High-throughput small-system NGS library construction method

Abstract

The invention relates to a construction method of a high-throughput small-system NGS library. According to the method, a library building system can be reduced to 0.1 *, the library building cost is greatly reduced, instruments and equipment such as Sciclon-384, Dragonfly and G PURE are integrated, the library building automation level is improved, the one-time library building flux is improved to 384 samples, meanwhile, a fragment sorting mode is optimized, fragment loss in the library building process is reduced, the library building period is shortened, and NGS library building is rapidly and automatically carried out with high flux and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of sequencing and relates to a high-throughput small-system NGS library construction method. Background Art

[0002] Next-Generation Sequencing (NGS) is the most commonly used technology in high-throughput sequencing technology. In conventional NGS library construction, the general process of NGS library construction is as follows: (1) Sample preparation: Extract DNA or RNA from biological samples (such as blood, tissue, etc.); (2) Nucleic acid extraction: Use appropriate methods (such as chemical methods, physical methods, etc.) to separate DNA or RNA from samples; (3) Library construction: 1) Fragmentation: Cut the DNA or RNA sample into short fragments of appropriate length; 2) End repair and ligation: Repair the ends of the DNA or RNA fragments, add appropriate linkers (a short DNA sequence), and connect the DNA fragments to the library; 3) Amplification and purification: Amplify the number of DNA fragments in the library through methods such as PCR, and remove impurities and unconnected DNA fragments through purification steps to improve the quality and purity of the library; (4) Library quality control: Perform quality control on the constructed library to ensure that it meets the sequencing requirements (5) Library storage: Store qualified libraries under appropriate conditions for subsequent sequencing.

[0003] For example, CN108866155A discloses a method for preparing a next-generation sequencing library, which is carried out according to the following steps: (1) DNA quantification: take 2-5 μL of the extracted genomic DNA and determine the concentration of the genomic DNA; (2) add DNA adapter sequences required for sequencing to both ends of the DNA fragment to be tested; (3) detect the concentration of the DNA library and the distribution pattern of the DNA fragment length; (4) calculate and mix the mixing ratio of the DNA library: according to the concentration of the target fragment, take the same total amount of target DNA from each library and mix them to obtain the final mixed library; (5) fragment selection of the mixed library: perform fragment selection on the mixed library; (6) detect and quantify the selected DNA library; (7) sequence the selected DNA library: according to the concentration of the library, according to the requirements of the relevant Illumina instrument, sequence it on the machine.

[0004] However, according to the existing library construction methods and processes, the library construction process takes a long time, the overall library construction throughput is low, and the cost of library construction kits is high, which requires a high level of technical skills from library construction personnel. Therefore, the development of a high-throughput, low-cost library construction method is of great significance to the field of second-generation sequencing. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for constructing a high-throughput small-system NGS library (including transcriptome and resequencing) in order to achieve rapid, high-throughput, low-cost and automated NGS library construction.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing a high-throughput small-scale resequencing library, the method comprising the following steps:

[0008] (1) DNA is sheared to obtain fragment products;

[0009] (2) ligating the fragmented product to a linker to obtain a linker-ligated product;

[0010] (3) purifying the adapter ligation product and performing fragment sorting to obtain a sorted product;

[0011] (4) performing PCR amplification on the sorted products to obtain amplified products;

[0012] (5) Purify the amplified product to obtain a sequencing library.

[0013] Preferably, the reagents for the interruption treatment in step (1) are dispensed by a non-contact dispenser Dragonfly.

[0014] Preferably, the shearing treatment comprises heating to 35-38°C for 6-30 min to randomly shear the DNA, followed by enzyme inactivation at 63-66°C for 25-35 min.

[0015] Preferably, the connector in step (2) is manipulated by an automatic liquid handling workstation Sciclone-384, and the buffer for connector connection is dispensed by a non-contact dispenser Dragonfly. The connection product is obtained by maintaining at 18-22°C for 12-18 minutes.

[0016] Preferably, the purified magnetic beads and the remelted water after purification in step (3) are controlled by the non-contact liquid dispenser Dragonfly, the supernatant discarding and ethanol washing steps in the purification process are completed by the non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed using the automatic liquid handling workstation Sciclone-384.

[0017] In the present invention, a new library construction process is designed, and instruments and equipment such as non-contact liquid dispensers (such as Dragonfly), automatic liquid handling workstations Sciclone-384, and non-contact microplate nucleic acid purification systems G.PURE are integrated to improve the automation level of library construction and the operation throughput. 384-well plate operations can be realized, and 384 samples can be built in a single library. Dragonfly non-contact liquid dispenser can achieve high-throughput and accurate liquid separation. Dragonfly relies on solid phase replacement gun tips to perform multi-channel non-contact liquid separation at the same time. The range is 200nL-4mL, and the accuracy can reach nanoliters, and it is not affected by liquid viscosity or environmental conditions. It solves the problem of too little and inaccurate sample volume in the miniaturized system, greatly saves library construction reagents, standardizes experiments, and is fast in sample addition. It only takes a few minutes to add samples to a 384-well plate, saving experimental time. G.PURE is an innovative automated solution launched by Cytena, Germany, for DNA purification in the next-generation sequencing (NGS) workflow. It uses non-contact technology to reversely remove liquid from 384-well plates, effectively reducing sample contamination and improving data quality. At the same time, ethanol can be distributed to 384-well plates for rinsing. Compared with traditional methods, a large number of gun tips are saved, and the G.PURE supernatant is discarded cleanly, avoiding the impact of ethanol residue on subsequent library yield. The entire operation process of G.PURE only takes a few minutes, reducing experimental costs and time. The automatic liquid handling workstation Sciclone-384 is a high-throughput automated library construction workstation Sciclone G3 NGSx iQ HT developed by Revvity. It is equipped with a 384-channel pipetting head, a built-in plate grabber and a desktop Inheco ODTC thermal cycler, which greatly improves the degree of automation.

[0018] Preferably, the enzyme and primers for PCR amplification in step (4) are mixed and then dispensed into a multi-well plate by a non-contact dispenser Dragonfly, mixed with the sorted product, and PCR amplification is performed. The PCR amplification procedure is:

[0019] 95-98℃, 3-5min pre-denaturation;

[0020] 95-98°C, 10-30s denaturation, 60-65°C, 15-30s annealing, 70-73°C, 30-45s extension, 8-10 cycles of amplification;

[0021] Extend at 70-73°C for 3-5 min.

[0022] Preferably, the purified magnetic beads and the remelted water after purification in step (5) are controlled by the non-contact liquid dispenser Dragonfly, the supernatant discarding and ethanol washing steps in the purification process are controlled by the non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed using the automatic liquid handling workstation Sciclone-384.

[0023] Preferably, the multi-well plate comprises a 384-well plate.

[0024] Preferably, the library construction system used in the high-throughput small-system sequencing library construction method is 0.1×.

[0025] In the present invention, the traditional 1× library construction system is reduced to 0.1×, and the reagent usage is reduced by 10 times without reducing the library output, which greatly reduces the cost of single sample library construction.

[0026] In a second aspect, the present invention provides a method for constructing a high-throughput small-system transcriptome library, the method comprising the following steps:

[0027] (S1) Enriching and purifying mRNA;

[0028] (S2) fragmenting the purified mRNA to obtain fragmented mRNA;

[0029] (S3) using the fragmented mRNA as a template, reverse transcription is performed to synthesize the first-strand cDNA;

[0030] (S4) synthesizing the second-strand cDNA using the first-strand cDNA, and performing end repair and A addition;

[0031] (S5) performing adapter ligation on the double-stranded cDNA and the adapter to obtain an adapter ligation product;

[0032] (S6) purifying the adapter ligation product and performing fragment sorting to obtain a sorted product;

[0033] (S7) performing PCR amplification on the sorted products to obtain amplified products;

[0034] (S8) Purify the amplified product to obtain a transcriptome library.

[0035] Preferably, the reagent for the interruption treatment in step (S2) is dispensed by a non-contact dispenser Dragonfly, specifically kept at 94° C. for 5-10 minutes to randomly interrupt the mRNA into small fragments.

[0036] Preferably, the synthesis buffer of the first-strand cDNA in step (S3) is dispensed by a non-contact dispenser Dragonfly. Specifically, the first-strand cDNA is synthesized according to the program of 25°C, 10 min, 42°C, 15 min, and 70°C, 15 min.

[0037] Preferably, the synthesis buffer of the second-strand cDNA in step (S4) is dispensed by a non-contact dispenser Dragonfly. Specifically, the second-strand cDNA is synthesized according to the program at 16°C, 30min, and 72°C, 15min.

[0038] Preferably, the connector in step (S5) is controlled by an automatic liquid handling workstation Sciclone-384, and the buffer for connector connection is controlled by a non-contact liquid dispenser Dragonfly. Specifically, the connection product is obtained by keeping it at 20°C for 15 minutes.

[0039] Preferably, the purified magnetic beads and the remelted water after purification in step (S6) are controlled by the non-contact liquid dispenser Dragonfly, and the supernatant discarding and ethanol washing steps in the purification process are completed by the non-contact microplate nucleic acid purification system G.PURE; and the liquid transfer between the multi-well plates is performed using the automatic liquid handling workstation Sciclone-384.

[0040] Preferably, the enzyme and primers for PCR amplification in step (S7) are mixed and then dispensed into a multi-well plate by a non-contact dispenser Dragonfly, mixed with the sorted product, and PCR amplification is performed. The PCR amplification procedure is:

[0041] 95-98℃, 1-3min pre-denaturation;

[0042] 95-98°C, 10-30s denaturation, 60-65°C, 15-30s annealing, 72°C, 30-45s extension, 13-15 cycles of amplification;

[0043] Extend at 72°C for 3-5 min.

[0044] Preferably, the purified magnetic beads and the remelted water after purification in step (S8) are controlled by the non-contact liquid dispenser Dragonfly, and the supernatant discarding and ethanol washing steps in the purification process are completed by the non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed using the automatic liquid handling workstation Sciclone-384.

[0045] Preferably, the multi-well plate comprises a 384-well plate.

[0046] In a third aspect, the present invention provides a small system mRNA enrichment and purification method, which is applied to the high-throughput small system transcriptome library construction method described in the second aspect, and comprises the following steps:

[0047] (S1-1) Mixing total RNA with mRNA purification magnetic beads, incubating, and binding mRNA to the magnetic beads;

[0048] (S1-2) placing the mixture of step (S1-1) on a magnetic rack, adsorbing the magnetic beads, and discarding the supernatant;

[0049] (S1-3) Add cleaning solution, pipette and mix well, place on the magnetic stand, and discard the cleaning solution;

[0050] (S1-4) adding elution buffer to elute the mRNA from the magnetic beads;

[0051] (S1-5) Add binding solution to rebind the mRNA to the magnetic beads;

[0052] (S1-6) Place on a magnetic stand, adsorb magnetic beads, and discard the supernatant;

[0053] (S1-7) Add cleaning solution, place on the magnetic rack, and discard the cleaning solution.

[0054] Preferably, the magnetic beads in step (S1-1) are separated by a non-contact liquid separator Dragonfly. Specifically, the mRNA is denatured at 65°C for 5 minutes.

[0055] Preferably, in step (S1-2), the supernatant is discarded by the non-contact microplate nucleic acid purification system G.PURE. Specifically, the mRNA purification magnetic beads are incubated with the denatured mRNA mixture at 25°C for 5 minutes, the mRNA is bound to the magnetic beads, placed on a magnetic stand for adsorption for 3-5 minutes, and the supernatant is discarded by the non-contact microplate nucleic acid purification system G.PURE.

[0056] Preferably, the cleaning solution in step (S1-3) is dispensed into a multi-well plate by a non-contact liquid dispenser Dragonfly, and the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE.

[0057] Preferably, the eluate in step (S1-4) is separated by a non-contact liquid separator Dragonfly, specifically at 80° C. for 2 min to elute the mRNA from the magnetic beads.

[0058] Preferably, in step (S1-6), the supernatant is discarded by the non-contact microplate nucleic acid purification system G.PURE. Specifically, the magnetic bead mixture is placed on a magnetic rack for adsorption for 3-5 minutes, and the supernatant is discarded by the non-contact microplate nucleic acid purification system G.PURE.

[0059] Preferably, the cleaning solution in step (S1-7) is dispensed into a multi-well plate by a non-contact liquid dispenser Dragonfly, and the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE. Specifically, after mixing, the mixture is placed on a magnetic stand for adsorption for 3-5 minutes, and the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE.

[0060] In a fourth aspect, the present invention provides a fragment sorting method, which is applied to the high-throughput small-system resequencing library construction method described in the first aspect or the high-throughput small-system transcriptome library construction method described in the second aspect, comprising the following steps:

[0061] (SS-1) taking the first DNA purification magnetic beads and mixing them with the purified adapter ligation product;

[0062] (SS-2) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and remove the supernatant;

[0063] (SS-3) Keep the multi-well plate on the magnetic stand and add ethanol to rinse the magnetic beads;

[0064] (SS-4) Keep the multi-well plate on the magnetic stand, dry the magnetic beads, remove the multi-well plate from the magnetic stand, add water for elution, and transfer the eluted product to a new multi-well plate;

[0065] (SS-5) adding a second DNA purification magnetic bead to the eluted product, mixing and incubating;

[0066] (SS-6) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and transfer the supernatant to a new multiwell plate;

[0067] (SS-7) add the third DNA purification magnetic beads, mix and incubate;

[0068] (SS-8) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and remove the supernatant;

[0069] (SS-9) Keep the multi-well plate on the magnetic stand and add ethanol to rinse the magnetic beads;

[0070] (SS-10) Keep the multi-well plate on the magnetic stand, dry the magnetic beads, remove the multi-well plate from the magnetic stand, and add water for elution;

[0071] (SS-11) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and transfer the supernatant to a new multiwell plate.

[0072] Most of the current library construction methods use a double-round magnetic bead sorting method to control the length of the library by controlling the amount of magnetic beads used. The basic principle is: in the first round, the magnetic beads bind to large fragments above the target fragment, and this part of the product is removed by discarding the magnetic beads, and the supernatant is retained; in the second round, the magnetic beads bind to the target fragment, and the DNA with a smaller molecular weight is removed by discarding the supernatant, and finally the target fragment is eluted from the magnetic beads. However, the currently known sorting methods will cause a large amount of fragment loss, seriously affecting the library yield and reducing the complexity of the final library. The present invention designs a new double-round magnetic bead sorting method, first removing small fragments and then discarding large fragments of DNA. The specific implementation method is: in the first round, the magnetic beads bind to small fragments below the target fragment, and the DNA with a smaller molecular weight is removed by discarding the supernatant; in the second round, the magnetic beads bind to large fragments above the target fragment, and this part of the product is removed by discarding the magnetic beads, and the supernatant is retained. The third round of magnetic beads purifies this part of the supernatant, and the target fragment is enriched by subsequent PCR amplification, which can reduce the fragment loss in the library construction process on the basis of controlling the length distribution range of the final library, and can significantly improve the yield of the transcriptome or resequencing library.

[0073] Preferably, the magnetic bead multiple of the first DNA purification magnetic beads in step (SS-1) is 0.6-0.7×, and the dosage is 6-7 μL, the magnetic bead multiple of the second DNA purification magnetic beads in step (SS-5) is 0.45-0.6×, and the dosage is 4.5-6 μL, and the magnetic bead multiple of the third DNA purification magnetic beads in step (SS-7) is 0.6-0.7×, and the dosage is 8.1-10.5 μL.

[0074] Preferably, the DNA purification magnetic beads in steps (SS-1), (SS-5) and (SS-7) are added to the multi-well plate via a non-contact liquid dispenser Dragonfly.

[0075] Preferably, the elution water in steps (SS-4) and (SS-10) is added to the multi-well plate via a non-contact liquid dispenser Dragonfly.

[0076] Preferably, the eluted products in steps (SS-3), (SS-4), (SS-5), (SS-6), (SS-10), and (SS-11) are transferred by an automated liquid handling workstation Sciclone-384.

[0077] Preferably, the removal of the supernatant in steps (SS-2) and (SS-8) is achieved by a non-contact microplate nucleic acid purification system G.PURE.

[0078] Preferably, the ethanol rinsing in steps (SS-3) and (SS-9) is achieved by a non-contact microplate nucleic acid purification system G.PURE.

[0079] Specifically, the fragment sorting method may include the following steps:

[0080] 1) After the DNA purification magnetic beads are equilibrated to room temperature, vortex the beads to mix;

[0081] 2) Pipette X μL DNA purification magnetic beads into the adapter-connected purified product, vortex or use a pipette to gently pipette 10 times to mix thoroughly, and incubate at room temperature (20-30°C) for 5 minutes;

[0082] 3) Vortex or pipette gently 10 times to mix thoroughly and incubate at room temperature for 5 minutes;

[0083] 4) Briefly centrifuge the 384-well plate and place it on a magnetic stand to separate the beads and liquid. After the solution becomes clear (about 5 minutes), carefully remove the supernatant.

[0084] 5) Keep the 384-well plate on the magnetic stand and add freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds and carefully remove the supernatant; repeat step 4) for a total of two rinses;

[0085] 6) Keep the 384-well plate on the magnetic rack all the time, remove the film and air-dry the magnetic beads for 1-3 minutes until no ethanol remains, remove the 384-well plate from the magnetic rack, add water for elution, incubate at room temperature for 5 minutes, and then transfer the eluted product to a new 384-well plate;

[0086] 7) Add X μL of DNA purification magnetic beads to the eluted product, vortex or pipette gently 10 times to mix thoroughly, and incubate at room temperature for 5 minutes;

[0087] 8) Briefly centrifuge the 384-well plate and place it on a magnetic stand to separate the beads and liquid. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new 384-well plate.

[0088] 9) Add X μL DNA purification magnetic beads, vortex or pipette gently 10 times to mix thoroughly, and incubate at room temperature for 5 minutes;

[0089] 10) Briefly centrifuge the 384-well plate and place it on a magnetic stand to separate the beads and liquid. After the solution becomes clear (about 5 minutes), carefully remove the supernatant.

[0090] 11) Keep the 384-well plate on the magnetic stand and add freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, carefully remove the supernatant, and repeat step 4) for a total of two rinses;

[0091] 12) Keep the 384-well plate on the magnetic rack, remove the film and air-dry the magnetic beads for 1-3 minutes until no ethanol remains, remove the 384-well plate from the magnetic rack, add water for elution, vortex or use a pipette to gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes;

[0092] 13) Briefly centrifuge the 384-well plate and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clarified (about 5 minutes), carefully remove the supernatant and transfer it to a new 384-well plate for subsequent PCR amplification.

[0093] Compared with the prior art, the present invention has at least the following beneficial effects:

[0094] The present invention designs a novel NGS library construction method, which can reduce the library construction system to 0.1×, greatly reduce the library construction cost, integrate the automatic liquid handling workstation Sciclone-384, the non-contact liquid dispenser Dragonfly and the non-contact microplate nucleic acid purification system G PURE and other instruments and equipment, improve the automation level of library construction, increase the throughput of one library construction to 384 samples, optimize the fragment sorting method, reduce the fragment loss in the library construction process, shorten the library construction cycle, and realize fast, high-throughput, low-cost and automated NGS library construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 Schematic diagram of the library construction process for resequencing and transcriptome construction;

[0096] Figure 2 The quality inspection results of the resequencing library for rice samples 0.1×-384 well plate construction;

[0097] Figure 3 The quality inspection results of the resequencing library for soil samples 0.1×-384 well plate construction;

[0098] Figure 4 The quality inspection results of the transcriptome library constructed for rice samples in 0.1×-384 well plates;

[0099] Figure 5 The quality inspection results of the transcriptome library constructed in 0.1×-384-well plate for human samples;

[0100] Figure 6 The results of the analysis of the difference in gene numbers between the 0.1×-384 system and the 1×-96 system for soil metagenomic resequencing;

[0101] Figure 7 Comparative analysis results of 0.1×-384 system and 1×-96 SNP genotype for rice sample resequencing;

[0102] Figure 8The results of SNP correlation analysis of 0.1×-384 system and 1×-96 system for rice sample resequencing library construction. DETAILED DESCRIPTION

[0103] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0104] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0105] In view of the problems of the existing library construction methods and processes, such as long library construction process, low overall library construction throughput, high cost of library construction kits, and high technical requirements for library construction personnel, the present invention designs a new NGS library construction process (resequencing and transcriptome, the process is as follows Figure 1 As shown in the figure, it integrates the automatic liquid handling workstation Sciclone-384, the non-contact dispenser Dragonfly and the non-contact microplate nucleic acid purification system G PURE and other instruments and equipment to achieve semi-automatic library construction. It uses a 0.1× system to build a 384-well plate library and controls the amount of magnetic beads to remove small fragments first and then discard large fragments of DNA, which can significantly improve the yield of transcriptome or resequencing libraries.

[0106] In the specific embodiments of the present invention, rice, soil and human samples are used as examples to verify the solution of the present invention. The non-contact liquid dispenser Dragonfly is the second-generation multi-functional liquid dispenser workstation launched by the British company SPT Labtech. discovery (DFD), the automatic liquid handling workstation Sciclone-384 is a high-throughput automated library construction workstation Sciclone G3 NGSx iQ HT developed by Revvity, and G.PURE is a non-contact microplate nucleic acid purification system launched by Cytena of Germany for DNA purification in the next-generation sequencing technology (NGS) workflow.

[0107] Example 1

[0108] This example constructs and analyzes a rice & soil resequencing library.

[0109] This embodiment uses Vazyme's The library was constructed using the Universal Plus DNA Library Prep Kit for Illumina, catalog number ND617.

[0110] 1. DNA sample preparation

[0111] Soil and rice samples were taken, 10 ng of genomic DNA was extracted (qubit quantification) into a 384-well plate, and the total volume was filled to 3.5 μL with nuclease-free water, shaken and mixed, and centrifuged instantly;

[0112] 2. Enzyme Cutting

[0113] Using Vazyme The Universal Plus DNA Library Prep Kit for Illumina (Cat. No. ND617) was used for DNA shearing reaction. According to Table 1, 0.5 μL of fragmentation reaction buffer FEA Buffer and 1 μL of fragmentation enzyme FEA Enzyme Mix were prepared into shearing Mix (operated on ice). The mixture was mixed by pipetting or oscillating, centrifuged instantly, and transferred to the Dragonfly sample slot of the non-contact dispenser. The shearing Mix was dispensed into the above-mentioned DNA sample plate. After completion, it was immediately transferred to ice, heat-sealed at 168°C for 2s, mixed and centrifuged instantly, and immediately placed in a PCR instrument to react according to the program in Table 2.

[0114] Table 1

[0115] Reagents Volume (μL) Genomic DNA 3.5 FEABuffer 0.5 FEAEnzyme Mix 1 total 5

[0116] Table 2

[0117] 1 4℃，15s 2 37℃,8min 3 65℃,30min 4 4℃maintain

[0118] 3. Connector connection

[0119] Using Vazyme Universal Plus DNA Library Prep Kit for Illumina (Cat. No. ND617) was used for adapter ligation reaction. 2.5 μL of Rapid Ligation Buffer 3, 0.5 μL of Rapid DNA Ligase, and 1 μL of deionized water ddH2O in Table 3 except for the DNA adapter were prepared in advance to form a mixed solution (Mix) (operated on ice). The mixture was mixed by pipetting or oscillating, centrifuged instantaneously, and transferred to the Dragonfly loading tank of the non-contact dispenser. 1 μL of DNA Adapter was added to the end repair plus A product after the shearing using the automatic liquid handling workstation Sciclone-384. Then the Dragonfly non-contact dispenser was turned on, and 4 μL of the ligation mixture Ligation Mix was added to the above product. The reaction was carried out according to the procedure in Table 4.

[0120] Table 3

[0121] Reagents Volume (μL) End repair after interruption plus A product 5 Rapid Ligation Buffer 3 2.5 DNA Adapter 1 Rapid DNA Ligase 0.5 <![CDATA[ddH2O]]> 1 total 10

[0122] Table 4

[0123] 1 Heat cover 2 20℃,15min 3 4℃maintain

[0124] 4. Purification of ligation product

[0125] Pay attention to the temperature of magnetic bead incubation and magnetic bead purification, try to maintain the uniformity of laboratory temperature (currently it is recommended to control the temperature at around 22°C), use Vazyme's VAHTSDNAClean Beads for purification, item number N411, and ensure that the magnetic beads have been equilibrated at room temperature for more than 30 minutes before use. The steps are as follows:

[0126] (1) Turn on the non-contact dispenser Dragonfly, add 6 μL VAHTS DNA Clean Beads (Cat. No. N411) to 10 μL of the ligation product in the above step, heat seal at 168°C for 2 seconds, mix and incubate at room temperature for 5 minutes; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5 minutes;

[0127] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0128] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 12 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, and incubate at room temperature for 5 minutes after centrifugation.

[0129] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 min. 10 μL of the supernatant was transferred to a new 384-well plate.

[0130] 5. Fragment sorting and purification

[0131] Refer to Table 5 for the process.

[0132] Table 5

[0133]

[0134] Use Vazyme's VAHTSDNAClean Beads for purification, catalog number N411. Make sure the magnetic beads have been equilibrated at room temperature for more than 30 minutes before use. The steps are as follows:

[0135] 5.1 Remove small fragments

[0136] (1) Open Dragonfly, add 6.4 μL (0.64×) of mixed VAHTS DNA Clean Beads (Cat. No. N411) to the above 384-well plate, heat seal at 168°C for 2 seconds, mix and incubate at room temperature for 5 minutes; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5 minutes;

[0137] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0138] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 11 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, centrifuge briefly, and incubate at room temperature for 5 minutes;

[0139] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automated liquid handling workstation for 5 min, and 10 μL of the supernatant was transferred to a new 384-well plate;

[0140] 5.2 Remove large fragments

[0141] (1) Turn on the non-contact dispenser Dragonfly, add 4.7 μL (0.47×) of mixed VAHTS DNA Clean Beads (Cat. No. N411) to the above 384-well plate, heat seal at 168°C for 2 seconds, mix and incubate at room temperature for 5 minutes;

[0142] (2) Place on the Sciclone-384 magnetic stand of the automated liquid handling workstation, let stand for 5 min, and transfer 13.2 μL of the supernatant to a new 384-well plate;

[0143] 5.3 Secondary purification

[0144] (1) Turn on the non-contact dispenser Dragonfly, add 8.6 μL (0.65×) of mixed VAHTS DNA Clean Beads (Cat. No. N411) to the above 384-well plate, heat seal at 168°C for 2 seconds, mix and incubate at room temperature for 5 minutes; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5 minutes;

[0145] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0146] (3) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly non-contact dispenser using Vazyme's Universal Plus DNA Library Prep Kit for Illumina (Cat. No. ND617) was used for PCR amplification reaction. According to Table 6, 5ul of nuclease-free water, 5ul of amplification enzyme buffer VAHTS HiFi Amplification Mix, and 1ul of amplification primer PCR Primer Mix 3 were prepared into a PCR Mix mixture. After mixing, 11μL of the mixture was added to a 384-well plate, and the plate was transferred to a magnetic rack and heat-sealed at 168℃ for 2s. After mixing, the plate was incubated at room temperature for 5min after instant centrifugation.

[0147] Table 6

[0148] Reagents Volume (μL) PCR Primer Mix 3 1 VAHTS HiFi Amplification Mix 5 Nuclease-free water 5 Total volume 11

[0149] 6. PCR amplification

[0150] (1) The above 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation, left to stand for 5 min, and 10 μL of the supernatant was transferred to a new 384-well plate for subsequent PCR amplification reaction.

[0151] (2) The reaction parameters are shown in Table 7.

[0152] Table 7

[0153]

[0154]

[0155] 7. PCR product purification

[0156] Use Vazyme's VAHTS DNAClean Beads for purification, catalog number N411. Make sure the beads have been equilibrated at room temperature for more than 30 minutes before use. The steps are as follows:

[0157] (1) Turn on the non-contact dispenser Dragonfly, add 6 μL VAHTS DNA Clean Beads (Cat. No. N411) to the 10 μL PCR amplification product in the above step, heat seal at 168°C for 2 seconds, mix and incubate at room temperature for 5 minutes; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5 minutes;

[0158] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0159] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 11 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, centrifuge briefly, and incubate at room temperature for 5 minutes;

[0160] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 min. 10 μL of the supernatant was transferred to a new 384-well plate. After the library quality inspection was qualified, subsequent Illumina sequencing was performed.

[0161] The library quality inspection results are shown in Table 8, and the Qsep400 peak results are shown in Figure 2 and Figure 3 As shown, the results show that the library quality inspection peak graph is qualified and conforms to the normal distribution, without front tailing and large and small fragments and other impurity peaks.

[0162] Table 8

[0163] sample Library concentration (ng / μL) Library volume (μL) Total library amount (ng) Detection fragment size (bp) Rice 28.06 10 280.6 546.6 soil 24.8 10 248 518.6

[0164] The statistics of sequencing data output are shown in Table 9. The results show that the library constructed by the 0.1× system method has good output, and the sequencing quality value is not significantly different from that of the library constructed by the 1× system method.

[0165] Table 9

[0166]

[0167] Example 2

[0168] This example constructs and analyzes rice and human transcriptome libraries.

[0169] This example uses Yeasen's mRNA Isolation Master Kit mRNA Purification Kit (Cat. No. 12603) and The Ultima Dual-mode mRNA Library Prep Kit is compatible with both Illumina and MGI platforms and the Dual-mode mRNA Library Prep Kit (Cat. No. 12309) for library construction.

[0170] 1. mRNA purification and shearing

[0171] 1.1 Sample preparation

[0172] Take rice and human RNA samples, place them on an ice box to melt, flick to mix and centrifuge briefly, take 300 ng of total RNA (Nanodrop quantification) to a 384-well plate, add nuclease-free water to the above RNA samples to make up the total volume to 5 μL, seal the film, centrifuge briefly, and place the 384-well plate on ice;

[0173] 1.2 mRNA and magnetic beads bind for the first time

[0174] (1) Yeasen's Place the mRNA Capture Beads in the mRNA Isolation Master Kit (Cat. No. 12603) on a four-dimensional rotator and mix thoroughly for more than 20 minutes, and adjust the PCR instrument or metal bath to 65°C or 80°C (maintained);

[0175] (2) Turn on the non-contact dispenser Dragonfly, dispense the mRNA capture beads into the RNA sample plate, heat seal at 165°C for 1.5 seconds, mix well, centrifuge briefly, and immediately place in a PCR instrument and incubate at 65°C for 5 minutes to denature the RNA;

[0176] (3) After denaturation, place the 384-well plate at room temperature for 5 min to allow the mRNA to bind to the magnetic beads;

[0177] (4) After instant centrifugation, place the 384-well plate on the non-contact microplate nucleic acid purification system G.PURE magnetic stand for 5 minutes. After observing that the magnetic beads are completely adsorbed, open the program and discard the supernatant to separate the mRNA from the total RNA;

[0178] (5) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly carrier of the non-contact dispenser and add 20 μL of Yeasen The beads wash buffer (Beads Wash Buffer) in the mRNA purification kit (Cat. No. 12603) was transferred to a magnetic rack and heat-sealed at 165°C for 1.5 seconds. After mixing and instant centrifugation, it was placed on the non-contact microplate nucleic acid purification system G.PURE magnetic rack for 5 minutes. After observing that the magnetic beads were completely adsorbed, the program was opened to remove the supernatant and washed once with ethanol, and the supernatant was completely discarded.

[0179] (6) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly carrier of the non-contact dispenser and add 5 μL of Yeasen mRNA Isolation Master Kit The Tris buffer in the mRNA purification kit (Cat. No. 12603) was transferred with a magnetic rack and heat-sealed at 165°C for 1.5 seconds. After mixing and instant centrifugation, the 384-well plate was placed at 80°C for 2 minutes to elute the mRNA.

[0180] 1.3 Second binding of mRNA to magnetic beads

[0181] (1) Add 5 μL of Yeasen's mRNA IsolationMaster Kit mRNA Purification Kit (Cat. No. 12603) contains the Beads Binding Buffer, heat-seal at 165°C for 1.5 seconds, mix well, centrifuge briefly, and leave at room temperature for 5 minutes to allow the mRNA to rebind to the magnetic beads.

[0182] (2) After instant centrifugation, place the 384-well plate on the G.PURE magnetic stand of the non-contact microplate nucleic acid purification system for 5 minutes. After observing that the magnetic beads are completely adsorbed, open the program and discard the supernatant to separate mRNA from total RNA.

[0183] (3) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly carrier of the non-contact dispenser and add 20 μL of Yeasen The magnetic beads washing buffer (Beads Wash Buffer) in the mRNA purification kit (Cat. No. 12603) was transferred to a magnetic rack and heat-sealed at 165°C for 1.5 seconds. After mixing and instant centrifugation, it was placed on the non-contact microplate nucleic acid purification system G.PURE magnetic rack for 5 minutes. After observing that the magnetic beads were completely adsorbed, the program was opened to remove the supernatant and washed once with ethanol, and the supernatant was completely discarded.

[0184] (4) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly carrier of the non-contact dispenser and add 3 μL of Yeasen The Ultima Dual-mode mRNA Library Prep Kit is compatible with the Illumina and MGI dual platforms and the fragmentation buffer (Frag / Prime Buffer) in the Dual-mode mRNA Library Construction Kit (Cat. No. 12309). Transfer with a magnetic rack, heat seal at 165°C for 1.5 seconds, mix and centrifuge instantly.

[0185] 1.4 mRNA elution and fragmentation

[0186] (1) Preheat the PCR machine to 94°C, place the 384-well plate on the PCR machine at 94°C and press the timer to start the timer, then cover the PCR machine with the hot cover and incubate for 7 minutes;

[0187] (2) After the timing is over, take out the plate and place it on an ice box immediately. Then centrifuge it and place it on the Sciclone-384 magnetic rack of the automatic liquid handling workstation. Let it stand for 4 minutes. Transfer all the supernatant (about 1.7 μL) to a new 384-well plate that has been filled with a single-strand synthesis reagent for the synthesis of the first-strand cDNA.

[0188] 2. Synthesize the first strand of cDNA

[0189] (1) When interrupting for 7 minutes, use Yeasen's Ultima Dual-mode mRNA Library Prep Kit is compatible with Illumina and MGI dual platforms and dual-mode mRNA library preparation kit (Cat. No. 12309) for single-strand synthesis. According to Table 10, 1st Strand Buffer 2 and 1st Strand Enzyme Mix 2 were prepared into a mixed solution of single-strand synthesis reagents. Mix was flicked to mix, centrifuged, and transferred to the Dragonfly sample slot of the non-contact dispenser. The single-strand reagent mixed solution Mix was dispensed into a new 384-well plate.

[0190] Table 10

[0191] Reagents Volume (μL) 1st Strand Buffer 2 0.6 1st Strand Enzyme Mix 2 0.2 Fragmented mRNA 1.7 Total volume 2.5

[0192] (2) After the disruption is completed, the 384-well plate is placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 minutes. All the supernatant (about 1.7 μL) is transferred to the 384-well plate containing the single-strand synthesis reagent, and heat-sealed at 165°C for 1.5 seconds. After mixing, the plate is centrifuged instantly.

[0193] (3) Set the reaction program according to Table 11 and run it. When the temperature of the PCR instrument reaches 25°C, immediately place the 384-well plate on the PCR instrument for reaction. After the reaction is completed, immediately proceed to the next step of second-strand synthesis.

[0194] Table 11

[0195] Heat cover 105℃ 1 25℃,10min 2 42℃,15min 3 70℃,15min 4 Keep

[0196] 3. Synthesis of the second strand of cDNA (including end repair and A-tailing)

[0197] Using Yeasen Ultima Dual-mode mRNA Library Prep Kit is compatible with Illumina and MGI dual platforms and dual-mode mRNA library preparation kit (Cat. No. 12309) for second-strand synthesis (including end repair and A-tailing). According to Table 12, the second-strand synthesis reaction buffer 2nd Strand Buffer (with dNTP) and the synthesizer 2nd Strand Enzyme Master Mix are prepared into a mixed solution Mix of second-strand synthesis reagents. The non-contact dispenser Dragonfly is turned on and the second-strand reagent mixed solution Mix is ​​dispensed into a 384-well plate containing the 1st strand cDNA. The plate is heat-sealed at 165°C for 1.5s, mixed and centrifuged instantly, and the reaction program is set and run according to Table 13. When the temperature reaches 16°C, the above 384-well plate is immediately placed in a PCR instrument and the PCR instrument hot cover is covered to perform second-strand cRNA synthesis.

[0198] Table 12

[0199] Reagents Volume (μL) 2nd Strand Buffer (with dNTP)* 3 2nd Strand Enzyme Master Mix 0.5 1st strand cRNA 2.5 Total volume 6

[0200] Table 13

[0201] Heat cover 105℃ 1 16℃,30min 2 72℃,15min 3 4℃maintain

[0202] 4. Connector connection

[0203] Using Yeasen Ultima Dual-mode mRNA Library Prep Kit is compatible with Illumina and MGI dual platforms and Dual-mode mRNA Library Construction Kit (Cat. No. 12309) for adapter ligation reaction. According to Table 14, prepare the ligation reaction buffer Ligation Enhancer and ligase Novel T4 RNA ligase except DNA Adapter into a mixed solution Mix of ligation reaction reagents (operate on ice), mix by pipetting or oscillating, centrifuge briefly, transfer to the Dragonfly non-contact dispenser sample tank, add 1μL DNA Adapter (connector) to the double-stranded product using the automatic liquid handling workstation Sciclone-384, then turn on the Dragonfly non-contact dispenser, add 3μL ligation mixture Ligation Mix to the above product, and react according to the procedure in Table 15.

[0204] Table 14

[0205] Reagents Volume (μL) Ligation Enhancer 2.6 Novel T4 RNA ligase 0.4 IDT Adaptor 1 dA-tailed dsDNA 6 total 10

[0206] Table 15

[0207] Heat cover close a 20℃,15min b 4℃maintain

[0208] 5. Purification of ligation product

[0209] Pay attention to the temperature of magnetic bead incubation and magnetic bead purification, try to keep the laboratory temperature uniform (currently it is recommended to control the temperature at around 22°C), use Yeasen's DNA Selection Beads are used for purification. Product No. 12601. Before using the magnetic beads, ensure that the magnetic beads have been equilibrated at room temperature for more than 30 minutes. The steps are as follows:

[0210] (1) Turn on the non-contact dispenser Dragonfly and add 6 μL of Yeasen's DNA Selection Beads (Cat. No. 12601), 168°C, heat seal for 2s, mix and incubate at room temperature for 5min; then place on the G.PURE magnetic rack of the non-contact microplate nucleic acid purification system and let stand for 5min;

[0211] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0212] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 12 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, and incubate at room temperature for 5 minutes after centrifugation.

[0213] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 min. 10 μL of the supernatant was transferred to a new 384-well plate.

[0214] 6. Fragment sorting and purification

[0215] Table 16

[0216]

[0217] 6.1 Remove small fragments

[0218] (1) Turn on the non-contact dispenser Dragonfly and add 6.8 μL (0.68×) of the mixed Yeasen DNA Selection Beads (Cat. No. 12601), 168°C, heat seal for 2s, mix and incubate at room temperature for 5min; then place on the G.PURE magnetic rack of the non-contact microplate nucleic acid purification system and let stand for 5min;

[0219] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0220] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 11 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, centrifuge briefly, and incubate at room temperature for 5 minutes;

[0221] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 min. 10 μL of the supernatant was transferred to a new 384-well plate.

[0222] 6.2 Remove large fragments

[0223] (1) Turn on the non-contact dispenser Dragonfly and add 5.6 μL (0.56×) of the mixed Yeasen DNA Selection Beads magnetic beads (Cat. No. 12601), 168°C, heat seal for 2 seconds, mix and incubate at room temperature for 5 minutes;

[0224] (2) Place on the Sciclone-384 magnetic stand of the automated liquid handling workstation, let stand for 5 min, and transfer 14.2 μL of the supernatant to a new 384-well plate;

[0225] 6.3 Secondary purification

[0226] (1) Turn on the non-contact dispenser Dragonfly and add 9.2 μL (0.65×) of the mixed Yeasen DNA Selection Beads sorting magnetic beads (Cat. No. 12601), 168°C, heat seal for 2s, mix and incubate at room temperature for 5min; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5min;

[0227] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0228] (3) Immediately transfer the 384-well plate with the magnetic stand to the Dragonfly non-contact dispenser using Yeasen's Ultima Dual-mode mRNA Library Prep Kit is compatible with Illumina and MGI dual platforms and Dual-mode mRNA Library Prep Kit (Cat. No. 12309) for PCR amplification reaction. According to Table 17, 5ul of nuclease-free water, 5ul of amplification enzyme buffer 2×Super II High-Fidelity Mix and 1ul of amplification primer PrimerMix were prepared into a PCR Mix mixture. After mixing, 11μL of the mixture was added to a 384-well plate, and the plate was transferred to a magnetic rack and heat-sealed at 168℃ for 2s. After mixing, the plate was incubated at room temperature for 5min.

[0229] Table 17

[0230]

[0231] 7. PCR amplification

[0232] (1) The above 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation, left to stand for 5 min, and 10 μL of the supernatant was transferred to a new 384-well plate for subsequent PCR amplification reaction.

[0233] (2) Reaction procedure as shown in Table 18

[0234] Table 18

[0235]

[0236] 8. PCR product purification

[0237] Using Yeasen DNA Selection Beads magnetic beads are used for purification. Product number is 12601. Before using the magnetic beads, ensure that the magnetic beads have been equilibrated at room temperature for more than 30 minutes. The steps are as follows:

[0238] (1) Turn on the non-contact dispenser Dragonfly and add 6 μL of Yeasen's 10 μL of the ligation product from the above step. DNA Selection Beads sorting magnetic beads (Cat. No. 12601), 168°C, heat seal for 2s, mix and incubate at room temperature for 5min; then place on the non-contact microplate nucleic acid purification system G.PURE magnetic stand and let stand for 5min;

[0239] (2) After observing that the magnetic beads are completely adsorbed, turn on the non-contact microplate nucleic acid purification system G.PURE, remove the supernatant, perform two rounds of 20 μL ethanol washing, and completely discard the ethanol after the end;

[0240] (3) Immediately transfer the 384-well plate with the magnetic rack to the Dragonfly carrier of the non-contact dispenser, add 11 μL of nuclease-free water, transfer with the magnetic rack, heat seal at 168°C for 2 seconds, centrifuge three times to mix, centrifuge briefly, and incubate at room temperature for 5 minutes;

[0241] (4) The 384-well plate was placed on the Sciclone-384 magnetic stand of the automatic liquid handling workstation and allowed to stand for 5 min. 10 μL of the supernatant was transferred to a new 384-well plate. After the library passed the quality inspection, it was used for subsequent Illumina sequencing.

[0242] The library quality inspection results are shown in Table 19, and the Qsep400 peak results are shown in Figure 4 and Figure 5 The results showed that the library quality inspection peak graph was qualified and conformed to the normal distribution, without front tailing and large and small fragments.

[0243] Table 19

[0244] sample Library concentration (ng / μL) Library volume (μL) Total library amount (ng) Detection fragment size (bp) Rice 15.54 10 155.4 485.2 people 22.82 10 228.2 528.7

[0245] The statistics of sequencing data output are shown in Table 20. The results show that the transcriptome library constructed by the 0.1× system method has good output, and the sequencing quality value is not significantly different from that of the library constructed by the 1× system method, indicating that it is completely feasible to reduce the system library construction.

[0246] Table 20

[0247]

[0248] Example 3

[0249] Compared with Example 1 (0.1×-384 system), this example uses 1×-96 system for library construction and analysis. The main difference is:

[0250] 1. Compared with the 0.1×-384 system, the reagent dosage of the 1×-96 system for library construction is amplified by 10 times. The reagent dosage in the steps of enzyme digestion, adapter ligation, ligation product purification, fragment sorting, PCR amplification, and PCR amplification product purification is amplified by 10 times, and the reagent dosage is greatly increased;

[0251] 2. The library construction of 0.1×-384 system is carried out on 384-well plates, and the single library construction throughput is 384 samples; the library construction of 1×-96 system is carried out on 96-well plates, and the single library construction throughput is 96 samples.

[0252] The results of the gene number difference analysis between the 0.1×-384 system and the 1×-96 system in soil metagenomic resequencing library construction are as follows Figure 6 As shown in the figure, DZ-10, DZ-11, and DZ-12 are three technical replicates of a sample constructed using the 1×-96 system, and CS-10, CS-11, and CS-9 are three technical replicates of the same sample constructed using the 0.1×-384 system. It can be seen that there is basically no difference in the number of genes between the two methods.

[0253] The results of the comparative analysis of the 0.1×-384 system and the 1×-96 SNP genotypes of rice sample resequencing library are as follows Figure 7 As shown, YF-DZ-2 is a rice sample constructed with the 1×-96 system, and YF-SJ-3, YF-SJ-4, and YF-SJ-5 are three technical replicates of the same rice sample constructed with the 0.1×-384 system. The consistency rate of pairwise comparison of SNP genotypes is 80%, which is consistent with the control group.

[0254] The results of SNP correlation analysis of rice sample resequencing library construction 0.1×-384 system and 1×-96 system are as follows Figure 8 As shown, DZ-3, DZ-4, and DZ-5 are three technical replicates of a rice sample constructed using the 1×-96 library system, and CS-2, CS-3, and CS-4 are three technical replicates of the same rice sample constructed using the 0.1×-384 library system. There is no problem with the correlation between the technical replicates.

[0255] In summary, the present invention takes the construction of rice and soil resequencing libraries and rice and human transcriptome libraries as examples to demonstrate the feasibility of the 0.1× small system 384-well plate library construction process designed by the present invention. The sequencing data is compared with the currently commonly used 1×-96-well plate library construction process and there is no abnormality, which provides a new idea for efficient, low-cost and automated NGS library construction.

[0256] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a high-throughput small-scale resequencing library, characterized in that: The high-throughput small-system sequencing library construction method comprises the following steps: (1) DNA is sheared to obtain fragment products; (2) ligating the fragmented product to a linker to obtain a linker-ligated product; (3) purifying the adapter ligation product and performing fragment sorting to obtain a sorted product; (4) performing PCR amplification on the sorted products to obtain amplified products; (5) Purify the amplified product to obtain a sequencing library.

2. The method for constructing a high-throughput small-scale resequencing library according to claim 1, characterized in that: The reagents for the interruption treatment in step (1) are dispensed by a non-contact dispenser Dragonfly; Preferably, the connector in step (2) is manipulated by an automated liquid handling workstation Sciclone-384, and the connector-connected buffer is dispensed by a non-contact dispenser Dragonfly.

3. The method for constructing a high-throughput small-scale resequencing library according to claim 1 or 2, characterized in that: The purified magnetic beads and the remelted water after purification in step (3) are controlled by the non-contact liquid dispenser Dragonfly, and the supernatant discarding and ethanol washing steps in the purification process are completed by the non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed using the automatic liquid handling workstation Sciclone-384; Preferably, the enzyme and primers for PCR amplification in step (4) are mixed and then dispensed into a multi-well plate by a non-contact dispenser Dragonfly, mixed with the sorted product, and PCR amplification is performed. The PCR amplification procedure is: 95-98℃, 3-5min pre-denaturation; 95-98°C, 10-30s denaturation, 60-65°C, 15-30s annealing, 70-73°C, 30-45s extension, 8-10 cycles of amplification; 70-73℃ for 3-5min; Preferably, the purified magnetic beads and the water melted after purification in step (5) are controlled by a non-contact liquid dispenser Dragonfly, the supernatant discarding and ethanol washing steps in the purification process are controlled by a non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed using an automatic liquid handling workstation Sciclone-384; Preferably, the multi-well plate comprises a 384-well plate.

4. A method for constructing a high-throughput small-system transcriptome library, characterized in that: The high-throughput small-system transcriptome library construction method comprises the following steps: (S1) Enriching and purifying mRNA; (S2) fragmenting the purified mRNA to obtain fragmented mRNA; (S3) using the fragmented mRNA as a template, reverse transcription is performed to synthesize the first-strand cDNA; (S4) synthesizing the second-strand cDNA using the first-strand cDNA, and performing end repair and A addition; (S5) performing adapter ligation on the double-stranded cDNA and the adapter to obtain an adapter ligation product; (S6) purifying the adapter ligation product and performing fragment sorting to obtain a sorted product; (S7) performing PCR amplification on the sorted products to obtain amplified products; (S8) Purify the amplified product to obtain a transcriptome library.

5. The method for constructing a high-throughput small-system transcriptome library according to claim 4, characterized in that: The reagents interrupted in step (S2) are dispensed by a non-contact dispenser Dragonfly; Preferably, in step (S3), the synthesis buffer of the first-strand cDNA is dispensed by a non-contact dispenser Dragonfly; Preferably, in step (S4), the synthesis buffer of the second-strand cDNA is dispensed by a non-contact dispenser Dragonfly; Preferably, the connector in step (S5) is controlled by an automatic liquid handling workstation Sciclone-384, and the buffer connected to the connector is controlled by a non-contact dispenser Dragonfly; Preferably, the purified magnetic beads and the water melted after purification in step (S6) are controlled by a non-contact liquid dispenser Dragonfly, and the supernatant discarding and ethanol washing steps in the purification process are completed by a non-contact microplate nucleic acid purification system G.PURE; the liquid transfer between the multi-well plates is performed using an automatic liquid handling workstation Sciclone-384; Preferably, the enzyme and primers for PCR amplification in step (S7) are mixed and then dispensed into a multi-well plate by a non-contact dispenser Dragonfly, mixed with the sorted product, and PCR amplification is performed. The PCR amplification procedure is: 95-98℃, 1-3min pre-denaturation; 95-98°C, 10-30s denaturation, 60-65°C, 15-30s annealing, 70-73°C, 30-45s extension, 13-15 cycles of amplification; 70-73℃ for 3-5min; Preferably, the purified magnetic beads and the water melted after purification in step (S8) are controlled by a non-contact liquid dispenser Dragonfly, and the supernatant discarding and ethanol washing steps in the purification process are completed by a non-contact microplate nucleic acid purification system G.PURE, and the liquid transfer between the multi-well plates is performed by an automatic liquid handling workstation Sciclone-384; Preferably, the multi-well plate comprises a 384-well plate.

6. A small system mRNA enrichment and purification method, characterized in that: The small system mRNA enrichment and purification method is applied to the high-throughput small system transcriptome library construction method according to claim 4 or 5, comprising the following steps: (S1-1) Mixing total RNA with mRNA purification magnetic beads, incubating, and binding mRNA to the magnetic beads; (S1-2) placing the mixture of step (S1-1) on a magnetic rack, adsorbing the magnetic beads, and discarding the supernatant; (S1-3) Add cleaning solution, pipette and mix well, place on the magnetic stand, and discard the cleaning solution; (S1-4) adding elution buffer to elute the mRNA from the magnetic beads; (S1-5) Add binding solution to rebind the mRNA to the magnetic beads; (S1-6) Place on a magnetic stand, adsorb magnetic beads, and discard the supernatant; (S1-7) Add cleaning solution, place on the magnetic rack, and discard the cleaning solution.

7. The method for enriching and purifying mRNA in a small system according to claim 6, characterized in that: The magnetic beads in step (S1-1) are separated by a non-contact liquid separator Dragonfly; Preferably, in step (S1-2), the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE; Preferably, the cleaning solution in step (S1-3) is dispensed into a multi-well plate by a non-contact dispenser Dragonfly, and the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE; Preferably, the eluent in step (S1-4) is separated by a non-contact liquid separator Dragonfly; Preferably, in step (S1-6), the supernatant is discarded by the non-contact microplate nucleic acid purification system G.PURE; Preferably, the cleaning solution in step (S1-7) is dispensed into a multi-well plate by a non-contact dispenser Dragonfly, and the supernatant is discarded by a non-contact microplate nucleic acid purification system G.PURE.

8. A fragment sorting method, characterized in that: The fragment sorting method is applied to the high-throughput small-system sequencing library construction method according to any one of claims 1 to 3 or the high-throughput small-system transcriptome library construction method according to claim 4 or 5, comprising the following steps: (SS-1) taking the first DNA purification magnetic beads and mixing them with the purified adapter ligation product; (SS-2) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and remove the supernatant; (SS-3) Keep the multi-well plate on the magnetic stand and add ethanol to rinse the magnetic beads; (SS-4) Keep the multi-well plate on the magnetic stand, dry the magnetic beads, remove the multi-well plate from the magnetic stand, add water for elution, and transfer the eluted product to a new multi-well plate; (SS-5) adding a second DNA purification magnetic bead to the eluted product, mixing and incubating; (SS-6) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and transfer the supernatant to a new multiwell plate; (SS-7) add the third DNA purification magnetic beads, mix and incubate; (SS-8) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and remove the supernatant; (SS-9) Keep the multi-well plate on the magnetic stand and add ethanol to rinse the magnetic beads; (SS-10) Keep the multi-well plate on the magnetic stand, dry the magnetic beads, remove the multi-well plate from the magnetic stand, and add water for elution; (SS-11) Centrifuge the multiwell plate and place it on a magnetic stand to separate the beads and liquid, and transfer the supernatant to a new multiwell plate.

9. The fragment sorting method according to claim 8, characterized in that: In step (SS-1), the magnetic bead multiple of the first DNA purification magnetic beads is 0.6-0.7×, and the dosage is 6-7 μL. In step (SS-5), the magnetic bead multiple of the second DNA purification magnetic beads is 0.45-0.6×, and the dosage is 4.5-6 μL. In step (SS-7), the magnetic bead multiple of the third DNA purification magnetic beads is 0.6-0.7×, and the dosage is 8.1-10.5 μL. Preferably, the DNA purification magnetic beads in steps (SS-1), (SS-5) and (SS-7) are added to the multi-well plate via a non-contact liquid dispenser Dragonfly; Preferably, the elution water in steps (SS-4) and (SS-10) is added to the multi-well plate via a non-contact liquid dispenser Dragonfly.

10. The fragment sorting method according to claim 8 or 9, characterized in that: The eluted products in steps (SS-3), (SS-4), (SS-5), (SS-6), (SS-10), and (SS-11) were transferred via the automated liquid handling workstation Sciclone-384; Preferably, the removal of the supernatant in steps (SS-2) and (SS-8) is achieved by a non-contact microplate nucleic acid purification system G.PURE; Preferably, the ethanol rinsing in steps (SS-3) and (SS-9) is achieved by a non-contact microplate nucleic acid purification system G.PURE.

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