Method for identifying plant brachyplast in nuclear element family and method for acquiring full-length members of family

Abstract

The invention belongs to the field of genetics data processing, and relates to a method for identifying a plant brachyplast in a nuclear element family and a method for acquiring full-length members of the family. The invention discloses a method for processing redundant candidate SINE sequences, confirming the SINE sequences, dividing SINE families and obtaining full-length members of the families. According to the method, the real SINE sequence can be accurately identified from the candidate sequences output by the SINE-finder, and the family of the SINE can be accurately divided. The method is suitable for researchers with a certain bioinformatics basis to carry out plant SINE research.

Description

Technical Field

[0001] The present invention belongs to the field of genetic data processing, and relates to the identification of plant short interspersed nuclear elements (SINEs) and the acquisition of full-length family members. Background Art

[0002] Transposons are one of the main sources of variation in the composition and structure of eukaryotic genomes. Transposons can provide regulatory sequences such as promoters, enhancers, and repressors for neighboring genes, and are one of the driving factors for gene expression variation. The variation of many important agronomic traits is causally related to transposons, which means that the study of transposons will play an important role in crop breeding.

[0003] There are many types of transposons, and SINE is one of them. It is a non-autonomous retrotransposon. It is small in length and numerous, but accounts for a small proportion in plant genomes. Most of the existing plant genome annotations lack information about SINEs, and there is also a lack of systematic research on SINEs. SINEs are often distributed near genes or in introns, and have a great impact on the structure of genes and genomes. SINEs are significantly enriched in the upstream region of citrus genes and have a great potential to affect the expression of neighboring genes, indicating that the study of plant SINEs has potential application value in breeding and deserves further study.

[0004] In 2011, a recognition software called SINE-finder was developed specifically for SINEs. The algorithm of this software is based on several sequence features of SINEs: two RNA polymerase III promoter motifs (boxA and boxB), target site duplication (TSD), and 3'-end Poly (A / T) tail, which is about 80 to 500 bp in length. These features, especially the key recognition features (boxA and boxB), are not particularly obvious, resulting in low recognition efficiency. Subsequently, a new software, SINEscan, appeared, which made some improvements to SINE-finder, but the false positive rate of these two softwares was as high as 95%. Later, a new software, AnnoSINE, appeared, but some tests that have not yet been officially published showed that the false positive rate of this tool in identifying SINEs was about 13.3%, indicating that these tools still have a lot of room for improvement. Judging from the research results of SINEs that have been published so far, the SINE candidate sequences output by the software still need further screening, especially manual screening by experts, to exclude false positive sequences.

[0005] SINEs can capture adjacent genomic regions by transduction to form new SINE families, or insert into another SINE to form dimers or even multimers, which can then amplify and form new SINE families. This leads to some complex evolutionary relationships between SINE families, adding obstacles to the division of SINE families.

[0006] In order to further optimize the process of processing redundant candidate sequences, confirming SINE sequences, dividing SINE families and obtaining full-length family members, the present application conducted the following research. Summary of the invention

[0007] Aiming at the technical problem that the false positive rate of the output results of the SINE identification program SINE-finder is too high and the obtained candidate SINE sequences need to be further screened, the present invention proposes a method for identifying a family of plant short interspersed nuclear elements and a method for obtaining full-length members of the family.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The present application provides a method for removing redundant sequences, which is implemented by an all-vs-all blast module, which removes redundant sequences in a set of sequences to be processed according to a preset standard, including the following contents:

[0010] a. Use cd-hit-est software to remove repeated sequences from candidate SINE sequences. When running cd-hit-est software, three parameters that deviate from the default values ​​are set as: -c 0.95 -G 0 -aS 0.95.

[0011] b. Construct a blast database with the candidate SINE sequences output in step a, using the makeblastdb command in the ncbi-blast+ software package. When the command is run, two parameters that deviate from the default values ​​are set: -dbtype nucl -parse_seqids.

[0012] c. Using the candidate SINE sequence output from step a as the query sequence, use the blastn command in the ncbi-blast+ software package to search the blast database constructed in step b. Each candidate SINE sequence used as a query and its homologous candidate SINE sequence will form a homologous sequence group. The screening criteria for homologous sequences are sequence identity greater than 80% and HSP length accounting for 80% of the query length.

[0013] d. Calculate the number of sequences in the homologous sequence group generated by each query sequence, retain the query sequence corresponding to the homologous sequence group with the largest number of sequences, and remove other sequences in this homologous sequence group.

[0014] e. Extract the remaining candidate SINE sequences and perform the next round of blast until the number of homologous sequences of all remaining candidate SINE sequences is zero. The remaining candidate SINE sequences, whose sequence similarity is lower than the preset standard, are regarded as non-redundant unique sequences for subsequent analysis.

[0015] A method for identifying a plant short dispersed nuclear element family comprises the following steps:

[0016] Step 1: Use the SINE-finder program to scan the genome of the target species, and the output of SINE-finder is the candidate SINE sequence.

[0017] Step 2: Use the all-vs-all blast module attached to the present invention to process the candidate SINE sequences output by SINE-finder, remove redundant candidate SINE sequences, and obtain unique candidate SINE sequences.

[0018] Step 3: Using the unique sequence obtained in step 2 as the query sequence, use the ncbi-blast+ software package to scan the genome sequence of the target species, obtain all homologous sequences and their flanking sequences for each unique sequence, and form their respective homologous sequence groups. The screening criteria for homologous sequences are that the sequence identity is higher than 80% and the HSP length accounts for 80% of the query length.

[0019] Step 4: Multiple sequence alignment of each homologous sequence group was performed using muscle software. The alignment results of each group of sequences were manually checked, and the homologous sequence group with typical characteristics of SINE was confirmed as the SINE homologous sequence group.

[0020] Step 5: Use Seaview software to generate consensus sequences for each SINE homologous sequence group. Merge these consensus sequences into one file, and use the all-vs-all blast module to further remove the redundancy in these consensus sequences. Each remaining consensus sequence represents a SINE family.

[0021] Step 6: Use the common sequence of each SINE family generated in step 5 as the query sequence, use the ncbi-blast+ software package to scan the genome sequence of the target species, obtain all homologous sequences and flanking sequences of each common sequence, and form their own homologous sequence groups. The output result screening criteria are the same as step 3. Each group of SINE homologous sequences obtained from each common sequence represents a SINE family and its members.

[0022] Step 7: Use muscle to compare the homologous sequence groups of each SINE family. Again, manually verify whether each SINE family has the typical characteristics of SINE. The sequence with complete characteristics is recorded as the full-length copy of the family. SINE families with more than 5 full-length copies in at least one species are retained and as the final result, the others are eliminated.

[0023] The present invention has the following beneficial effects:

[0024] 1. This patent provides a method for screening SINE candidate sequences. SINE-finder is a program for identifying plant SINEs based on structural features, and provides core algorithms for a variety of SINE identification software packages. However, the false positive rate of the software is as high as 95%, and its output results need to be screened before subsequent work can be carried out. SINE-finder can output up to thousands of candidate SINE sequences, which makes the screening process of candidate SINE sequences very cumbersome and labor-intensive, and also has certain requirements for programming capabilities. The method provided by the present invention can optimize the screening of candidate sequences. As long as the genome of the target species is provided, the method provided by the present invention is used to remove duplicate and redundant candidate SINE sequences, and obtain a homologous sequence group of the candidate SINE sequences, combined with a graphical manual inspection, the SINE sequence in the SINE-finder output results can be confirmed.

[0025] 2. This patent provides a method for dividing SINE families and obtaining full-length family members. Since SINEs can capture genomic regions to generate new SINE families, and can also generate new SINE families through multimerization, there are complex evolutionary relationships between SINE families, which makes the division of SINE families more difficult. The method provided by the present invention can merge various redundant SINE sequences and divide them into SINE families. With the representative sequences of these families as references, the full-length members of each SINE family can be easily obtained through blastn software. Using this method, through two graphical manual inspections before and after, the work of SINE candidate sequence screening and family division that previously required a lot of manual operations and was cumbersome can be completed, which is of great significance for the identification, function and evolution of SINEs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1Schematic diagram of the process of identifying and dividing plant SINEs into families according to the present invention.

[0028] Figure 2 Schematic diagram of the process of the all-vs-all blast module.

[0029] Figure 3 Examples of typical features of plant SINE sequences (used for manual inspection of the multiple alignment results in steps 4 and 7). DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for identifying a plant short dispersed nuclear element family comprises the following steps:

[0033] Step 1: Use SINE-finder to scan the target genome. SINE-finder is run with default parameters. Each sequence in the output file of SINE-finder software is used as a candidate SINE sequence for subsequent identification and family division analysis.

[0034] Step 2: Use the all-vs-all blast module to further process the output of SINE-finder. The module first uses cd-hit-est to remove repeated sequences in the candidate SINE sequences (the criteria for repeated sequences are: sequence similarity is higher than 95%, and the alignment region accounts for 95% of the length of the longer sequence). Then multiple rounds of all-vs-all blast are performed. When running all-vs-all blast, if the similarity between candidate SINE sequences is higher than the preset standard (sequence consistency is higher than 80%, and HSP length accounts for 80% of the query length), a homologous sequence group will be formed. After each round of all-vs-all blast, the candidate SINE sequence with the most homologous sequences is retained, and all sequences homologous to it are deleted. The remaining candidate SINE sequences undergo the next round of all-vs-all blast until no homologous sequences are found for all the remaining candidate SINE sequences. The remaining candidate SINE sequences are analyzed in the next step as unique sequences.

[0035] Step 3, uses the makeblastdb command in the ncbi-blast+ software package to build the blast database of the target species genome, and the command parameter is set to: -dbtype nucl-parse_seqids. Take the unique sequence obtained in step 2 as the query sequence, use the blastn command in the ncbi-blast+ software package to scan the blast database of the genome. In the parameters of the blastn command, two parameters that deviate from the default value are set to: -perc_identity 80 and -qcov_hsp_perc 80 (blastn command adopts this parameter setting in the present invention). The blast output result that each unique sequence obtains represents its homologous sequence and constitutes its original homologous sequence group. Use the blastdbcmd command in the ncbi-blast+ software package to extract the sequences of each original homologous sequence group and the flanking 100bp sequences on both sides thereof to constitute its homologous sequence group. This command adopts default parameters.

[0036] Step 4: Use muscle software to perform multiple sequence alignment on each homologous sequence group obtained. The alignment results of each homologous sequence group are manually checked, and the homologous sequence group with typical characteristics of SINE is confirmed as the SINE homologous sequence group.

[0037] When manually checking the alignment results of homologous sequence groups, the screening criteria ( Figure 3 The reference examples of typical characteristics of SINE are as follows: 1. After multiple alignments, the homologous sequence group should be clearly divided into three parts. The two flanking regions at both ends, about 100bp, should be highly variable; the middle region, about 80 to 500bp in length, should be relatively conservative. 2. The conserved region in the middle contains two motifs, box A and box B, both of which are 11bp long and 30-35bp apart. Note that the degenerate motifs that box A and box B can recognize are (box A: RVTGG and box B: GTTCRA). 3. There is a poly(A / T) tail at the 3' end of the middle conserved region, and the poly(A / T) contains at least 5 consecutive A / Ts.

[0038] Step 5: Use Seaview software to generate consensus sequences for each SINE homologous sequence group confirmed in step 4. The relevant parameters of the software are set to allow gaps and use IUPAC symbols. Merge these consensus sequences into one file and use the all-vs-all blast module to remove redundant sequences in these consensus sequences. Each sequence in the output of the all-vs-all blast module is a consensus sequence of an independent SINE family.

[0039] Step 6: Use the common sequence of each SINE family generated in step 5 as the query sequence and use the blastn command to scan the blast database of the genome. The blast output result obtained for each unique sequence represents its homologous SINE sequence and constitutes its original homologous sequence group. Use the blastdbcmd command to extract each sequence of each original homologous sequence group and the flanking 100bp sequences on both sides to form its homologous sequence group. This command uses default parameters.

[0040] Step seven, using muscle software, perform multiple sequence alignment on each SINE homologous sequence group generated in step six, and manually check the alignment results again (see step four for the inspection criteria) to confirm again whether each family has the typical characteristics of SINE. The sequence with complete characteristics is recorded as the full-length copy of the family. The SINE families that pass the second manual inspection are further screened. The screening condition is: there must be at least 5 full-length copies in one species. Families that do not meet the conditions are eliminated, and the SINE sequences of each remaining group represent a SINE family and its full-length members.

[0041] The all-vs-all blast module involved in the above method is to remove redundant sequences from a set of sequences to be processed according to a preset standard. The specific operation is as follows:

[0042] a. Use cd-hit-est software to remove repeated sequences from candidate SINE sequences. When running cd-hit-est software, three parameters that deviate from the default values ​​are set as: -c 0.95 -G 0 -aS 0.95.

[0043] b. Construct a blast database with the candidate SINE sequences output in step a, using the command makeblastdb in the ncbi-blast+ package. When this command is run, two parameters that deviate from the default values ​​are set: -dbtype nucl -parse_seqids.

[0044] c. Using the candidate SINE sequence output from step a as the query sequence, use the blastn command in the ncbi-blast+ software package to search the blast database constructed in step b. When the command is run, two parameters that deviate from the default values ​​are set to: -perc_identity80 -qcov_hsp_perc 80. Candidate SINE sequences with similarities higher than the preset standard will form a homologous sequence group.

[0045] d. Calculate the number of sequences in the homologous sequence group generated by each query sequence, retain the query sequence corresponding to the homologous sequence group with the largest number of sequences, and remove other sequences in this homologous sequence group.

[0046] e. Extract the remaining candidate SINE sequences and perform the next round of blast until the number of homologous sequences of all remaining candidate SINE sequences is zero. The remaining candidate SINE sequences, whose sequence similarity is lower than the preset standard, are regarded as non-redundant unique sequences for subsequent analysis.

[0047] Application Examples

[0048] Screening of kiwifruit SINE candidate sequences and division of SINE families.

[0049] In this example, three kiwifruit genomes, namely, 'Hongyang' (Actinidia chinensis Planch., hongyang_rv3), 'Red5' (Actinidia chinensis Planch., Red5_rv1), and 'white' (Actinidia eriantha Benth., white_rv1), were selected as examples to screen SINE candidate sequences and divide SINE families.

[0050] (1) Download the genome sequences of ‘Hongyang’, ‘Red5’ and ‘white’ from the kiwifruit genome website (http: / / kiwifruitgenome.org / ).

[0051] (2) The genome sequences of three kiwifruit species were scanned using SINE-finder, and the default parameters were used when running SINE-finder. The 'Hongyang' genome produced 1690 candidate SINE sequences, and the 'Red5' genome produced 917 candidate SINE sequences.

[0052] The genome of 'white' produced 1623 candidate SINE sequences. After merging the candidate SINE sequences of the three kiwifruit species, 4230 candidate sequences were obtained for subsequent processing.

[0053] (3) The 4230 candidate SINE sequences obtained in step (2) were processed using the all-vs-all blast module to generate 3553 deduplicated candidate SINE sequences. After removing redundant sequences, 2698 unique candidate SINE sequences were generated.

[0054] (4) Using the 2698 unique candidate SINE sequences obtained in step (3) as query sequences, the blastn command was used to scan the blast database of the three genomes, and a total of 2698 original homologous sequence groups were obtained. The blastdbcmd command in the ncbi-blast+ software package was used to extract each sequence of the original homologous sequence group and the flanking 100 bp sequences on both sides, and 2698 homologous sequence groups were obtained.

[0055] (5) Perform multiple sequence alignment on the 2698 homologous sequence groups obtained in step (4), and manually check the alignment results (refer to Figure 3 The 42 homologous sequence groups have typical characteristics of SINEs and are confirmed as SINE homologous sequence groups. The positions of the 42 candidate SINE sequences on chromosomes and the lengths of target site duplications (TSDs) corresponding to them are listed as follows:

[0056]

[0057]

[0058]

[0059] (6) Using Seaview software, we generated consensus sequences for each of the 42 SINE homologous sequence groups. We used the all-vs-all blast module to remove redundant sequences from these consensus sequences, and obtained 16 non-redundant consensus sequences, representing the 16 SINE families in the kiwifruit genome. These 16 consensus sequences are as follows.

[0060]

[0061]

[0062] (7) Using the common sequences of the 16 SINE families as query sequences, the blastn and blastdbcmd commands were used to scan the three genomic blast databases to obtain the respective SINE homologous sequence groups. The 16 homologous sequence groups obtained all represent the 16 SINE families and their members.

[0063] (8) Using muscle software, multiple sequence alignment was performed on these 16 homologous sequence groups, and the alignment results were manually checked again, confirming that the 16 homologous sequence groups have typical characteristics of SINEs. Since the full-length copies of Kiwi-2 in the three species are less than 5, Kiwi-2 and its homologous sequence groups were eliminated, and the remaining 15 groups of SINEs each represent a SINE family and its full-length family members.

[0064] The tool software used in this method and the website where it is obtained are as follows.

[0065] 1.SINE-finder: https: / / doi.org / 10.1105 / tpc.111.088682

[0066] 2.cd-hit-est: https: / / github.com / weizhongli / cdhit / releases.

[0067] 3.ncbi-blast+: https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / .

[0068] 4.muscle: https: / / github.com / rcedgar / muscle / releases / latest.

[0069] 5.Seaview: https: / / doua.prabi.fr / software / seaview_data / seaview5.exe.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for removing redundant sequences, characterized in that: The steps are: a. Use cd-hit-est software to remove duplicate sequences in the candidate SINE sequences to obtain duplicate-free candidate SINE sequences; b. Using the deduplicated candidate SINE sequences of step a as the object, use the makeblastdb command in the ncbi-blast+ software package to construct a blast database; c. Using the candidate SINE sequence of step a as the query sequence, using the blastn command in the ncbi-blast + software package, searching the blast database constructed in step b, a plurality of homologous sequence groups corresponding to the query sequence were obtained; d. Calculate the number of sequences in each homologous sequence group in step c, retain the query sequence corresponding to the homologous sequence group with the largest number of sequences, and remove other sequences in this homologous sequence group; e. Repeat steps bd until the number of homologous sequences of all remaining candidate SINE sequences is zero and the sequence similarity between them is lower than the preset standard, thus completing the removal of redundant sequences.

2. The method for removing redundant sequences according to claim 1, characterized in that: When the cd-hit-est software is run in step a, three parameters that deviate from the default values ​​are set as: -c 0.95, -G 0, and -aS 0.

95.

3. The method for removing redundant sequences according to claim 2, characterized in that: When the makeblastdb command is run in step b, two parameters that deviate from the default values ​​are set as: -dbtype nucl, -parse_seqids.

4. The method for removing redundant sequences according to claim 3, characterized in that: When the blastn command is run in step c, two parameters that deviate from the default values ​​are set as: -perc_identity 80, -qcov_hsp_perc 80.

5. The method for removing redundant sequences according to claim 4, characterized in that: The preset standards in step d are: sequence identity is higher than 80%, and the HSP length accounts for 80% of the query sequence length.

6. A method for identifying a plant short interspersed nuclear element (SINE) family, characterized in that: The identification method is implemented based on the method described in any one of claims 1 to 5, and the steps are: (1) Use SINE-finder to scan the target genome to obtain sequences as candidate SINE sequences; (2) The candidate SINE sequences in step (1) are processed by the method described in any one of claims 1 to 5 to obtain unique sequences; (3) The unique sequence in step (2) was subjected to blastn and blastdbcmd commands to obtain the homologous sequence and the flanking 100 bp sequence as the homologous sequence group; (4) The homologous sequence group in step (3) was multiple aligned by muscle software and then manually checked to obtain the SINE homologous sequence group; (5) The SINE homologous sequence group of step (4) is processed by Seaview software to obtain a common sequence, and the common sequence of the SINE family is obtained by the method described in any one of claims 1 to 5; (6) The common sequence of the SINE family in step (5) was subjected to blastn and blastdbcmd commands to obtain homologous SINE sequences and flanking 100 bp sequences as a homologous sequence group of a SINE family; (7) The homologous sequence groups of each SINE family in step (6) were multiple aligned using muscle software and manually checked and confirmed again. The SINE sequences with complete features were recorded as full-length copies and then screened for the second time. Finally, each group of SINE sequences obtained was a SINE family.

7. The method for identifying the plant short dispersed nuclear element family according to claim 6, characterized in that: The parameters of blastn in step (3) and step (6) are set as: -perc_identity 80 and qcov_hsp_perc 80.

8. The method for identifying the plant short scattered nuclear element family according to claim 6, characterized in that: The manual inspection and confirmation method in step (4) and step (7) is applicable to the multiple comparison results of homologous sequence groups, and its standard is: ① After multiple alignment, a set of sequences should be clearly divided into three parts. The two flanking regions at both ends, about 100 bp, should be highly variable; the middle region, about 80-500 bp in length, should be relatively conservative; ② The conserved region in the middle contains two motifs, box A and box B. The length of these two motifs is 11 bp, and the interval between them is 30-35 bp; ③ There is a poly(A / T) tail at the 3' end of the middle conserved region, and the poly(A / T) contains at least 5 consecutive A / Ts; The degenerate motif recognizable by the above box A is box A: RVTGG, and the degenerate motif recognizable by box B is box B: GTTCRA.

9. The method for identifying the plant short dispersed nuclear element family according to claim 6, characterized in that: The condition for the second screening is that there must be at least 5 full-length copies in one species.

10. A method for obtaining full-length members of a plant short interspersed nuclear element family, characterized in that: Each group of SINE sequences with complete characteristics obtained by the identification method according to any one of claims 6 to 9 is a full-length member of a SINE family.

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