METHOD FOR HIGH-THROUGHPUT DETECTION OF DIFFERENTIAL EXPRESSION OF PLANT CIRCRNA ALLELIC LOCI

CROSS-REFERENCE TO RELATED APPLICATIONS AND CLAIM TO PRIORITY

This application claims priority to Chinese application number 201811582470.8, filed Dec. 24, 2018 with a title of METHOD FOR HIGH-THROUGHPUT DETECTION OF DIFFERENTIAL EXPRESSION OF PLANT CIRCRNA ALLELIC LOCI, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the technical field of gene expression detection, and in particular, to methods for high-throughput detection of differential expression of plant circRNA allelic loci.

BACKGROUND OF THE INVENTION

An allele (also called allelomorph) generally refers to a pair of genes that regulate phenotypic traits at the same position on homologous chromosomes

Allelic Expression Imbalance (AEI) is in the same cell, and each gene generally has two copies. The cis-acting or trans-acting shifts the expression ratio of the two copies of the gene by 1:1. The phenomenon of AEI is ubiquitous. In addition to the absolute imbalance expression of genetic imprinted gene, a considerable number of genes have AEI in different individuals and in the same individual at different developmental phase and tissues. It is also associated with polymorphic loci in specific regions of the genome.

At present, the commonly used AEI detection mainly focuses on the genes coding for proteins. However, there is no high-throughput accurate analysis method for the allelic expression of circRNA widely existed in transcriptome data.

SUMMARY OF THE INVENTION

In view of the above, an objective of the present invention is to provide a method for high-throughput detection of differential expression of plant circRNA allelic loci.

To achieve the above purpose, the present invention provides the following technical solution.

A method for high-throughput detection of differential expression of plant circRNA allelic loci, which includes the following steps:

1) extracting total RNA of a plant sample, and constructing a strand-specific library with the total RNA;

2) performing paired-end sequencing on the strand-specific library in step 1) with Illumina HiSeq to obtain raw sequencing data;

3) screening circRNAs data from the raw sequencing data obtained in step 2);

4) extracting reverse splicing reads at a circRNAs looping position from the circRNAs data obtained in step 3);

5) performing single nucleotide variation detection on the reverse splicing reads to obtain SNP loci in the reverse splicing reads; and

6) collecting statistics about the number of reads of different genotypes, which are aligned to the SNP loci in step 5, in the reverse splicing reads in step 4, to align the proportion of the number of reads of different genotypes to the proportion of expression quantities of different genotypes.

Preferably, the screening circRNAs data in step 3) includes the following steps: 3.1) performing transcript splicing on the raw sequencing data according to a reference genome;

3.2) extracting 18-22 nt (nucleotides) from both ends of each read in the raw sequencing data that is not aligned to the reads on the reference genome, and forming a pair of anchor sequences, the anchor sequence including a 5′-terminal sequence and a 3′-terminal sequence;

3.3) re-aligning the anchor sequence to a reference genome, where the 5′-terminal sequence of the anchor sequence is aligned to the 3′-terminal of the reference sequence, and the 3′-terminal sequence of the anchor sequence is aligned to the upstream of a matching locus of the 5′-terminal sequence of the anchor sequence in the reference sequence, and a splicing locus GT-AG exists between the matching locus of the 5′-terminal sequence of the anchor sequence and a matching locus of the 3′-terminal sequence of the anchor sequence in the reference sequence, and this splicing locus read is used as circRNA data.

Preferably, the screening circRNAs data is implemented by means of the find_circ software and the CIRIexplorer software.

Preferably, the circRNAs are screened by using the find_circ software and the CIRIexplorer software, respectively, to obtain candidate data of circRNAs screened by the find_circ software and candidate data of circRNAs screened by the CIRIexplorer software, and an intersection of the candidate data of circRNAs screened by the find_circ software and the candidate data of circRNAs screened by the CIRIexplorer software is used as the circRNAs data.

Preferably, the extracting reverse splicing reads at a circRNAs looping position in the circRNAs obtained in step 4) is implemented using a samtools view—R instruction in the find_circ software.

Preferably, the single nucleotide variation detection in step 5) is performed using a SNP calling in the GATK software.

Preferably, after extracting total RNA of a plant sample and before constructing a strand-specific library in step 1), the method further includes a step of removing rRNA and a step of linear RNA digestion that are performed sequentially.

Preferably, a reaction system of the linear RNA digestion is 50 μL, including the following components: 5 μg of RNA, 5 μL of 10× Reaction Buffer, 20 U of RNase R, and the balance of RNase-Free water.

Preferably, the temperature for the linear RNA digestion is 36-38° C., and the time for the linear RNA digestion is 1-2 h.

Preferably, the plant is a forest tree.

The advantageous effects of the present invention: the method of the present invention can perform high-throughput and accurate analysis of differential expression of allelic loci for circRNAs widely existing in transcriptome data and provide a new research strategy for systematic analysis of the transcriptome data.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart showing the differential expression analysis of plant circRNA allelic loci.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for high-throughput detection of differential expression of plant circRNA allelic loci, including the following steps:

1) total RNA of a plant sample is extracted, and a strand-specific library is constructed with the total RNA;

2) paired-end sequencing is performed on the strand-specific library in step 1) with Illumina HiSeq to obtain raw sequencing data;

3) circRNAs data is screened from the raw sequencing data obtained in step 2);

4) reverse splicing reads at a circRNAs looping position are extracted from the circRNAs data obtained in step 3);

5) single nucleotide variation detection is performed on the reverse splicing reads to obtain SNP loci in the reverse splicing reads; and

6) the number of reads of different genotypes, which are aligned to the SNP loci in step 5, in the reverse splicing reads in step 4 are counted to align the proportion of the number of reads of different genotypes to the proportion of expression quantities of different genotypes.

The present invention extracts total RNA of the plant sample and constructs a strand-specific library with the total RNA. The present invention has no special requirement for the types of plant samples, and conventional plants may be used, preferably forest trees. In the specific implementation process of the present invention, Populus tomentosa is selected. The plant sample in the present invention is preferably a leaf tissue. The present invention has no particular limitation on the method for extracting total RNA of the plant sample, and the conventional total RNA extraction method in the art may be used. In the specific implementation process of the present invention, the total RNA is extracted by using an RNA extraction kit (e.g., MagJ ET Plant RNA Purification Kit, No. K2772).

After extracting the total RNA and before constructing the strand-specific library, the present invention preferably further includes a step of removing rRNA and a step of linear RNA digestion that are performed sequentially. The step of removing rRNA is preferably performed by using Ribo-Zero™ rRNA Removal Kits (Plant) (No. MRZPL116). In the present invention, the method for removing rRNA preferably includes: mixing 30-50 μl of total RNA with 50-70 μl of magnetic beads, vortexing for 8-12 s, standing at room temperature for 4-6 min, incubating at 49-51° C. for 4-6 min, placing on a magnetic frame until a supernatant is clarified, and collecting the supernatant; and more preferably, mixing 40 μl of total RNA with 60 μl of magnetic beads, vortexing for 10 s, standing at room temperature for 5 min, incubating at 50° C. for 5 min, placing on a magnetic frame until a supernatant is clarified for 2 min, and collecting the supernatant.

The present invention obtains a Poly (A)-RNA sample, i.e., a linear RNA, after the step of removing the rRNA, preferably digests the obtained linear RNA. The digestion is preferably performed by using RNase Rd. A reaction system of the linear RNA digestion is preferably 50 μL, including the following components: 5 μg of RNA, 5 μL of 10× Reaction Buffer, 20 U of RNase R, and the balance of RNase-Free water. The temperature for the linear RNA digestion is preferably 36-38° C., and more preferably 37° C. The time for the linear RNA digestion is preferably 1-2 h, and more preferably 1.5 h. In the present invention, after the digestion, a strand-specific library is constructed using the digested RNA. In the present invention, a SMART Kit (SMART cDNA Library Construction Kit, NO. 634901) is preferably used to construct the strand-specific library.

After obtaining the strand-specific library, the present invention performs paired-end sequencing on the strand-specific library with Illumina HiSeq to obtain raw sequencing data. The read length of the sequencing described in the present invention is preferably 150 nt. The amount of data for sequencing is preferably greater than 12 G. The sequencing in the present invention may be entrusted to Novogene Company.

After obtaining the raw sequencing data, the present invention screens circRNAs data from the obtained raw sequencing data. In the particular implementation process of the present invention, a linker and the redundant sequence in the raw sequencing data are first removed. In the present invention, the screening circRNAs data includes the following steps:

3.1) transcript splicing is performed on the raw sequencing data;

3.2) 18-22 nt are extracted from both ends of each read in the raw sequencing data that is not aligned to the reads on the reference genome, and a pair of anchor sequences is formed, the anchor sequence including a 5′-terminal sequence and a 3′-terminal sequence; and

3.3) the anchor sequence is re-aligned to a reference genome, where the 5′-terminal sequence of the anchor sequence is aligned to the 3′-terminal of the reference sequence, and the 3′-terminal sequence of the anchor sequence is aligned to the upstream of a matching locus of the 5′-terminal sequence of the anchor sequence in the reference sequence, and a splicing locus GT-AG exists between the matching locus of the 5′-terminal sequence of the anchor sequence and a matching locus of the 3′-terminal sequence of the anchor sequence in the reference sequence, and this splicing locus read is used as circRNA data.

In the present invention, the transcript splicing is performed preferably using default parameters of the Cufflinks software. Steps 3.2) and 3.3) are preferably implemented by the find_circ software and the CIRIexplorer software. More preferably, the circRNAs are screened by using the find_circ software and the CIRIexplorer software, respectively, to obtain candidate data of circRNAs screened by the find_circ software and candidate data of circRNAs screened by the CIRIexplorer software, and an intersection of the candidate data of circRNAs screened by the find_circ software and the candidate data of circRNAs screened by the CIRIexplorer software is used as the circRNAs data.

In the specific implementation process of the present invention, the screening parameters of the find_circ software and the CIRIexplorer software for screening circRNAs include -q 5, -a 20, -m 2, -d 2, and --noncanonical. The screening criteria of the foregoing parameters are preferably: (1) -q 5: the minimum support number of anchor sequence alignment is 5; (2) -a 20: the anchor sequence is 20 bp; (3) -m 2: a branch point cannot occur elsewhere in the range of two nucleic acids in the anchor sequence; (4) -d 2: sequence alignment only supports two mismatches; and (5) --noncanonical: GU/AG appears on both sides of a splicing locus and a clear breakpoint can be detected.

Since the find_circ software and the CIRIexplorer software generate false positive data in the process of screening circRNAs, the intersection of the candidate data of circRNAs screened by the find_circ software and the candidate data of circRNAs screened by the CIRIexplorer software can greatly reduce the false positives, thereby ensuring the authenticity and accuracy of the screened circRNAs data.

After obtaining the circRNAs data, the present invention extracts reverse splicing reads at a circRNAs looping position in the circRNAs data. In the present invention, the extracting reverse splicing reads at the circRNAs looping position from the obtained circRNAs data is preferably implemented using a samtools view—R instruction in the find_circ software.

After obtaining the reverse splicing reads, the present invention performs single nucleotide variation detection on the reverse splicing reads to obtain SNP loci in the reverse splicing reads. The single nucleotide variation detection is preferably performed using a SNP calling in the GATK software.

After obtaining the SNP loci in the reverse splicing reads, the present invention collects statistics about the number of reads of different genotypes in the reverse splicing reads that are aligned to the SNP loci, to align the proportion of the number of reads of different genotypes to the proportion of the expression quantities of different genotypes.

The method of the present invention can realize high-throughput and high-accuracy analysis of the differential expression of the circRNAs allelic loci through the foregoing steps: provide technical support for the analysis of the allelic expression patterns of subsequent circRNAs, and lay a foundation for comprehensively deciphering the regulation effect of gene plant genome allelic expression and the genetic effect of genomic imprinting. Thus, the present invention has a great application value in the analysis of the genetic effects of plant complex traits and molecular design breeding.

The technical solution provided by the present invention will be described below in detail with reference to examples. However, the examples should not be construed as limiting the protection scope of the present invention.

EXAMPLE 1

Fresh leaves of P. tomentosa are used. The total RNA is extracted using an RNA extraction kit (MagJ ET Plant RNA Purification Kit, No. K2772). The rRNA is removed using the Ribo-Zero™ rRNA Removal Kits (Plant) (No. MRZPL116) to obtain a Poly (A)-RNA sample. The linear RNA is digested with RNase Rd (the reaction system includes 5 μg of RNA, 5 μL of 10× Reaction Buffer, 20 U of RNase R, and RNase-Free water supplemented to 50 μL) to obtain the Poly (A)-/Ribo-RNA sample. The strand-specific cDNA library is constructed using a SMART kit (SMART cDNA Library Construction Kit, NO. 634901).

Paired-end sequencing is performed using Illumina HiSeq™ 2500 with a sequencing data volume of 12 G. A linker and a redundant sequence are removed; and the transcript is spliced through the default parameters of the Cufflinks software. 20-nt is extracted, with find_circ, at either end of a sequence that is not aligned to a reference sequence (the reference sequence is the populus trichocarpa V3.0 genomic sequence https://phytozome.jgi.doe.gov/pz/portal.html) as a pair of anchor sequences. Each pair of anchor sequences is re-aligned to the reference sequence. If the 5′ terminal of the anchor sequence is aligned to the reference sequence (the starting and terminating loci are denoted as A3 and A4, respectively), and the 3′ terminal of the anchor sequence is aligned to the upstream of a matching locus of the 5′ terminal of the anchor sequence (the starting and ending loci are denoted as A1 and A2, respectively), and a splicing locus (GT-AG) exists between A2 and A3 of the reference sequence, then this read is used as a candidate circRNA. The screening parameters are -q 5, -a 20, -m 2, -d 2, and --noncanonical. The screening criteria are (1) 1-q 5: yjr minimum support number of the anchor sequence alignment is 5; (2) -a 20: the anchor sequence is 20 bp; (3) -m 2: a branch point cannot occur elsewhere in the range of two nucleic acids in the anchor sequence; (4) -d 2: sequence alignment only supports two mismatches; and (5) --noncanonical: GU/AG appears on both sides of a splicing locus and a clear breakpoint can be detected. Moreover, the circRNAs are screened using the default parameters of the CIRIexplorer software. 887 circRNAs are obtained through the find_circ software analysis, and 920 circRNAs are obtained by the CIRIexplorer software. According to the reverse splicing reads of the circRNAs, the intersection of the two prediction results is obtained, and 97 circRNAs are obtained (Table 1).

TABLE 1

Candidate circRNAs of P. tomentosa leaves

circRNA number
Expression quantity
P value

pto_circ_0000008
699.0198
1.52E−143

pto_circ_0000057
854.3575
3.73E−58

pto_circ_0000187
310.6755
1.38E−67

pto_circ_0000221
1009.695
1.29E−102

pto_circ_0000227
1087.364
6.28E−156

pto_circ_0000284
388.3443
3.11E−71

pto_circ_0000345
466.0132
1.62E−72

pto_circ_0000349
310.6755
4.48E−76

pto_circ_0000521
1165.033
3.96E−73

pto_circ_0000587
310.6755
7.09E−60

pto_circ_0000617
310.6755
7.09E−60

pto_circ_0000674
388.3443
3.11E−71

pto_circ_0000776
621.3509
3.01E−77

pto_circ_0000871
543.6821
2.58E−92

pto_circ_0000892
388.3443
3.11E−71

pto_circ_0000981
310.6755
7.09E−60

pto_circ_0001002
310.6755
7.09E−60

pto_circ_0001090
233.0066
3.67E−156

pto_circ_0001278
310.6755
7.09E−60

pto_circ_0001301
310.6755
7.09E−60

pto_circ_0001411
310.6755
7.09E−60

pto_circ_0001488
310.6755
7.09E−60

pto_circ_0001506
233.0066
8.78E−85

pto_circ_0001508
776.6887
7.50E−58

pto_circ_0001558
699.0198
8.32E−112

pto_circ_0001566
699.0198
2.29E−135

pto_circ_0001622
388.3443
1.28E−149

pto_circ_0001628
2252.397
6.33E−149

pto_circ_0001741
388.3443
3.11E−71

pto_circ_0001816
310.6755
7.09E−60

pto_circ_0002027
466.0132
5.21E−82

pto_circ_0002055
155.3377
7.96E−77

pto_circ_0002058
466.0132
5.21E−82

pto_circ_0002106
621.3509
8.89E−168

pto_circ_0002110
388.3443
3.11E−71

pto_circ_0002122
388.3443
3.11E−71

pto_circ_0002159
1553.377
3.33E−203

pto_circ_0002237
388.3443
1.28E−149

pto_circ_0002256
388.3443
3.11E−71

pto_circ_0002264
1087.364
6.28E−156

pto_circ_0002291
310.6755
7.09E−60

pto_circ_0002292
388.3443
3.11E−71

pto_circ_0002322
310.6755
7.09E−60

pto_circ_0002328
1242.702
3.03E−172

pto_circ_0002377
310.6755
7.09E−60

pto_circ_0002407
2951.417
1.60E−191

pto_circ_0002469
1631.046
2.31E−116

pto_circ_0002472
233.0066
2.64E−66

pto_circ_0002480
621.3509
3.15E−102

pto_circ_0002503
932.0264
8.10E−72

pto_circ_0002610
1165.033
3.64E−164

pto_circ_0002658
233.0066
2.64E−66

pto_circ_0002659
699.0198
8.32E−112

pto_circ_0002730
310.6755
7.99E−85

pto_circ_0002748
155.3377
2.47E−130

pto_circ_0002749
854.3575
3.73E−58

pto_circ_0002750
1786.384
6.41E−281

pto_circ_0002919
233.0066
2.09E−75

pto_circ_0002941
854.3575
3.56E−108

pto_circ_0002982
699.0198
8.32E−112

pto_circ_0003064
155.3377
1.27E−97

pto_circ_0003093
621.3509
3.49E−101

pto_circ_0003157
854.3575
1.73E−88

pto_circ_0003159
310.6755
2.20E−59

pto_circ_0003160
776.6887
4.30E−121

pto_circ_0003167
310.6755
7.09E−60

pto_circ_0003179
621.3509
3.15E−102

pto_circ_0003206
699.0198
8.32E−112

pto_circ_0003256
466.0132
5.21E−82

pto_circ_0003489
155.3377
1.27E−97

pto_circ_0003498
233.0066
2.64E−66

pto_circ_0003579
621.3509
5.15E−65

pto_circ_0003608
1087.364
3.49E−101

pto_circ_0003617
388.3443
3.11E−71

pto_circ_0003620
310.6755
7.09E−60

pto_circ_0003811
543.6821
2.58E−92

pto_circ_0003942
388.3443
3.11E−71

pto_circ_0003970
310.6755
7.09E−60

pto_circ_0004016
1398.04
5.61E−188

pto_circ_0004037
155.3377
3.94E−57

pto_circ_0004047
1087.364
3.49E−101

pto_circ_0004133
2174.728
4.16E−247

pto_circ_0004142
155.3377
8.06E−67

pto_circ_0004203
776.6887
7.50E−58

pto_circ_0004208
543.6821
4.88E−83

pto_circ_0004218
310.6755
7.09E−60

pto_circ_0004238
2407.735
3.85E−280

pto_circ_0004273
543.6821
2.58E−92

pto_circ_0004277
543.6821
2.58E−92

pto_circ_0004278
388.3443
3.11E−71

pto_circ_0004280
2563.073
8.51E−198

pto_circ_0004282
621.3509
3.15E−102

pto_circ_0004284
1708.715
4.39E−175

pto_circ_0004288
543.6821
2.64E−82

pto_circ_0004315
1242.702
3.03E−172

pto_circ_0004318
1398.04
3.04E−166

pto_circ_0004528
699.0198
2.19E−74

Based on the results of the find_circ analysis, the reverse splicing reads at the circRNAs looping position are extracted using the samtools view—R instruction for subsequent nucleic acid variation analysis.

SNP calling is performed on the extracted reads sequence using the GATK (version: 4.0.1.0) software in the following steps: first, variation detection is performed on two samples by using a HaplotypeCaller tool in the software; --pair-hmm-gap-continuation-penalty parameter is set as 10, and the remaining parameters are set as default values to obtain variation information of each sample; the variation files of each sample are combined by a CombineGVCFs tool; and finally, allelic variation detection is performed on each sample by using a GenotypeGVCFs tool, and a vcf file is generated, where the vcf file contains the mutation loci and genotype information of all samples (Table 2).

The SNPs in the reverse splicing reads are used as markers, and the number of reverse splicing reads on the SNPs is collected and aligned as the expression quantity of the candidate circRNA allelic loci (Table 2).

TABLE 2

Expression patterns of

candidate circRNAs allelic loci of P. tomentosa leaves

Reference
Variant

circRNA number
genotype
genotype
Alt
Ref
Alt/Ref

pto_circ_0000057
G
T
76
124
0.612903

pto_circ_0000187
T
C
83
83
1

pto_circ_0000221
C
T
705
705
1

pto_circ_0000227
A
G
229
47
4.87234

pto_circ_0000284
A
G
31
309
0.100324

pto_circ_0000345
A
G
14
43
0.325581

pto_circ_0000349
C
G
264
264
1

pto_circ_0000521
A
C
11
56
0.196429

pto_circ_0000587
G
A
82
391
0.209719

pto_circ_0000617
G
A
147
229
0.641921

pto_circ_0000674
T
A
13
13
1

pto_circ_0000776
T
C
49
49
1

pto_circ_0000871
T
C
91
415
0.219277

pto_circ_0000892
T
A
53
75
0.706667

pto_circ_0000981
A
G
91
37
2.459459

pto_circ_0001002
T
G
52
857
0.060677

pto_circ_0001090
T
C
6
75
0.08

pto_circ_0001278
G
A
15
86
0.174419

pto_circ_0001301
A
G
763
66
11.56061

pto_circ_0001411
T
A
66
59
1.118644

pto_circ_0001488
G
A
89
949
0.093783

pto_circ_0001506
G
A
845
845
1

pto_circ_0001508
T
A
95
66
1.439394

pto_circ_0001558
G
A
53
53
1

pto_circ_0001566
A
T
286
508
0.562992

pto_circ_0001622
G
A
3
3
1

pto_circ_0001628
G
A
624
624
1

pto_circ_0001741
G
A
205
205
1

pto_circ_0001816
A
G
984
671
1.466468

pto_circ_0002027
T
A
531
65
8.169231

pto_circ_0002055
C
T
519
519
1

pto_circ_0002058
C
T
92
92
1

pto_circ_0002106
T
C
542
228
2.377193

pto_circ_0002110
A
G
89
31
2.870968

pto_circ_0002122
A
G
45
8
5.625

pto_circ_0002159
T
C
31
69
0.449275

pto_circ_0002237
G
T
31
31
1

pto_circ_0002256
G
T
589
589
1

pto_circ_0002264
C
T
903
903
1

pto_circ_0002291
T
A
438
2
219

pto_circ_0002292
T
C
368
71
5.183099

pto_circ_0002322
A
C
461
333
1.384384

pto_circ_0002328
A
T
182
124
1.467742

pto_circ_0002377
T
C
379
69
5.492754

pto_circ_0002407
T
C
45
9
5

pto_circ_0002469
T
C
69
44
1.568182

pto_city 0002472
C
T
69
69
1

pto_circ_0002480
T
C
9
1
9

pto_circ_0002503
C
G
123
123
1

pto_circ_0002610
A
T
7
717
0.009763

pto_circ_0002658
A
G
42
42
1

pto_circ_0002659
T
C
7
8
0.875

pto_circ_0002730
G
A
8
8
1

pto_circ_0002748
T
C
101
427
0.236534

pto_circ_0002749
A
G
47
47
1

pto_circ_0002750
T
A
47
786
0.059796

pto_circ_0002919
C
G
822
822
1

pto_circ_0002941
T
C
46
19
2.421053

pto_circ_0002982
T
A
85
85
1

pto_circ_0003064
C
G
19
19
1

pto_circ_0003093
C
T
85
85
1

pto_circ_0003157
T
A
123
96
1.28125

pto_circ_0003159
A
C
24
24
1

pto_circ_0003160
G
C
96
96
1

pto_circ_0003167
G
T
879
879
1

pto_circ_0003179
T
C
798
798
1

pto_circ_0003206
T
C
716
298
2.402685

pto_circ_0003256
C
G
612
612
1

pto_circ_0003489
T
C
6
24
0.25

pto_circ_0003498
T
C
298
88
3.386364

pto_circ_0003579
G
T
4
4
1

pto_circ_0003608
C
G
24
24
1

pto_circ_0003617
G
A
88
88
1

pto_circ_0003620
A
G
23
23
1

pto_circ_0003811
T
C
29
33
0.878788

pto_circ_0003942
C
G
29
29
1

pto_circ_0003970
G
A
33
33
1

pto_circ_0004016
T
C
33
217
0.152074

pto_circ_0004037
C
T
356
356
1

pto_circ_0004047
A
C
54
54
1

pto_circ_0004133
A
C
217
217
1

pto_circ_0004142
C
T
135
135
1

pto_circ_0004203
G
A
635
635
1

pto_circ_0004208
C
G
762
275
2.770909

pto_circ_0004218
C
T
542
193
2.80829

pto_circ_0004238
C
T
54
437
0.12357

pto_circ_0004273
T
A
72
287
0.250871

pto_circ_0004277
T
C
27
62
0.435484

pto_circ_0004278
C
T
554
74
7.486486

pto_circ_0004280
C
T
681
6
113.5

pto_circ_0004282
G
A
17
17
1

pto_circ_0004284
T
G
473
39
12.12821

pto_circ_0004288
T
C
972
89
10.92135

pto_circ_0004315
A
C
72
72
1

pto_circ_0004318
T
A
193
59
3.271186

pto_circ_0004528
G
A
275
275
1

The results show that only 44.7% of the circRNA allelic loci in the leaves of P. tomentosa are balanced and the remaining loci are unbalanced.

EXAMPLE 2

High-temperature treated leaves of P. simonii are used for total RNA extraction using the RNA extraction kit (MagJ ET Plant RNA Purification Kit, No. K2772); and rRNA is removed using the Ribo-Zero™ rRNA Removal Kits (Plant) (No. MRZPL116). Then, the Poly (A) RNAs are combined by using a magnetic bead method, to obtain a Poly (A)-RNA sample; and the linear RNA is digested with RNase Rd (the reaction system includes 5 μg of RNA, 5 μL of 10× Reaction Buffer, 20 U of RNase R, and RNase-Free water supplemented to 50 μL), to obtain a Poly (A)-/Ribo-RNA sample. The strand-specific cDNA library is constructed using a SMART Kit (a SMART cDNA Library Construction Kit, NO. 634901).

Paired-end sequencing is performed using Illumina HiSeq™2500 with a sequencing data volume of 12 G. The linker and the redundant sequence are removed, and the transcript is spliced by the Cufflinks software. 20-nt of anchor sequence is extracted, with find_circ, from either end of the reads that are not aligned to the reference sequence. Each pair of anchor sequences is re-aligned to the reference sequence. If the 5′ terminal of the anchor sequence is aligned to the reference sequence (the starting and terminating locus are denoted as A3 and A4, respectively), and the 3′ terminal of the anchor sequence is aligned to the upstream of this locus (the starting and terminating loci are denoted as A1 and A2, respectively), and a splicing locus (GT-AG) exists between A2 and A3 in the reference sequence, then this read is used as a candidate circRNA. The screening parameters are -h, -v, -s, -G, -n, -p, -q, -a, -m, -d, --noncanonical, --randomize, --allhits, --stranded, --strandpref, and --halfunique. The screening parameters include -q 5, -a 20, -m 2, -d 2, and --noncanonical. The screening criteria of the foregoing parameters are preferably: (1) -q 5: the minimum support number of anchor sequence alignment is 5; (2) -a 20: the anchor sequence is 20 bp; (3) -m 2: a branch point cannot occur elsewhere in the range of two nucleic acids in the anchor sequence; (4) -d 2: sequence alignment only supports two mismatches; and (5) --noncanonical: GU/AG appears on both sides of the splicing locus, and a clear breakpoint can be detected. The circRNAs are screened using the default parameters of the CIRIexplorer software. 804 circRNAs are obtained by the find_circ software analysis, and 670 circRNAs are obtained by the CIRIexplorer software. The intersection of the two prediction results is obtained according to the reverse splicing reads of the circRNAs to obtain 121 circRNAs (Table 3).

TABLE 3

High-temperature response circRNA of P. simonii

circRNA number
Expression quantity
P value

psi_circ_0000763
3841.211
0

psi_circ_0000764
4207.041
0

psi_circ_0000919
4207.041
0

psi_circ_0001048
0
0

psi_circ_0001049
0
0

psi_circ_0001050
0
0

psi_circ_0001082
3475.381
0

psi_circ_0001184
0
0

psi_circ_0001188
182.9148
0

psi_circ_0001197
731.6593
0

psi_circ_0001482
2469.35
0

psi_circ_0001487
5395.987
0

psi_circ_0001572
3658.296
0

psi_circ_0001628
3201.009
0

psi_circ_0001655
3292.467
0

psi_circ_0001670
7865.337
0

psi_circ_0001986
4481.413
0

psi_circ_0002068
7316.593
0

psi_circ_0002110
4024.126
0

psi_circ_0002133
1737.691
0

psi_circ_0002149
25242.24
0

psi_circ_0002561
11340.72
0

psi_circ_0002570
3201.009
0

psi_circ_0002675
731.6593
0

psi_circ_0002676
640.2018
0

psi_circ_0002840
3658.296
0

psi_circ_0003374
7956.794
0

psi_circ_0003500
5853.274
0

psi_circ_0003621
4572.87
0

psi_circ_0003998
3749.754
0

psi_circ_0004007
3475.381
0

psi_circ_0004131
4298.498
0

psi_circ_0004232
2560.807
0

psi_circ_0004510
7773.88
0

psi_circ_0004511
17468.36
0

psi_circ_0004574
1829.148
0

psi_circ_0004602
2469.35
0

psi_circ_0004604
0
0

psi_circ_0001194
914.5741
1.7E−302

psi_circ_0001180
0
1.9E−297

psi_circ_0004576
2377.893
6.9E−297

psi_circ_0001146
2377.893
4.1E−289

psi_circ_0000292
2286.435
1.4E−288

psi_circ_0001987
2286.435
1.4E−288

psi_circ_0001765
1829.148
1.1E−275

psi_circ_0000460
1554.776
1.3E−275

psi_circ_0001416
2103.52
1.1E−271

psi_circ_0000295
2012.063
4.4E−263

psi_circ_0001775
2012.063
4.4E−263

psi_circ_0001977
1920.606
2.4E−254

psi_circ_0004235
1737.691
7.3E−252

psi_circ_0000760
1737.691
1.8E−236

psi_circ_0004005
1737.691
1.8E−236

psi_circ_0004036
1737.691
1.8E−236

psi_circ_0000180
0
5.7E−230

psi_circ_0002557
1646.233
1.9E−228

psi_circ_0001020
1646.233
2.5E−227

psi_circ_0003449
1646.233
2.5E−227

psi_circ_0004601
1646.233
2.5E−227

psi_circ_0000308
1554.776
5E−218

psi_circ_0001470
1554.776
5E−218

psi_circ_0001940
1554.776
5E−218

psi_circ_0002839
1554.776
5E−218

psi_circ_0004453
2286.435
3.4E−217

psi_circ_0004299
0
1.5E−215

psi_circ_0001346
1463.319
1.5E−208

psi_circ_0004247
1463.319
1.5E−208

psi_circ_0004367
1463.319
1.5E−208

psi_circ_0001228
1554.776
1.7E−205

psi_circ_0002735
0
9.6E−201

psi_circ_0000311
1371.861
6.5E−199

psi_circ_0000476
1371.861
6.5E−199

psi_circ_0004006
1371.861
6.5E−199

psi_circ_0000787
1280.404
4.5E−189

psi_circ_0003832
1280.404
4.5E−189

psi_circ_0003942
1280.404
4.5E−189

psi_circ_0004454
1280.404
4.5E−189

psi_circ_0004456
1280.404
4.5E−189

psi_circ_0001134
1188.946
5E−179

psi_circ_0003450
1188.946
5E−179

psi_circ_0004035
1188.946
5E−179

psi_circ_0004575
1188.946
5E−179

psi_circ_0001566
1188.946
1.8E−176

psi_circ_0001182
0
9.1E−170

psi_circ_0002674
0
9.1E−170

psi_circ_0003631
0
9.1E−170

psi_circ_0000368
1097.489
9.6E−169

psi_circ_0001874
1097.489
9.6E−169

psi_circ_0003244
1097.489
9.6E−169

psi_circ_0003656
1097.489
9.6E−169

psi_circ_0003929
1097.489
9.6E−169

psi_circ_0002987
182.9148
3.8E−161

psi_circ_0001046
1006.031
3.3E−158

psi_circ_0001129
1006.031
3.3E−158

psi_circ_0001133
1006.031
3.3E−158

psi_circ_0001139
1006.031
3.3E−158

psi_circ_0002968
1006.031
3.3E−158

psi_circ_0003452
1006.031
3.3E−158

psi_circ_0004248
1006.031
3.3E−158

psi_circ_0003699
0
1.8E−153

psi_circ_0001217
1097.489
6.1E−153

psi_circ_0001568
1097.489
6.1E−153

psi_circ_0003419
1554.776
5.4E−152

psi_circ_0000367
914.5741
2.2E−147

psi_circ_0000789
914.5741
2.2E−147

psi_circ_0000790
914.5741
2.2E−147

psi_circ_0001130
914.5741
2.2E−147

psi_circ_0001211
914.5741
2.2E−147

psi_circ_0001961
914.5741
2.2E−147

psi_circ_0002736
914.5741
2.2E−147

psi_circ_0003385
914.5741
2.2E−147

psi_circ_0004034
914.5741
2.2E−147

psi_circ_0000989
274.3722
2.2E−144

psi_circ_0002909
1280.404
6.2E−140

psi_circ_0003015
1280.404
6.2E−140

psi_circ_0000010
0
1.7E−136

psi_circ_0003066
0
1.7E−136

psi_circ_0000004
823.1167
3E−136

psi_circ_0000202
823.1167
3E−136

psi_circ_0000575
823.1167
3E−136

psi_circ_0001159
823.1167
3E−136

The reverse splicing reads data tag files at the circRNAs looping position are sorted according to the result of the find_circ analysis, and the reverse splicing reads at the circRNAs looping position are extracted using the samtools view—R instruction for subsequent nucleic acid variation analysis.

SNP calling is performed on the extracted reads sequence using the GATK (version: 4.0.1.0) software in the following steps: first, variation detection is performed on two samples by using a HaplotypeCaller tool, --pair-hmm-gap-continuation-penalty parameter is set as 10, and the remaining parameters are default values to obtain variation information of each sample; the variation files of each sample are combined by a CombineGVCFs tool; and finally, allelic variation detection is performed on each sample by using a GenotypeGVCFs tool, and a vcf file is generated, where the vcf file contains the mutation loci and genotype information of all samples (Table 4).

The SNPs in the reverse splicing reads are used as markers, the number of reverse splicing reads on the SNPs is collected and aligned as the expression quantity of the candidate circRNA allelic loci (Table 4).

TABLE 4

Expression pattern of high-temperature response

circRNA allelic loci of P. simonii

Reference
Variant

circRNA number
genotype
genotype
Alt
Ref
Alt/Ref

psi_circ_0000004
A
C
24
786
0.030534

psi_circ_0000010
C
T
85
857
0.099183

psi_circ_0000180
A
G
47
75
0.626667

psi_circ_0000202
G
C
96
949
0.101159

psi_circ_0000292
A
T
182
124
1.467742

psi_circ_0000295
T
C
9
1
9

psi_circ_0000308
C
G
19
309
0.061489

psi_circ_0000311
G
T
4
437
0.009153

psi_circ_0000367
C
G
123
217
0.56682

psi_circ_0000368
T
A
72
287
0.250871

psi_circ_0000460
T
C
69
44
1.568182

psi_circ_0000476
C
G
24
86
0.27907

psi_circ_0000575
G
T
879
56
15.69643

psi_circ_0000760
T
C
7
8
0.875

psi_circ_0000763
G
T
76
124
0.612903

psi_circ_0000764
T
C
83
391
0.212276

psi_circ_0000787
A
G
23
415
0.055422

psi_circ_0000789
A
T
7
717
0.009763

psi_circ_0000790
A
G
42
427
0.098361

psi_circ_0000919
C
T
705
427
1.651054

psi_circ_0000989
T
C
46
19
2.421053

psi_circ_0001020
C
G
822
24
34.25

psi_circ_0001046
T
C
972
89
10.92135

psi_circ_0001048
A
G
229
47
4.87234

psi_circ_0001049
A
G
31
309
0.100324

psi_circ_0001050
A
G
14
43
0.325581

psi_circ_0001082
C
G
264
287
0.919861

psi_circ_0001129
A
C
72
43
1.674419

psi_circ_0001130
T
C
7
8
0.875

psi_circ_0001133
T
A
193
59
3.271186

psi_circ_0001134
C
T
356
62
5.741935

psi_circ_0001139
G
A
275
69
3.985507

psi_circ_0001146
A
C
461
333
1.384384

psi_circ_0001159
T
C
798
508
1.570866

psi_circ_0001180
T
A
438
2
219

psi_circ_0001182
C
G
762
275
2.770909

psi_circ_0001184
A
C
11
56
0.196429

psi_circ_0001188
G
A
82
391
0.209719

psi_circ_0001194
C
T
903
124
7.282258

psi_circ_0001197
G
A
147
229
0.641921

psi_circ_0001211
G
A
8
229
0.034934

psi_circ_0001217
T
C
69
44
1.568182

psi_circ_0001228
T
C
6
24
0.25

psi_circ_0001346
T
C
798
75
10.64

psi_circ_0001416
C
T
69
8
8.625

psi_circ_0001470
C
T
85
8
10.625

psi_circ_0001482
T
A
13
33
0.393939

psi_circ_0001487
T
C
49
49
1

psi_circ_0001566
G
A
635
635
1

psi_circ_0001568
C
T
69
69
1

psi_circ_0001572
T
C
91
415
0.219277

psi_circ_0001628
T
A
53
75
0.706667

psi_circ_0001655
A
G
91
37
2.459459

psi_circ_0001670
T
G
52
857
0.060677

psi_circ_0001765
T
C
45
9
5

psi_circ_0001775
C
G
123
123
1

psi_circ_0001874
T
C
27
62
0.435484

psi_circ_0001940
T
A
123
96
1.28125

psi_circ_0001961
T
C
101
427
0.236534

psi_circ_0001977
A
T
7
717
0.009763

psi_circ_0001986
T
C
6
75
0.08

psi_circ_0001987
T
C
379
69
5.492754

psi_circ_0002068
G
A
15
86
0.174419

psi_circ_0002110
A
G
763
66
11.56061

psi_circ_0002133
T
A
66
59
1.118644

psi_circ_0002149
G
A
89
949
0.093783

psi_circ_0002557
T
A
47
786
0.059796

psi_circ_0002561
G
A
845
845
1

psi_circ_0002570
T
A
95
66
1.439394

psi_circ_0002674
C
T
542
193
2.80829

psi_circ_0002675
G
A
53
53
1

psi_circ_0002676
A
T
286
508
0.562992

psi_circ_0002735
T
C
298
88
3.386364

psi_circ_0002736
A
G
47
47
1

psi_circ_0002839
A
C
24
24
1

psi_circ_0002840
G
A
3
3
1

psi_circ_0002909
T
A
85
85
1

psi_circ_0002968
A
C
461
333
1.384384

psi_circ_0002987
T
G
473
39
12.12821

psi_circ_0003015
C
G
19
19
1

psi_circ_0003066
T
A
123
96
1.28125

psi_circ_0003244
C
T
554
74
7.486486

psi_circ_0003374
G
A
624
624
1

psi_circ_0003385
T
A
47
786
0.059796

psi_circ_0003419
T
C
9
1
9

psi_circ_0003449
T
C
46
19
2.421053

psi_circ_0003450
A
C
54
54
1

psi_circ_0003452
A
T
182
124
1.467742

psi_circ_0003500
G
A
205
205
1

psi_circ_0003621
A
G
984
671
1.466468

psi_circ_0003631
C
T
54
437
0.12357

psi_circ_0003656
C
T
681
6
113.5

psi_circ_0003699
T
C
45
9
5

psi_circ_0003832
T
C
29
33
0.878788

psi_circ_0003929
G
A
17
17
1

psi_circ_0003942
C
G
29
29
1

psi_circ_0003998
T
A
531
65
8.169231

psi_circ_0004005
G
A
8
8
1

psi_circ_0004006
G
A
88
88
1

psi_circ_0004007
C
T
519
519
1

psi_circ_0004034
C
G
822
822
1

psi_circ_0004035
A
C
217
217
1

psi_circ_0004036
T
C
101
427
0.236534

psi_circ_0004131
C
T
92
92
1

psi_circ_0004232
T
C
542
228
2.377193

psi_circ_0004235
A
G
42
42
1

psi_circ_0004247
T
C
716
298
2.402685

psi_circ_0004248
T
C
379
69
5.492754

psi_circ_0004299
G
T
879
879
1

psi_circ_0004367
C
G
612
612
1

psi_circ_0004453
G
C
96
96
1

psi_circ_0004454
G
A
33
33
1

psi_circ_0004456
T
C
33
217
0.152074

psi_circ_0004510
A
G
89
31
2.870968

psi_circ_0004511
A
G
45
8
5.625

psi_circ_0004574
T
C
31
69
0.449275

psi_circ_0004575
C
T
135
135
1

psi_circ_0004576
T
C
368
71
5.183099

psi_circ_0004601
T
A
85
85
1

psi_circ_0004602
G
T
31
31
1

psi_circ_0004604
G
T
589
589
1

The results show that only 25.8% of the circRNA allelic loci in the leaf tissues of P. simonii treated by high temperature stress are of balance expression, and the remaining loci are of imbalance expression.

It can be seen from the above examples that the method provided by the present invention uses strand-specific library RNA sequencing, combines with circRNA analysis software and nucleic acid variation analysis software, to accurately analyze the expression pattern of plant circRNA allelic loci in a high-throughput manner. It can be seen from the results in the examples that the method can achieve high-throughput analysis of allelic expression of plant circRNAs, and provides a new research approach for systematically analyzing the allelic expression pattern of the circRNAs by fully utilizing the results of transcriptome sequencing.

The foregoing descriptions are only preferred implementation manners of the present invention. It should be noted that for a person of ordinary skill in the art, several improvements and modifications may further be made without departing from the principle of the present invention. These improvements and modifications should also be deemed as falling within the protection scope of the present invention.

METHOD FOR HIGH-THROUGHPUT DETECTION OF DIFFERENTIAL EXPRESSION OF PLANT CIRCRNA ALLELIC LOCI

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