Single-stranded oligonucleotide molecule and method for detecting synthesis quality of oligonucleotide pool

Abstract

The invention discloses a single-stranded oligonucleotide molecule and a method for detecting the synthesis quality of an oligonucleotide pool, the single-stranded oligonucleotide molecule provided by the invention can detect the synthesis quality of the oligonucleotide pool during construction of a high-throughput library, and during detection, universal sequences are respectively added at two ends of Oligo Pool, so that the synthesis quality of the oligonucleotide pool is detected. And a single-stranded oligonucleotide molecule containing a UMI sequence is added for each Oligo by utilizing a polymerase extension reaction. A target product is released through enzyme digestion, and sequencing linker sequences are added to the two ends of the Oligo sequence through PCR amplification and used for subsequent high-throughput sequencing. By utilizing the single-stranded oligonucleotide molecule and the detection method disclosed by the invention, the influence on the homogeneity of the Oligo Pool in a library building process can be reduced to the greatest extent, and the homogeneity and error rate of a single sequence can be corrected by using UMI in a signal generation analysis stage, so that the accuracy and homogeneity of synthesis of the Oligo Pool can be effectively evaluated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a single-stranded oligonucleotide molecule and method for detecting the quality of oligonucleotide pool synthesis. Background Technology

[0002] Oligo pools are mixed libraries composed of a large number of short-chain DNA or RNA sequences (typically 20-200 nt) with different sequences. High-throughput, large-scale synthesis technology of oligo pools has been widely used in gene editing (such as CRISPR libraries), directed evolution, and DNA storage. However, the accurate assessment of their synthesis quality faces two core challenges: how to effectively evaluate the uniformity of the oligo pool and the accuracy of the synthesis.

[0003] Currently, the best approach is to use high-throughput sequencing technology, which requires library construction of the OligoPool before high-throughput sequencing. Due to the low concentration and short fragments in the OligoPool, library construction typically involves amplification of the OligoPool, adapter ligation, and then sequencing. However, amplification of the OligoPool and the amplification process during library construction can introduce significant bias. Furthermore, steps such as purification during library construction and amplification can cause changes in homogeneity. In addition, errors are introduced during amplification and sequencing themselves, leading to inaccurate assessments of final homogeneity and error rate.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a single-stranded oligonucleotide molecule and method for detecting the quality of oligonucleotide pool synthesis. The single-stranded oligonucleotide molecule and detection method of this invention can minimize the impact on the uniformity of the Oligo Pool during amplification and library construction. Furthermore, UMI can be used to correct the uniformity and error rate of a single sequence during the bioinformatics analysis stage, which can effectively evaluate the accuracy and uniformity of Oligo Pool synthesis.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a single-chain oligonucleotide molecule comprising: A 5' universal primer sequence, which is formed at the 5' end of a single-stranded oligonucleotide molecule; The 3' universal primer sequence is formed at the 3' end of the single-stranded oligonucleotide molecule; The UMI sequence is formed between the 5' universal primer sequence and the 3' universal primer sequence; The single-stranded oligonucleotide molecule has a blocking group modified at the 3' end and a biotin modified at the 5' end; and it contains an enzyme cleavage site.

[0007] Secondly, this invention provides the application of the above-mentioned single-stranded oligonucleotide molecules in sequencing and library construction.

[0008] Thirdly, the present invention provides a kit for quality detection of oligonucleotide library synthesis, comprising the above-mentioned single-stranded oligonucleotide molecules.

[0009] Fourthly, the present invention provides a method for detecting the quality of oligonucleotide pool synthesis, comprising: A universal sequence was added to both ends of each synthesized Oligo, with the 3' universal sequence matching the 5' universal primer sequence in the single-stranded oligonucleotide molecule; Then, single-stranded oligonucleotide molecules were added to Oligo molecules with universal sequences attached to both ends, and a complementary new chain was synthesized at the 3' end of Oligo using polymerase; streptavidin magnetic beads were then added to the reaction system, and after mixing, nucleic acid molecules trapped on the magnetic beads were obtained by magnetic separation. The magnetic beads are separated from the captured nucleic acid molecules by removing the portion of the single-streptavidin-bound oligonucleotide molecule from the enzyme cleavage site. The isolated nucleic acid molecules are denatured and destranded into single strands by PCR, and sequencing adapter sequences are added to both ends of the Oligo sequence of one of the single strands by amplification. The resulting product is purified and then subjected to high-throughput sequencing.

[0010] The present invention has the following beneficial effects: The single-stranded oligonucleotide molecule in this invention consists of a 5' universal primer sequence, a UMI sequence, and a 3' universal primer sequence. The 3' end is specially modified, and the 5' end is modified with biotin. The single-stranded oligonucleotide molecule contains restriction enzyme sites. The special modification at the 3' end prevents the UMI sequence from influencing subsequent PCR processes, ensuring that each oligo sequence has exactly one UMI. The biotin modified at the 5' end binds to streptavidin magnetic beads during detection, capturing and purifying the target product and minimizing the heterogeneity that may occur with traditional magnetic bead purification. The restriction enzyme sites in the oligonucleotide molecule can cleave the magnetic beads, reducing the influence of the magnetic beads on amplification.

[0011] The single-stranded oligonucleotide molecules provided by this invention can be used to detect the quality of oligonucleotide pool synthesis during high-throughput library construction. During detection, universal sequences are added to both ends of the oligo pool, and a polymerase extension reaction is used to add an independent single-stranded oligonucleotide molecule containing a UMI sequence to each oligo. The target product is then released by enzyme digestion, and sequencing adapter sequences are added to both ends of the oligo sequences via PCR amplification for subsequent high-throughput sequencing. Using the single-stranded oligonucleotide molecules and detection method of this invention minimizes the impact on the uniformity of the oligo pool during library construction, and UMI can be used to correct the uniformity and error rate of individual sequences during bioinformatics analysis, effectively evaluating the accuracy and uniformity of oligo pool synthesis. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a structural diagram of the single-stranded oligonucleotide molecule in this invention; Figure 2 This is a schematic flowchart of the method for detecting the quality of oligonucleotide pool synthesis in this invention; Figure 3 This is a schematic diagram of the method for detecting the quality of oligonucleotide pool synthesis in this invention; Figure 4 This refers to the database construction result in Example 1; Figure 5 This is the database construction result in Example 2. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] The uniformity of an Oligo Pool refers to whether the concentration or abundance distribution of different oligonucleotide sequences in the Oligo Pool is uniform. High uniformity means that the proportion of all designed oligonucleotides in the mixture is close to the same, while low uniformity means that some sequences are significantly excessive or insufficient.

[0016] Oligo pool accuracy refers to the consistency between each oligonucleotide sequence in the pool and its target design sequence, i.e., whether errors introduced during synthesis or amplification (such as base mismatches, insertions, deletions, or abnormal chemical modifications) exist. High accuracy indicates that all sequences closely match their design template, while low accuracy implies the presence of many erroneous sequences, which may affect the reliability of downstream experiments.

[0017] Accuracy focuses on the correctness of the sequence itself, while uniformity focuses on the consistency of sequence abundance; both jointly determine the quality of the Oligo Pool. However, if high-throughput sequencing technology is used to detect the accuracy and uniformity of the synthesized Oligo Pool, the method itself will affect the judgment of uniformity and error rate. To improve the amplification bias and purification selectivity problems existing in the implementation of this technology, this invention inserts and ligates functional fragments into the Oligo sequence after the Oligo Pool is synthesized and before high-throughput sequencing. This minimizes the impact on uniformity in subsequent synthesis quality detection, effectively evaluating the accuracy and uniformity of the synthesized Oligo Pool.

[0018] This invention provides a single-chain oligonucleotide molecule, the structure of which is as follows: Figure 1 As shown, the sequence represents the nucleotide sequence from the 3' end to the 5' end from left to right. Specifically, the single-stranded oligonucleotide molecule includes a 5' universal primer sequence and a 3' universal primer sequence. The 5' universal primer sequence is formed at the 5' end of the single-stranded oligonucleotide molecule, and the 3' universal primer sequence is formed at the 3' end of the single-stranded oligonucleotide molecule. It also includes a UMI sequence, which is formed between the 5' universal primer sequence and the 3' universal primer sequence.

[0019] A UMI sequence (Unique Molecular Identifier) ​​is a short nucleotide tag widely used in molecular biology and high-throughput sequencing to track and distinguish original molecules, eliminating the effects of PCR amplification bias and sequencing errors. It is a short nucleotide sequence synthesized randomly or semi-randomly, with a length of 4–40 bp, more preferably 8–20 bp, and is typically added to each original molecule (i.e., Oligo) before library preparation. In some embodiments of this invention, the UMI sequence length is 18 bp.

[0020] A universal sequence refers to a standardized DNA or RNA sequence fragment that is pre-designed and reused in various application scenarios in molecular biology experiments. These sequences themselves do not carry specific experimental information, but they support key steps in the experimental process, such as amplification, sequencing, cloning, or functionalization. The 5' and 3' universal primer sequences in the single-stranded oligonucleotide molecules of this invention can serve as binding sites for PCR primers. The length and sequence information of the universal primer sequences at both ends are not limited, and those skilled in the art can design them according to actual conditions.

[0021] In the single-stranded oligonucleotide molecule of the present invention, the 3' end is modified with a blocking group and the 5' end is modified with biotin; and the single-stranded oligonucleotide molecule contains an enzyme cleavage site.

[0022] The blocking group at the 3' end serves to prevent elongation. This blocking group can be a known blocking group in the art, such as iDT, C3 spacer, 3' phosphorylation, and ddNTP, or other groups with similar functions, such as NH2, -N3, DBCO, DIG, THS, 2-F-RC, 2-F-RU, 2'-O-Methyl C / G / U / A, ddC, m5C / m5dC, HMC, LNA (AT CG), m6A / m6dA, THO, and CHO. In some embodiments, the blocking group modified at the 3' end of the single-stranded oligonucleotide molecule is iDT.

[0023] The 5' end of biotin is modified to bind with streptavidin in subsequent processes, thereby achieving purification of the target product. Purification of the target product through the highly specific and strong binding of the biotin-streptavidin system avoids the fragment size selectivity of traditional purification methods, improving purification efficiency and ensuring purification uniformity.

[0024] After purifying the target product using the biotin-streptomycin avidin system, in order to reduce the influence of magnetic beads on amplification, this invention also designs an enzyme cleavage site at the end of the single-stranded oligonucleotide molecule that links biotin. Specifically, it is located on the 5' universal primer sequence of the single-stranded oligonucleotide molecule. By designing this enzyme cleavage site, the magnetic beads can be removed after purification, thereby reducing the influence of magnetic beads on subsequent processes.

[0025] The aforementioned restriction enzyme site is located on the 5' end universal primer sequence. It can be a uracil insertion site, a restriction endonuclease cleavage site, or a combination of both. In some embodiments of the present invention, the restriction enzyme site on the 5' end universal primer sequence is a uracil insertion site. This site can be cleaved by the USER enzyme to remove a biotin-linked sequence, thereby separating the magnetic beads from the nucleic acid molecules.

[0026] The above-mentioned single-stranded oligonucleotide molecule can be used to connect universal sequences to both ends of the Oligo to be detected. The universal sequence at the 3' end of the Oligo matches the universal sequence at the 3' end of the single-stranded oligonucleotide molecule, and can form a double strand through complementary pairing, which can then be used for Oligo Pool synthesis quality detection.

[0027] Based on the above-mentioned single-stranded oligonucleotide molecules, the present invention can provide both a kit for detecting the quality of oligonucleotide pool synthesis in sequencing and library construction, and a method for detecting the quality of oligonucleotide pool synthesis.

[0028] The kit includes the above-mentioned single-stranded oligonucleotide molecules. The specific sequence is not limited by this invention. Any single-stranded oligonucleotide molecule that is suitable for a specific detection target and is obtained by using the above design concept is within the scope of protection of this invention.

[0029] A schematic flowchart of a method for detecting the quality of oligonucleotide pool synthesis provided by this invention is shown below. Figure 2 As shown, the specific principle is as follows: Figure 3 As shown, the detection method includes the following steps: S1. Dilute the samples in the Oligo Pool with universal sequences added to both ends.

[0030] The universal sequence at the 3' end of each Oligo molecule matches the universal primer sequence at the 5' end of the single-stranded oligonucleotide molecule, enabling them to form a double strand through complementary pairing. The universal sequence at the 5' end of the Oligo molecule can be a conventional universal sequence in the art, and this invention does not limit it.

[0031] S2. The diluted sample is mixed with the above single-stranded oligonucleotide molecules, and new chains are synthesized by using the above single-stranded oligonucleotide molecules as templates under the action of polymerase.

[0032] S3. The extended product is mixed with streptavidin magnetic beads, and then the nucleic acid molecules trapped on the magnetic beads are obtained by magnetic separation.

[0033] The 5' end of a single-stranded oligonucleotide molecule is attached to biotin, which can bind to streptavidin with high specificity and strength, thereby capturing nucleic acid molecules onto streptavidin-linked magnetic beads. The magnetic beads can then be separated from other substances through magnetic separation, thereby enriching the nucleic acid molecules captured by the magnetic beads and achieving the purpose of purification.

[0034] S4. The nucleic acid molecules captured by the magnetic beads are separated from the captured nucleic acid molecules by removing the portion of the single-stranded oligonucleotide molecule that is bound to streptavidin through the enzyme cleavage site.

[0035] In this invention, the enzyme cleavage site on the single-stranded oligonucleotide molecule is located on the universal primer sequence at its 5' end. Using this site, the biotin-linked part (i.e. the part that binds to streptavidin) can be cleaved off under the action of enzymes such as restriction endonucleases, thereby separating the magnetic beads from the nucleic acid molecules.

[0036] S5. The isolated nucleic acid is denatured and destranded into single strands by the first PCR. One single strand is the extended Oligo sequence, and the other is the single-stranded oligonucleotide molecule with the biotin-linked end removed. In a reaction system with added polymerase and primer molecules, the sequencing adapter sequence is ligated to both ends of the extended Oligo sequence and its complementary strand in two steps.

[0037] S6. Purify the product obtained after two PCRs.

[0038] In some embodiments, the purification method includes magnetic bead purification.

[0039] S7. The purified product is then sequenced.

[0040] After high-throughput sequencing, the detection results of the products are obtained and further analyzed to obtain the accuracy and uniformity results of the batch of products.

[0041] The reaction system and procedure for extension and two PCR amplification steps described above can be adjusted according to specific sequences and reagents from different sources, and this invention does not limit such adjustments.

[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0043] The primer and reagent information used in the embodiments of this invention is as follows: Table 1 Primer Information

[0044] Note: NNN represents the UMI sequence; the above sequences are compatible with the Illumina sequencing platform; [i5] and [i7] are index sequences. In the sequence listing, T (thymine) at positions 4, 9, and 17 of SEQ ID NO.1 represents U (uracil); the index sequence [i5] is inserted between positions 29 and 30 of SEQ ID NO.3; the index sequence [i7] is inserted between positions 24 and 25 of SEQ ID NO.4.

[0045] Table 2 Component Information

[0046] Example 1 This embodiment describes a method for detecting the quality of oligonucleotide pool synthesis, the steps of which are as follows: S1. Sample Dilution 1. Quantify Oligo samples using the Qubit ssDNA kit.

[0047] Note: The Oligo sample is a template sample synthesized by Qingke Biotechnology, and its specific sequence is shown in SEQ ID NO.9.

[0048] 2. Use the tool on the webpage https: / / nebiocalculator.neb.com / #! / ssdnaamt to convert the Oligo sample quality.

[0049] 3. Based on the mass calculated in S1.2, take the corresponding volume of Oligo sample into a new PCR tube and add ddH2O to 9 μL.

[0050] S2. Extension 1. Add 1 μL of hyb-UMI-i7-3U (10μM) to the above sample, vortex 3 times for 3 seconds each time, and collect the reaction solution to the bottom of the tube by instantaneous centrifugation.

[0051] 2. Add 10 μL of 2×Phanta Flash Master Mix to S2.1, vortex 3 times for 3 seconds each time, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0052] 3. Place the above samples into the PCR instrument and perform the reaction according to the reaction procedure in Table 3: Table 3 Extended Reaction Procedure

[0053] S3 streptavidin magnetic bead purification 1. Place the xCapSeq™ affinity bead bottle on a vortex mixer for 20 s to resuspend the beads. Transfer 50 μL of the beads to a new centrifuge tube using a pipette. Place the centrifuge tube on a magnetic separator and let it stand for 1 min (this operation will be referred to as magnetic separation below). Remove the supernatant with a pipette and remove the centrifuge tube from the magnetic separator. 2. Add 500 μL of Buffer I to the centrifuge tube, cap the tube, and shake well to resuspend the magnetic beads. Magnetic separation will occur; remove the supernatant. 3. Repeat step 2 once. 4. Add 250 μL of biotinylated nucleic acid diluted with Buffer I, and vortex thoroughly to resuspend the magnetic beads. Place the centrifuge tube on a rotary mixer and mix at room temperature for 30 min. 5. Magnetic separation: Transfer the supernatant to a new centrifuge tube. 6. Wash the magnetic beads three times following the method in step 2. 7. Add 25 μL of DEPC water and resuspend the magnetic beads. This completes the biotinylated nucleic acid binding step.

[0054] S4 Capture Product Release 1. Transfer the sample and magnetic beads from S3.7 to a new 0.2 mL PCR tube, add 25 μL of 2×Phanta Flash Master Mix and 1 μL of Uracil-Specific Excision Reagent (1 U / μL), vortex 3 times for 3 seconds each time, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0055] 2. Place the above samples into a PCR instrument and incubate at 37°C for 30 minutes.

[0056] S5: First PCR 1. Centrifuge the PCR tube in S4.2 briefly, place it on a magnetic rack, and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate the supernatant into a new 0.2 mL PCR tube using a pipette, and discard the magnetic beads.

[0057] 2. Add 2 μL primer-i7-13 (10 μM) and 2 μL primer-5common-i5 (10 μM), vortex 3 times for 3 seconds each time, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0058] 3. Place the above samples on a PCR instrument and perform the reaction according to Table 4: Table 4. Reaction Procedure for the First PCR

[0059] S6 Second PCR 1. Take 1 μL of the above sample and add 7 μL of ddH2O, 1 μL of primer-i5-13, 1 μL of primer-i7-13, and 10 μL of 2×Phanta Flash Master Mix. Vortex 3 times for 3 seconds each time, and then centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0060] 2. Place the above samples on a PCR instrument and perform the reaction according to Table 5: Table 5 Reaction Procedure for the Second PCR

[0061] 3. After the reaction is complete, transfer all the above samples to a new 1.5 mL centrifuge tube.

[0062] S7 magnetic bead purification 1. Remove the DNA Clean Beads 30 minutes in advance and place them at room temperature. Shake well before use.

[0063] 2. Use a pipette to aspirate 20 μL of DNA Clean Beads into the adapter ligation product in S4.5, and gently pipette at least 10 times until all magnetic beads are suspended. On the last pipette, make sure that all liquid in the pipette tip and magnetic beads are transferred into the centrifuge tube.

[0064] 3. Incubate at room temperature for 5 min. 4. Centrifuge the centrifuge tube briefly, place it on a magnetic rack, and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate the supernatant with a pipette and discard it.

[0065] 5. Keep the centrifuge tubes on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0066] 6. Repeat step S5.5 once, and try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge the tube briefly. After separating the tubes on a magnetic rack, use a small-capacity pipette to remove the liquid from the bottom of the tube.

[0067] 7. Keep the centrifuge tubes on the magnetic rack, open the centrifuge tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked.

[0068] 8. Remove the centrifuge tube from the magnetic rack, add 25 μL of DEPC water (DNase, RNase free) to elute the DNA, and gently pipette at least 10 times until all magnetic beads are suspended.

[0069] 9. Incubate at room temperature for 5 min. 10. Centrifuge the centrifuge tube briefly, place it on a magnetic rack, and let it stand for 2-5 minutes until the liquid is clear. Use a pipette to transfer 23 μL of the supernatant to a new 0.2 mL centrifuge tube.

[0070] S8. Sequencing The supernatant obtained from S7.10 was subjected to high-throughput sequencing, and the sequencing results were then analyzed for uniformity and accuracy. The analysis method was as follows: 10M reads were directly extracted from the sequencing data, and BLAST was used for sequence alignment. The sequence alignment rate was calculated, and the specific proportion of the first 4 NNNN and the last 4 NNNN combination sequences in the alignment template sequence was compared.

[0071] Database construction results are as follows Figure 4 As shown, channel 1 is the negative control sample for the first PCR; channel 2 is the result of the first PCR; channel 3 is the negative control for the second PCR; and channel 4 is the result of the second PCR.

[0072] The sequencing results are shown in Table 6: Table 6 Sequencing Results

[0073] The analysis results are shown in Table 7: Table 7 Analysis Results

[0074] Since the sequence results are almost identical and the library construction uses single-chain capture, the uniformity is good with Q95 / Q5 = 6.2 before UMI correction. After UMI correction, Q95 / Q5 decreases from 6.2 to 1.4, and the uniformity is still significantly improved, indicating that the library construction method has achieved the expected results.

[0075] Example 2 The difference from Example 1 is that the Oligo samples in this example are a different batch of samples. Specifically, compared with Example 1, Example 2 mixes ten different sequences with a CG content of 25%-70%. The specific sequence information is shown in SEQ ID NO.5-14.

[0076] All the database construction processes in this embodiment are the same as in Embodiment 1, except for the samples; since the number of templates has increased from 1 to 10, the amount of data to be analyzed has increased to 100M.

[0077] Database construction results are as follows Figure 5 As shown, channel 1 is the negative control sample for the first PCR; channel 2 is the result of the first PCR; channel 3 is the negative control for the second PCR; and channel 4 is the result of the second PCR.

[0078] The sequencing results are shown in Table 8: Table 8 Sequencing Results

[0079] The analysis results are shown in Table 9: Table 9 Analysis Results

[0080] Due to the different CG content in the sequences, amplification can lead to bias. In the uncorrected UMI, the Q95 / Q5 ratio is 473, indicating a significant difference in uniformity. After UMI correction, the Q95 / Q5 ratio decreases from 473 to 10.2, showing a very significant improvement in uniformity, indicating that the library construction method has achieved the expected results.

[0081] Currently, the conventional detection technology generally involves PCR amplification of the Oligo Pool, followed by WGS library construction and high-throughput sequencing. This method has significant shortcomings. For example, during Oligo Pool amplification, inherent sequence differences (such as CG content and secondary structure) can lead to significant amplification bias, greatly impacting uniformity. Furthermore, during WGS library construction, the selectivity of purification fragments can also select for Oligo molecules of different lengths, causing variations in uniformity. Therefore, amplification uniformity is affected by factors such as the composition of the Oligo Pool and differences in CG content, resulting in a Q95 / Q5 ratio of approximately 10-10000 or even higher.

[0082] According to the QIAGEN experimental manual (Optimization of library amplification for next-generation sequencing, Katja Heitz, Ioanna Andreou, Peter Hahn, Annika Piotrowski, Frank Reinecke, Dirk Löffert, and Nan Fang, QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany), PCR-based WGS library preparation typically exhibits significant non-uniformity. For example, some conventional amplification mixtures may result in only about 70% base coverage at or above 0.8-fold normalization, far below the homogeneity level of over 90% achievable in this invention. Quantitative analysis shows that the homogeneity under conventional PCR methods is poor; for example, the amount of DNA at different loci may differ by up to 2,000-fold after amplification, and in some regions of the genome (especially extreme GC regions), coverage differences of more than 2-fold or even close to zero coverage may occur. This non-uniformity has a ripple effect on downstream analyses, leading to false positives / false negatives in variant detection, obscuring structural variations, fragmenting of genome assembly, and distortion of gene expression estimates, ultimately potentially misleading scientific findings and biological interpretations. Even commonly used methods such as "duplicate removal," designed to correct for biases, can introduce new, subtle biases, further highlighting the complexity of the problem.

[0083] Compared with the above detection methods, the present invention directly constructs the Oligo Pool library without the need for Oligo Pool amplification. At the same time, it uses biotin purification to replace traditional purification methods and employs UMI uniformity correction and other methods to effectively solve the above problems.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A single-stranded oligonucleotide molecule, characterized in that, include: A 5' universal primer sequence is formed at the 5' end of the single-stranded oligonucleotide molecule; A 3' universal primer sequence is formed at the 3' end of the single-stranded oligonucleotide molecule; The UMI sequence is formed between the 5' universal primer sequence and the 3' universal primer sequence; The single-stranded oligonucleotide molecule is modified with a blocking group at its 3' end and biotin at its 5' end; the single-stranded oligonucleotide molecule contains an enzyme cleavage site.

2. The single-chain oligonucleotide molecule according to claim 1, characterized in that, The UMI sequence is a short nucleotide sequence synthesized randomly or semi-randomly, containing 4 to 40 bases; Preferably, the UMI sequence contains 8 to 20 bases.

3. The single-chain oligonucleotide molecule according to claim 1, characterized in that, The blocking groups include: iDT, C3Spacer, 3' phosphorylation group, ddNTP, NH2, -N3, DBCO, DIG, THS, 2-F-RC, 2-F-RU, 2'-O-Methyl C / G / U / A, ddC, m5C / m5dC, HMC, LNA(ATCG), m6A / m6dA, THO, and CHO.

4. The single-chain oligonucleotide molecule according to claim 1, characterized in that, The restriction enzyme site is located on the 5' universal primer sequence of the single-stranded oligonucleotide molecule; The restriction enzyme sites include uracil insertion sites and / or restriction endonuclease cleavage sites.

5. The use of the single-stranded oligonucleotide molecule according to any one of claims 1 to 4 in sequencing and library construction.

6. The application according to claim 5, characterized in that, The application includes detecting the synthesis quality of oligonucleotide pools during the construction of high-throughput libraries.

7. A kit for quality control of oligonucleotide library synthesis, characterized in that, Includes the single-chain oligonucleotide molecule as described in any one of claims 1 to 4.

8. A method for detecting the quality of oligonucleotide pool synthesis, characterized in that, include: A universal sequence was added to both ends of each synthesized Oligo, and the universal sequence at the 3' end of the 3' end matched the universal primer sequence at the 5' end of the single-stranded oligonucleotide molecule. Then, single-stranded oligonucleotide molecules were added to Oligo molecules with universal sequences attached to both ends, and a complementary new chain was synthesized at the 3' end of Oligo using polymerase; streptavidin magnetic beads were then added to the reaction system, and after mixing, nucleic acid molecules trapped on the magnetic beads were obtained by magnetic separation. The magnetic beads are separated from the captured nucleic acid molecules by removing the portion of the single-streptavidin-bound oligonucleotide molecule from the enzyme cleavage site. The isolated nucleic acid molecules are denatured and destranded into single strands by PCR, and sequencing adapter sequences are added to both ends of the Oligo sequence of one of the single strands by amplification. The resulting product is purified and then subjected to high-throughput sequencing.

9. The detection method according to claim 8, characterized in that, Sequencing adapter sequences were added to both ends of the Oligo sequence through amplification by a first PCR and a second PCR.

10. The detection method according to claim 8, characterized in that, The purification method includes magnetic bead purification.

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