Next-generation sequencing primer pair combined with DNA bar code and sequencing method

Abstract

The invention relates to the field of biological gene engineering, in particular to a DNA bar code combined next-generation sequencing primer pair and a sequencing method, the primer pair is suitable for a large number of species, the detection result is accurate and reliable, the primer pair comprises a left primer and a right primer, the primer comprises a TA [barcode] region and a conserved sequence combination region, and the conserved sequence combination region comprises a DNA bar code and a DNA bar code. The left primer conserved sequence binding region is AGGTGAGTN from the 5'to the 3 'end, the corresponding right primer conserved sequence binding region is CCTTCTN from the 5' to the 3 'end, or the left primer conserved sequence binding region is CCTTCTN from the 5' to the 3 'end, the corresponding right primer conserved sequence binding region is AGGTGAGTN from the 5' to the 3 'end, N is a random basic group, and the TA [barcode] region is connected to the 5' end of the left primer or the right primer. Sample mixing sequencing is carried out after PCR amplification, and the amount of data generated by sequencing can be uniform.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic engineering, and in particular to a second-generation sequencing primer pair combined with a DNA barcode and a sequencing method. Background Art

[0002] Today's biological genetic engineering field is almost dealing with countless sequences, and the source of these sequences is the popular high-throughput sequencing technology. Today's sequencing, whether it is RNA, DNA, miRNA or ChIP-Seq, is based on NGS (next-generation sequencing) technology. The most commonly used one is the sequencing technology of Illumina. Of course, there are other second-generation sequencing technologies besides Illumina. ABI also has SOLiD sequencing technology. It is called second-generation sequencing because it is much better than the first-generation sequencing technology, specifically in terms of speed, accuracy, efficiency and so on.

[0003] The general process of next-generation sequencing is as follows: 1) Library preparation: randomly fragment the DNA into small fragments of a few hundred bases or shorter using atomization or ultrasound. Use polymerase and exonuclease to cut the DNA fragments into blunt ends, followed by phosphorylation and adding a nucleotide sticky end. Then connect the sequencing adapter to the fragments; 2) Cluster creation, adding template molecules to the chip for generating clone clusters and sequencing cycles. The chip has a silicon substrate with 8 longitudinal lanes. There are countless fixed single-stranded adapters on the surface of the chip in each lane. The DNA fragments with adapters obtained in the above steps are denatured into single strands and then combined with the adapter primers on the sequencing channel to form a bridge structure for subsequent pre-amplification. Through continuous cycles, millions of clustered double-stranded fragments to be tested are obtained; 3) Sequencing, DNA polymerase binds to the fluorescent reversible terminator, the fluorescent labeled cluster is imaged, and the bound nucleotides are cleaved and decomposed before the next cycle begins; 4) Data analysis: The raw data obtained by sequencing is a sequence of dozens of bases in length. These short sequences must be assembled into long contigs or even the framework of the entire genome through bioinformatics tools, or these sequences must be aligned to existing genomes or genome sequences of similar species for further analysis to obtain biologically meaningful results. Polymerase chain reaction (PCR) is a technology widely used in molecular genetics and diagnosis. It is used to amplify specific DNA fragments and can be regarded as a special DNA replication outside the body. Any short DNA sequence can be analyzed. The biggest feature of PCR is that it can significantly increase trace amounts of DNA, even if the sample contains only low levels of DNA. PCR technology can be used to amplify selected fragments of DNA or RNA for analysis.

[0004] A DNA barcode refers to a relatively short, standard, sufficiently variable, easily amplified DNA fragment in an organism that can represent the species. It enhances human ability to monitor, understand and utilize biodiversity resources. It has broad application prospects in life sciences, forensic medicine, epidemiology, medicine, food quality control and other fields.

[0005] Currently, the second-generation sequencing on the market still has the disadvantages of long sequencing time and uneven amount of sequencing output data, which seriously increases time and economic costs. Summary of the invention

[0006] In view of this, the purpose of the invention is to provide a second-generation sequencing primer pair combined with a DNA barcode designed according to the Chambon rule, the primer pair is applicable to a large number of species, and the detection results are accurate and reliable. Specifically: The primer pair comprises a left primer and a right primer; the primer comprises a TA[barcode] region and a conserved sequence binding region; the conserved sequence binding region of the left primer is AGGTGAGTN from 5' to 3' end, and the corresponding conserved sequence binding region of the right primer is CCTTCTN from 5' to 3' end, or the conserved sequence binding region of the left primer is CCTTCTN from 5' to 3' end, and the corresponding conserved sequence binding region of the right primer is AGGTGAGTN from 5' to 3' end, wherein N is a random base; the TA[barcode] region is connected to the 5' end of the left primer or the right primer.

[0007] Another object of the present invention is to provide a method for second-generation genome sequencing, comprising the following steps: Extract DNA from each sample to be sequenced; the sample to be tested is an animal, plant or fungus; Perform PCR amplification on the DNA of each sample to be sequenced, the amplification primer pair includes a left primer and a right primer, the primer includes a TA[barcode] region and a conserved sequence binding region, the conserved sequence binding region of the left primer from 5' to 3' end is AGGTGAGTN, and the corresponding conserved sequence binding region of the right primer from 5' to 3' end is CCTTCTN, or the conserved sequence binding region of the left primer from 5' to 3' end is CCTTCTN, and the corresponding conserved sequence binding region of the right primer from 5' to 3' end is AGGTGAGTN, wherein N is a random base, and the TA[barcode] region is connected to the 5' end of the left primer or the right primer; the barcode in the primer pair used for each sample to be sequenced is different; Quantify the PCR products to keep them at a consistent concentration and then mix them; The second-generation library construction and sequencing analysis results are used, and the samples to be sequenced are distinguished by barcodes. The sequencing data are mapped to the reference genome or assembled separately to mine population variation sites.

[0008] As a more optimal technical solution of the present invention, the DNA extraction method of the sample to be sequenced is CTAB method, SDS method or magnetic bead method.

[0009] As a better technical solution of the present invention, the PCR amplification process of the sample to be sequenced includes binding amplification and enrichment amplification; binding the primers to the genome is binding amplification, and the first 9 cycles are performed at 94°C, 45 seconds, 30°C, 1 minute, and 72°C, 20 seconds respectively; enrichment amplification is to increase the annealing temperature to 50°C for 30 cycles.

[0010] As a more optimal technical solution of the present invention, the concentration for quantifying the PCR product is 200 ng / ul.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The primer pair provided by the present invention is designed by finding the region near the target gene according to the Chambon rule, is applicable to a large number of species, and has accurate and reliable detection results.

[0012] The sequencing method provided by the present invention reduces the library preparation time, utilizes multiple barcodes for simultaneous sequencing, reduces costs and increases the rate, and performs mixed sequencing based on the results after PCR amplification, which can make the amount of sequencing output data more uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings: Figure 1 This is the agarose gel electrophoresis diagram of the PCR products after DNA from soybean and corn leaves was selected for library construction.

[0014] Figure 2 This is the agarose gel electrophoresis diagram of the PCR products after DNA libraries were constructed from rice, sugarcane, litchi, shrimp, oat and deer.

[0015] Figure 3 This is the agarose gel electrophoresis diagram of the PCR product after DNA of Fusarium oxysporum was selected for library construction.

[0016] Figure 4 This is the base content distribution diagram obtained in Example 4.

[0017] Figure 5 This is a graph showing the sequencing result comparison of primer pair 1.

[0018] Figure 6 This is a diagram showing the sequencing results of primer pair 1 compared with the sequence of corn.

[0019] Figure 7 This is a diagram showing the sequencing results of primer pair 1 and the alignment results of soybean sequences.

[0020] Figure 8 This is a graph showing the sequencing result comparison of primer pair 2.

[0021] Fig. 9 This is a diagram showing the comparison between the sequencing results of primer pair 2 and the sequence of corn.

[0022] Fig.10 This is a diagram showing the sequencing results of primer pair 2 and the alignment results of soybean sequences. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by ordinary technicians in the field without making creative work, such as modification, deformation or simple replacement, should belong to the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the raw materials, reagents, instruments, etc. used are all commonly used in the art, available to the public or commercially available, unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art.

[0025] In the embodiments of the present invention, soybean, corn, rice, sugar cane, litchi, shrimp, oat, deer and Fusarium oxysporum are used to extract DNA, and Fusarium oxysporum is a fungus.

[0026] The following two pairs of primers were designed for the experiment.

[0027] Primer pair 1: Left primer: TAGACACTAAAGGTGAGTN (SEQ ID NO. 1); Right primer: CCTTCTGGAGTNNN (SEQ ID NO. 2); Primer pair 2: Left primer: TAGGCATGAAACCTTCTN (SEQ ID NO. 3); Right primer: AGGTGAGTCTTNNN (SEQ ID NO. 4).

[0028] Then, the library is constructed. The reagents used for the library construction include dNTP, magnesium ions, Taq buffer, Taq enzyme, left primer and right primer.

[0029] The amplification program was as follows: the first 9 cycles were performed at 94°C for 45 seconds, 30°C for 1 minute, and 72°C for 20 seconds; the enrichment amplification was performed by raising the annealing temperature to 50°C for 30 cycles.

[0030] Example 1: Design and synthesize PCR amplification primers according to Chambon's rule.

[0031] Chambon's rule refers to the fact that the 5' boundary sequence of introns in most nuclear mRNA precursors is GU, and the 3' boundary sequence is AG. The conservative sequence pattern is considered a rule, that is, GU represents the 5' end of the donor junction, and AG represents the 3' end of the acceptor junction. Two characteristics of Chambon's rule are that there is no homology or complementarity at the two ends of the intron, and that the junction has a very short conserved sequence, also known as the boundary sequence.

[0032] The PCR amplification primers are as follows 1) or 2) 1) Universal left primer (5' to 3' end): TA[barcode]AAAGGTGAGTN The barcode is 6 bases, with a total of 384 barcodes (which can be expanded as needed), and N is a random base; Universal right primer (5' to 3' end): CCTTCTN 2) Universal left primer (5' to 3' end): AGGTGAGTN Universal right primer (5' to 3' end): TA[barcode]AAACCTTCTN The barcode is 6 bases, with a total of 384 barcodes (which can be expanded as needed).

[0033] Example 2: Extraction of DNA from samples to be sequenced.

[0034] The samples to be sequenced are soybeans and corn. The DNA extraction method of the samples to be sequenced can be one of the CTAB method, SDS method or magnetic bead method. Steps for extracting DNA using the magnetic bead method: Step 1: Take about 100 mg of fresh plant tissue or about 20 mg of dry weight tissue, add liquid nitrogen and grind thoroughly. In this embodiment, the magnetic bead method is used to extract DNA from tender leaves of soybean and corn.

[0035] Step 2: immediately add 600 μL of buffer GEA1 to the ground powder, quickly invert and mix, and heat in a 65°C water bath for 15 min or in a 75°C oven for 30 min. In this embodiment, heating in a 65°C water bath for 15 min is selected. Mix the sample several times during this period to increase the nucleic acid yield.

[0036] Step 3: Add 200 μL of buffer GEA2, mix thoroughly by inversion, and leave at room temperature for 15 min.

[0037] Step 4: Centrifuge at 4000 rpm for 45 minutes and transfer 400 μL of supernatant to a new deep-well plate.

[0038] Step 5. Add 5 μL of RNase A (10 mg / mL) and mix by inversion.

[0039] Step 6, nucleic acid binding: add 400 μL of binding solution to each reaction well in the 96-well plate, shake and mix the magnetic beads for 1 min before use, add 10 μL of magnetic beads, and after adding the magnetic beads, shake at 1200 rpm for 5 min.

[0040] Step 7. Magnetic separation: Place the 96-well plate on a magnetic rack and let it stand for 120 seconds until the magnetic beads are completely adsorbed, then discard the supernatant.

[0041] Step 8. Cleaning: add 600 μL of rinsing solution GEW1 (rinsing solution GEW1: anhydrous ethanol = 1:1) to each reaction well in the 96-well plate, shake at 1200 rpm for 2 min, perform magnetic separation as in step 7, and discard the supernatant; add 600 μL of rinsing solution GEW2 (rinsing solution GEW2: anhydrous ethanol = 1:4) to each well in the 96-well plate, shake at 1200 rpm for 2 min, perform magnetic separation as in step 7, and discard the supernatant.

[0042] Step 9, alcohol removal: After the supernatant is completely removed, dry at room temperature for 7 minutes.

[0043] Step 10, nucleic acid elution: add 200 μL of elution buffer GEB to each reaction well in the 96-well plate and shake at 1200 rpm for 10 min.

[0044] Step 11. Nucleic acid transfer: Place the 96-well plate on a magnetic stand and let it stand for 120 seconds until the magnetic beads are completely adsorbed. Transfer 190 μL of the nucleic acid solution to a clean 2 mL 96-well plate. The extraction process is complete.

[0045] Example 3: Using primer pair 1 to perform PCR amplification on each DNA sample to be sequenced, and using primer pair 2 to perform PCR amplification on each DNA sample to be sequenced.

[0046] Primer pair 1 and primer pair 2 were added to each sample DNA to be sequenced. Different samples used different barcodes, barcode 1 was GACACT; barcode 2 was GGCATG. Primer pair 1 and primer pair 2 were combined with the genome of the sample to be sequenced for amplification: the first 9 cycles were performed at 94°C for 45 seconds; 30°C for 1 minute; 72°C for 20 seconds for denaturation, annealing and extension. Then the annealing temperature was raised to 50°C for 30 cycles for enrichment amplification.

[0047] Agarose gel electrophoresis detection of PCR products: prepare 2% agarose gel (1.2g agarose and 60ml 1xTAE), heat in a microwave oven to dissolve agarose, add 4ul dye EB, mix well and pour into the gel plate to solidify, add 4ul PCR product to each well, add 5ul Marker 2000bp, adjust the voltage to 60V and run the electrophoresis for 45min. After the electrophoresis, use a gel imager to observe the results, and the size of the DNA fragment can be estimated based on the position of the DNA marker.

[0048] Quantify the PCR products of all samples to be sequenced to a concentration of 200 ng / ul, keep the concentration consistent, and mix the quantified PCR products.

[0049] like Figure 1 Shown are the agarose gel electrophoresis results of PCR products after library construction of DNA from soybean and corn tender leaves. It can be seen that the primers can randomly bind to different species and amplify fragments of uniform size and concentration. Figure 1 The first row in the figure uses the primer sequence pair: TAGACACTAAAGGTGAGTN (left primer) and CCTTCTGGAGTNNN (right primer); GACACT on the left primer is the barcode1 sequence, the first blank in the first row is Marker 2000bp, blanks 1 to 3 are soybean DNA samples, and blanks 4 to 6 are corn DNA samples; Figure 1 The second line in the sequence uses the primer sequence pair: TAGGCATGAAACCTTCTN (left primer) and AGGTGAGTCTTNNN (right primer); the base AAA in the sequence is to make the part that binds to the barcode tilt up for subsequent sequencing and sample separation, and each sample is separated according to the barcode used. The GGCATG on the left primer is the barcode2 sequence, the first space in the second line is Marker 2000bp, spaces 1 to 3 are soybean DNA samples, and spaces 4 to 6 are corn DNA samples.

[0050] In other embodiments, primer pair 1 and primer pair 2 (barcode 1 sequence on the left primer) are used to perform PCR amplification on DNA of other samples to be sequenced (rice, sugarcane, litchi, shrimp, oat, deer and Fusarium oxysporum), and agarose gel electrophoresis is performed, such as Figure 2 and Figure 3 As shown, the first line is an agarose gel electrophoresis diagram of PCR amplification of DNA from rice, sugarcane, litchi, shrimp, oat, deer and Fusarium oxysporum using primer pair 1, and the second line is an agarose gel electrophoresis diagram of PCR amplification of DNA from rice, sugarcane, litchi, shrimp, oat, deer and Fusarium oxysporum using primer pair 2. Figure 2 and Figure 3 It can be seen that primer pair 1 and primer pair 2 can also randomly combine in other plants, animals and fungi and amplify fragments with uniform size and concentration.

[0051] Example 4: Using second-generation library construction and sequencing (Illumina sequencing); Step 1, sample preparation: Ultrasonic waves randomly break the DNA chain to obtain DNA fragments, use enzymes to fill the ends, add A at the 3' end, and then use ligase to add adapters to both ends of the DNA fragments. The adapters mainly include: sequencing primer binding site (green), index (yellow / red) and sequences complementary to oligos on the flowcell chip (purple / blue).

[0052] Step 2: Cluster generation: DNA fragments are amplified through bridge PCR to form thousands of clusters at the same time. The clusters are basically synchronized, thus amplifying the signal for subsequent sequencing.

[0053] Step 3, sequencing: Load the single-stranded library into the flowcell and place it in the sequencer. The DNA fragments are amplified and automatically clustered. This process can produce millions of copies of single-stranded DNA. Then sequencing by synthesis occurs: each nucleotide has a fluorescent marker and a reversible terminator. The presence of the terminator means that only one base can be extended in one cycle, and the fluorescent signal can indicate which nucleotide is added. Then add chemicals to cut off the terminator, and the next base can be attached. This cycle repeats to obtain sequence data.

[0054] The purified library was sequenced directly on the sequencing platform Novasek-PE150. The quality of the data was controlled and the base quality distribution was as follows: Figure 4 As shown in the figure, the average quality is above Q30, indicating that the sequencing quality is good. The mixed sample data is separated into experimental samples by specific barcode sequences, and the BLAST function on NCBI is used for sequence alignment. The sequence alignment results of some data of the library sequencing of primer pair 1 and primer pair 2 are shown in the figure. Figures 5 to 10 As shown, the sequence information of the experimental samples corn and soybean can be compared and the consistency of the sequence comparison is high, averaging above 90%, proving that the primer pair of the present invention is capable of relatively accurate and random amplification of multiple species.

[0055] The above results indicate that the sequencing results obtained using the primer pair used in this application have reliable accuracy.

[0056] 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 second generation sequencing primer pair combined with a DNA barcode, characterized in that: The primer pair includes a left primer and a right primer, and the primer includes a TA[barcode] region and a conserved sequence binding region; The conserved sequence binding region of the left primer from 5' to 3' end is AGGTGAGTN, and the corresponding conserved sequence binding region of the right primer from 5' to 3' end is CCTTCTN; Or the conservative sequence binding region of the left primer from 5' to 3' end is CCTTCTN, and the corresponding conservative sequence binding region of the right primer from 5' to 3' end is AGGTGAGTN, where N is a random base. The TA[barcode] region is connected to the 5' end of the left primer or the right primer, The sample to be tested is an animal, plant or fungus.

2. A genome second generation sequencing method, characterized in that: The steps include: Extract DNA from each sample to be sequenced; the sample to be tested is an animal, plant or fungus; PCR amplification is performed on the DNA of each sample to be sequenced, and the amplification primer pair includes a left primer and a right primer; the primer includes a TA[barcode] region and a conserved sequence binding region; the conserved sequence binding region of the left primer is AGGTGAGTN from 5' to 3' end, and the corresponding conserved sequence binding region of the right primer is CCTTCTN from 5' to 3' end, or the conserved sequence binding region of the left primer is CCTTCTN from 5' to 3' end, and the corresponding conserved sequence binding region of the right primer is AGGTGAGTN from 5' to 3' end, wherein N is a random base; the TA[barcode] region is located at the 5' end of the left primer or the right primer; the barcode in the primer pair used for each sample to be sequenced is different; Quantify the PCR products and mix them to maintain a consistent concentration; The second-generation sequencing was used for library construction.

3. The method for second generation genome sequencing according to claim 2, characterized in that: The DNA extraction method of the sample to be sequenced is CTAB method, SDS method or magnetic bead method.

4. The method for second generation genome sequencing according to claim 2, characterized in that: The PCR amplification process of the sample to be sequenced includes binding amplification and enrichment amplification; binding the primers to the genome is binding amplification, and the first 9 cycles are performed at 94°C, 45 seconds, 30°C, 1 minute, and 72°C, 20 seconds respectively; enrichment amplification is to increase the annealing temperature to 50°C for 30 cycles.

5. The method for second generation genome sequencing according to claim 2, characterized in that: The concentration for quantification of PCR products was 200 ng / ul.

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