4. Correction of assembly errors

When the chloroplast genome is assembled with NGS data, gaps or unspecified nucleotides 'N' appear, including false gaps, errors due to repetition of tandem repeats, errors due to monopolymers, And single nucleotide polymorphism (SNP) type errors due to genomic DNA interference. Before correcting errors in the creation of chloroplast genomes with NGS data, you should first find error loci, which can be accomplished by mapping the raw data after completing the draft chloroplast genome sequence and using the raw data trends mapped through the CLC assembly viewer It is necessary to look at the whole. Areas with assembly errors can be calibrated as follows, since there are many mis-mapped primitive leads (sequence fragments).

4-1. False gap

Gaps caused by assembly errors, although not actually gaps, usually contained N in the assembled sequence. As shown in FIG. 4, although there are overlapping sequences on the right and left sides of the misaligned sequence centered on N, a fake gap having one N in the assembly process is created. The overlapping portions are artificially combined with one sequence, , And the modified sequences were clearly mapped to native leads. This result confirmed that the modified sequence and reference sequence coincided with the sequence of the rice Nippon barley variety, and in the case of ginseng, the PCR and sequencing were also able to confirm the method of resolving the fake gap through the modification process .

4-2. Tandem repeats

NGS produces significantly more data than lenticular sequencing, but the likelihood of assembly errors due to short lead lengths (around 100bp) has increased. In particular, in the de novo genome assembly, tandem duplication regions often experienced assembly errors due to changes in the number of copies. Repetition with longer repeat units than the read length or tandem or interspersed within the genome resulted in repeat collapse and rearrangement. If the length of the repeat unit is smaller than the analytical quantity, the error can be solved by adjusting the k-mer value according to the repeat length. As shown in FIG. 5, the repeated collapse of the column redundancy in units of 18 bp was corrected from 2 copies to 4 copies when the k-mer value was set to 64 at maximum. When primitive data mapping is performed on a draft chlorophyll sequence assembled with less than the number of column redundancies, not only can the mis-mapped leads be identified where repeat collapse occurs, but also because the mis- In addition, since it is significantly higher than the surrounding area, it is possible to predict and correct duplication error of repetition. Since the unit size of column redundancy in most plant chloroplast genomes is less than 100bp, almost all errors can be detected and removed by this method.

4-3. Monopolymer

Monopolymers cause many problems in the genome of chloroplasts as well as genomic DNA. In the chloroplast genome of rice and ginseng, the regions of 8 mer or more homopolymer were found to be 95 and 91, respectively. Among them, adenine (A) or Thymine (T) repetition accounted for the majority (Table 6).

Although errors in this single polymer region may be caused by sequence errors, a large number of mutations accumulate in the homopolymer region, especially in the chloroplast sequence fragments inserted into mitochondria or nuclear DNA, and these sequences interfere with the assembly causing chloroplast genome assembly errors Of the cases. In rice, DNA fragment sequences derived from the chloroplast genome were distributed throughout chromosomes. In the 78,424bp (NCBI accession No. GU592207.1) of the rice chloroplast genome, 17 Ts are homopolymers, which are distributed in 10 chromosomes of rice, especially in the T polymer region (Fig. 6 And b). In the initial context assembled with the Os3 data set, it was assembled into T8, which could be judged to be misassembled due to a similar chloroplast sequence present on chromosomes 5, 6, 7 and 9 in the nucleus. As a correction method, it was possible to correct the shape by randomly selecting a single polymer having a high depth by mapping the raw data after randomly generating sequences according to the number of homopolymers T existing in the raw data. Since the chloroplast genome sequence exists at a high depth in the NGS data, it can be concluded that the selection is the most accurate chloroplast genome sequence. Actually, a draft chloroplast genome sequence was constructed with sequences having sequences T, 7, 8, 9, 10, 11, 12, 15 and 17 T with repeating T homopolymer combinations, followed by pair- end), the chloroplast genome sequence was confirmed to be the highest in the reference chloroplast genome sequence with 17 single T polymers at 33.14 (Fig. 6c). Thus, the degree of chloroplast-derived sequences in the nucleus DNA affects the assemblage specifically in the rice chloroplast genome assembly, indicating that the amount of WGS data used was more than five times that of rice genome coverage, The error probability increased and the formation of chimeric assembly increased.

4-4. Spurious single nucleotide polymorphisms (SNPs) due to interference of homologous mitochondria and nuclear DNA

When the amount of WGS used in the initial assembly increases, chloroplast-derived DNA fragments inserted into the mitochondrion and nuclear genome may mistakenly participate in assembly, resulting in single nucleotide polymorphism (SNP) errors. The de-bruin graph (Compeau et al. 2011, Nat Biotechnol, 29: 987-991), such errors have occurred very rarely (Figure 7b). This type of error appeared as if it were a SNP and was confirmed through the identification of primitive leads mapped to the draft assembly. Although the original chloroplast genome using the Os5 set shows guanine (G) and thymine (T) at 51,940bp and 51,944bp, most primitive leads (186 out of 212) have T and A and are mapped incorrectly (Fig. 7A). On the other hand, 24 out of 212 sequences with G and T were found to be present in the mitochondria. This allowed us to compensate for false SNPs with T and A, the major sequences of 186 of the total 212 leads.

Example  5. Optimization of the conditions for the sequencing of the complete chloroplast genome

Genomic DNA was prepared from plant leaves and a 300-500 bp pair of at least 1 μg of the genomic DNA was prepared. Using the HiSeq2000 or MySeq (Illumina, USA) platform, WGS 1Gbp data was generated. In order to remove the sequences having low quality value among the sequences of the generated data set and to know the contamination rate of the chloroplast sequences, a relational sequence is found in a public database and is mapped using the CLC reference assembly tool, The chloroplast contamination rate of the set was determined, and WGS containing about 100 to 500 times the amount of chloroplast genome coverage was extracted and assembled using a CLC assembler. It is helpful to set the k-mer value to 64 and proceed with the assembly to prevent incorrect assembly of the column redundancy. After a gap filling process after assembly, the existing known chloroplast sequences are compared using the BLAST function, the chloroplast sequences are discriminated in the assembled contig data set, the sequence is determined, the overlap between the contig sequence is found, Chloroplast cotig (Fig. 8). Through the mapping of the generated chloroplast contig with the raw data, the part with the error is found, and through the raw data mapping, the fake gap, the column redundancy error, the single polymer error, and the SNP error, Respectively.

Example  6. Complete nuclear ribosome DNA  How to assemble unit sequences

The 45s transcriptional gene region, ITS1 (internal transcribed spacer 1) and ITS2 region have relatively stable structure and have been used as a major target of plant evolution and differentiation studies. One unit of 45s nrDNA of plant is known to be about 6-18kb in length and has been reported to be homogenized rapidly in a plant species by concerted evolution but also exists in some heterogeneous form. The difference in the length of the nrDNA units is mainly due to the length diversity of the tandem subrepeat elements present in the intergenic spacer (IGS). In addition, the column sub-repeating elements present in IGS generate heterogeneous forms in the genome by unequal crossing over, and therefore, most of them do not contain complete nrDNA units even in the plants in which the whole genome is detoxified.

In the present invention, together with the chloroplast genome assembly, a simple and accurate method for completing the 45S nrDNA repeat unit, which is an important object of plant evolution and differentiation studies, has been developed. The protocol presented is the most representative sequence of the nrDNA unit in a way that completes the IGS sequence together with the 45s nrDNA transcription unit, but does not mean that there is no different kind of nrDNA.

Contigs assembled in a random set and identified as nrDNA sequences had an almost complete 45s sequence and contained all or part of the IGS sequence. Errors in nrDNA sequence assemblies include heterozygosity in the genome, difficulty in identifying a representative sequence, occurrence of N due to repetitive collapse due to the presence of tandem arrays, . However, almost all of the complete nrDNA sequences could be completed through the following steps.

First, in the assembly of conserved sequences within 45s, SNP could occur due to the presence of heterologous forms. In this case, mainly in ITS1 and ITS2, the selection of nucleotides with the highest depth was advantageous in finding a representative form. In addition, we were able to find different types of seeds at the same time by selecting heterogeneous sequence leads.

Second, IGS repetition collapses due to the array of sub - repeating elements in IGS. IGS of ginseng showed various repetition sizes ranging from 8bp to 641bp. The 641bp repetition unit was 3.5 copies, which again were arranged in columns of 337bp, 149bp, and in many cases the wrong assembly occurred. Since there are several repeats larger or smaller than the read length and there are some sequence differences between the repeating units, it is possible to solve the collapse of repeats based on the mapping information of the pair ends.

Third, when one congener produced by a de novo assembly contains only the 45s rDNA gene region and some IGS sequences, the two contigs generated using the 45s rDNA feature that is arranged in columns are connected in parallel, Filled up with 50-200 nucleotides and artificially created a new contig. The generated new contig file was subjected to the process of removing the nucleotide through the Gapcloser (SOAP de novo package) program, and the final unit was completed through the raw data mapping.

Example  However, Nippon Bare 'Chloroplasts and nuclear ribosomes of varieties DNA Verification using

Rice genomic DNA was isolated by using the standard cultivar Nipponbare, which has almost complete genome and chloroplast sequence, and confirmed the chloroplast and nrDNA by the above method. Additional PCR and ABI sequencing re-confirmation experiments were performed to compare the reference sequence with the completed sequence to see if there was an assembly error.

As a result, the chloroplast sequence of 134,591 bp and the 7,928 bp rDNA sequence completed by the method of the present invention exactly coincided with the reference sequence, showing that the method of the present invention is correct (Fig. 9). The nrDNA repeat unit including rice IGS consists of 5,877 bp of 45s (18s-5.8s-26s) and 2,051bp of IGS including external transcribed spacer (ETS) and non-transcribed spacer (NTS) 254bp sub-units known as < RTI ID = 0.0 > a < / RTI > In the completed rice genome standard information, it was confirmed that about 4.5 copies corresponding to 100% of the unit of the present invention exist at the upper end of chromosome 9 (GenBank No. OSJNBb0013K10; AP008245.2).

Example  8. Chloroplast genomes for various plant species de novo  assembly

Variety of plant species according to the foregoing method of the present invention moss, rice plant and closely related species genome size is relatively large ginseng and American ginseng (Panax quinquefolius ) and onion. The size of the ginseng 'Chunpoong' was 156,248bp and the number of ginseng was 156,088bp. The nucleotide sequence variation between the species was about 0.1% (Fig. 12). We confirmed that the correct chloroplast genome was generated by PCR and ABI sequencing for error - prone regions such as column redundancy. On the other hand, mutations of 127 SNPs and 71 insertions (indel) were found in the GenBank genomic sequence (GenBank No. AY582139.1, 66) in GenBank, which may be caused by differences in materials or the possibility of sequencing errors . On the other hand, when the chloroplast genome sequence was completed for the 12 ginseng cultivars according to the present invention, the mutation regions of all the ginseng cultivars were within 10 SNPs and 7 Indels, and the previously reported chloroplast genome sequences were PCR- (Fig. 13).

In addition, seven WGS varieties and nine Arizona Genomics Institute WGSs for the Oryza Map Alignment Project and nine or five varieties of Leersia varieties, Some application examples in which the 4Gbp WGS data were distributed and the chloroplast genome was completed by the method of the present invention are shown in the tables and figures (Table 7 and FIG. 14).

In addition, a phylogenetic tree was examined based on the decoded chloroplast genome sequence, and it was confirmed that the degree of similarity in the vicinity of rice (Fig. 14). In rice, Japonica and Indica cultivars were clearly distinguished, and no variation was observed among the varieties belonging to the same subspecies group. In addition, Indica and Japonica hybrid varieties Tongil, Dasan and Milyang 23 showed the same chloroplast type as the final model (Fig. 15).

Example  9. Complete nuclear ribosome for rice and ginseng species DNA  Unit of de novo Assembly Blury

The lengths of the 45s transcriptional units were 5,856bp and 5,853bp, respectively, and the lengths of IGS were 5,235bp and 5,316bp, respectively, longer than that of rice. There was almost no homology (Figs. 10B and 10C). PCR confirmed the product of the expected length and predicted that the assembly was correct (Figures 10d and 10e). However, an additional band of about 500 bp in length was amplified (Fig. 10 (e) and (10 f)) in the amplification of ginseng IGS, indicating that a variant type of nrDNA unit exists or a region other than the NOR region in the nuclear genome Derived rDNA fragments present in E. coli. The repeat number of the lower repetition known as the enhancer of the 45s transcription unit located in the 5 'ETS in the IGS is a serial arrangement of 3.5 copies of 641bp in chestnut and 3.5 copies of 640bp in 3.5 copies (variable: 95%). Of the 17 rice species, the wild rice ( Oryza nivara ) was not able to complete the genome differently from other species, presumably because of the heterogeneous rDNA in the wild rice genome. When the 45S nrDNA regions of 17 rice species were compared, although some mutations were observed particularly in the regions of ITS1 and ITS2 (Fig. 16), the mutation among the ginseng cultivars showed a SNP in the region of 5.8s. Ginseng and USS showed significant differences in IGS and SNP in some gene regions and ITS regions. Phylogenetic analysis of the 15S rDNA region sequences of 13 kinds of ginseng and 16 kinds of rice (except O. nivara ) obtained by the present invention was confirmed (FIGS. 17 and 18).

Example  10. Identification of species and variety after completion of chloroplast sequence Marker  Development verification

Using the method of the present invention, it was possible to complete chloroplasts and rDNA for more than 100 kinds of plants. In case of lichen, mitochondria could also be completely completed. Based on the completed sequence, various PCR markers can be efficiently developed that can identify the species from each other and can be used effectively for the development of barcoding markers for identification, classification and classification of herbal medicines. (Fig. 19). In addition, when the chloroplast sequence was completed for other varieties of the same species, a variety specific marker could be developed. As shown in FIG. 20, three specific markers could be developed between the two ginseng varieties, thereby utilizing them for breed identification and protection of breed rights It is very useful as a means of developing a marker that can be used (Fig. 21).

In addition, it has been confirmed that the marker can be efficiently used for marker development that can be used for species classification and breed identification by developing a unique markers for identifying interspecies species and cultivars through rDNA sequences (FIG. 22).

Claims (8)

(a) decoding a nucleotide sequence of a whole genome of an organism by a next generation sequencing (NGS) method;
(b) generating an NGS data set based on the genomic coverage amount of chloroplast, mitochondrial or nuclear ribosomal DNA using the leads (sequence fragments) generated through the nucleotide sequence decode of step (a);
(c) assembling leads of the generated NGS data set of step (b) using assembly software;
(d) isolating contigs comprising at least one sequence selected from the group consisting of chloroplasts, mitochondria, and nuclear ribosomal DNA (nrDNA) sequences in the contig produced after assembly in step (c); And
(e) linking the separated contigs of step (d) using a nucleotide sequence comparison program and correcting errors occurring in the assembly, wherein the complete genomic sequence of the chloroplast, mitochondrial or nuclear ribosomal DNA of the organism Either alone or simultaneously. The method according to claim 1, wherein the step (e) of decrypting the complete nucleotide sequence of the chloroplast or mitochondrion comprises aligning and ligating the cotig containing the chloroplast sequence among the isolated contigs of step (d) Then mapping the generated raw data sequence and eliminating assembly errors. The method according to claim 1, wherein the step (e) of decrypting the complete nucleotide sequence of the nuclear ribosomal DNA comprises artificially arranging two contigs comprising the 45s rDNA sequence of the separated contigs of step (d) , And using a gap closer program to fill the physical gaps in the IGS (intergenic spacer) region, complete a complete 45s rDNA unit, and complete the complete 45s rRNA unit primitive Mapping the data sequence and eliminating assembly errors. The method according to claim 1, wherein the organism is a microorganism, a lower photosynthetic organism, a mushroom, a higher plant having a large genome, an insect, a fish or an animal. 2. The method of claim 1, wherein the NGS data set is an amount that can cover 50 to 500 times the chloroplast genome. 2. The method of claim 1, wherein the assembly software is CLC de novo assembly software or SOAP de novo assembly software. 2. The method of claim 1, wherein the assembly error is a base sequence error, a false gap, a tandem repeat error, a monopolymer error, or a single nucleotide polymorphism (SNP) error. A recording medium on which a computer-readable program for performing the method according to any one of claims 1 to 7 is recorded.

Images