Free DNA library building method suitable for nanopore sequencing
By optimizing the library preparation process for nanopore sequencing and extending the end repair and barcode ligation time, the problems of short and low concentration of cfDNA fragments were solved, achieving efficient and stable sequencing data output and meeting the high-precision analysis requirements of cfDNA.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU COWIN BIOTECH CO LTD
- Filing Date
- 2026-01-22
- Publication Date
- 2026-04-14
AI Technical Summary
Existing nanopore sequencing technologies face challenges in processing circulating cell-free DNA (cfDNA), including low library construction efficiency and insufficient sequencing data output due to short fragments and low concentrations. In particular, they struggle to effectively accommodate the natural methylation information of cfDNA when PCR amplification is not required.
By optimizing the library preparation process for nanopore sequencing, extending the end repair time to 1 hour and the barcode ligation time to 2 hours, and combining it with magnetic bead purification technology, a high-efficiency cfDNA library was constructed, ensuring the stability and purity of sequencing adapter ligation.
It significantly improved the pore occupancy rate of nanopores, stabilizing it at over 80%, increasing the amount of sequencing data and ensuring efficient and high-precision analysis of cfDNA.
Smart Images
Figure CN121852512A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a free DNA library suitable for nanopore sequencing. Background Technology
[0002] Circulating cell-free DNA (cfDNA) is fragmented DNA found in bodily fluids such as blood. As an important non-invasive biomarker, it has great value in in vitro diagnostic fields such as non-invasive prenatal testing, early tumor diagnosis, and monitoring of treatment efficacy (liquid biopsy). However, cfDNA itself presents challenges such as short fragments, low concentrations, and complex backgrounds, placing extremely high demands on the sensitivity and accuracy of its detection technologies.
[0003] Currently, sequencing analysis of cfDNA mainly relies on second-generation sequencing technology. While this technology is mature and offers high throughput, its library preparation process, which depends on PCR amplification, is prone to introducing sequence bias, affecting quantitative accuracy; furthermore, its relatively long detection cycle makes it difficult to meet the needs of rapid clinical diagnosis. In contrast, third-generation sequencing technologies, represented by Oxford nanopore sequencing, exhibit significant advantages: they can achieve real-time sequencing of single molecules without PCR amplification, not only avoiding amplification bias and ensuring accurate quantification, but also greatly shortening the detection time. Simultaneously, their ultra-long read lengths facilitate the analysis of complex genomic regions.
[0004] The most promising application of combining cfDNA with nanopore sequencing technology is direct methylation sequencing. DNA methylation is a key gene expression regulatory mechanism closely related to cancer development. Unlike traditional methods that require bisulfite treatment that severely damages DNA, nanopore technology can directly and non-destructively read methylation modification information on the natural DNA strand during sequencing. This means that gene sequence variations and whole-genome methylation profiles can be obtained simultaneously in a single sequencing run, providing a more powerful tool for cancer tracing and early diagnosis. However, developing a dedicated library preparation method that is highly compatible with nanopore sequencing and perfectly preserves its natural methylation information, tailored to the characteristics of cfDNA, has become a key bottleneck driving the development of this field. This invention is proposed to solve this problem. Summary of the Invention
[0005] This invention aims to provide a free DNA library preparation method suitable for nanopore sequencing, focusing on solving the problems of low efficiency and insufficient sequencing data output caused by short and low concentration of cfDNA fragments in conventional library preparation.
[0006] The basic principle of nanopore sequencing is as follows: 1. Double-stranded DNA is unwound by a motor enzyme before passing through the nanopore, with one strand binding to the enzyme; 2. Driven by the motor enzyme, the single-stranded DNA linearly passes through the nanopore; 3. Different bases generate specific electrical signals as they pass through the nanopore, enabling real-time sequencing. Generally, the longer the DNA fragment, the longer it takes to pass through the nanopore, resulting in higher sequencing efficiency. Therefore, nanopore sequencing often uses long DNA fragments (tens of kb) as templates. However, the average length of cfDNA is only about 170 bp. If a conventional library preparation process is used, short DNA fragments passing through the nanopore too quickly can easily lead to premature termination of sequencing, making it difficult to obtain complete and accurate sequencing information.
[0007] The library preparation process before nanopore sequencing mainly includes DNA end preparation, adapter ligation, and purification. DNA end preparation involves end repair and A-tailing; adapter ligation connects the sequencing adapter to the pre-treated DNA fragment and then to the motor protein; finally, purification removes unligated adapters, enzymes, salt ions, and other impurities to obtain the final sequencing library. Unlike next-generation sequencing, nanopore library preparation does not involve PCR amplification, thus preserving the original DNA sequence to the greatest extent possible and avoiding mutation bias introduced by amplification.
[0008] Conventional nanopore library construction (such as the standard procedure using the ONT official SQK-NBD kit) is mainly designed for long, high-concentration genomic DNA fragments. Its key steps typically include: end repair for 15 minutes, A-tailing and barcode ligation for 15 minutes, followed by adapter ligation and purification. However, the low concentration and short fragments of cfDNA make it difficult for this standard procedure to guarantee library construction success with low input volumes, and it is also prone to generating byproducts such as adapter dimers.
[0009] In preliminary experiments, the inventors found that cfDNA libraries constructed using the standard procedure had low yields, were prone to nanopore blockage during sequencing, had low pore occupancy, and limited data output. Through system optimization, extending the end repair time to 1 hour and barcode ligation to 2 hours, while maintaining standard kit procedures for the remaining steps, the inventors unexpectedly discovered that libraries constructed using the optimized method showed significantly improved data output after sequencing, with nanopore occupancy consistently reaching over 80%.
[0010] To further increase the amount of sequencing data, the preferred implementation scheme is as follows: after purifying the cfDNA library with magnetic beads, take 20–50 ng for sequencing. When the total library volume is within this range, the loading volume can be appropriately reduced while still maintaining high well occupancy and high data output.
[0011] The technical solution of this invention to solve the technical problem is as follows:
[0012] This invention provides a method for preparing free DNA libraries suitable for nanopore sequencing, comprising the following steps:
[0013] 1) End repair and A-tail addition: Take the extracted cfDNA, add end repair enzyme and buffer to perform end repair, repair the extracted DNA double strand into a flat 5' end with a phosphate group; add an adenine deoxynucleotide to the 3' end of the end-repaired DNA fragment, i.e., an A-tail, to achieve complementary ligation with the T tail of the barcode.
[0014] 2) Barcode ligation: Using ligase, the barcode is ligated to the A tail of the repaired DNA fragment. A specific barcode is ligated to the end of each DNA fragment, and after ligation, the DNA fragment carries a unique "sample ID".
[0015] 3) Adapter ligation: Mix the sequencing adapters for the nanopore sequencer with the DNA fragments obtained in the previous step, and perform adapter ligation under the action of DNA ligase to connect the sequencing adapters to the DNA fragments.
[0016] 4) Purification: After the ligation reaction, the DNA library obtained in the previous step is purified using magnetic beads. The product obtained after elution is the free DNA library, which can be directly used for ONT sequencing.
[0017] The ligase used in the above steps was from Kangwei Century (product number CW2701), the end-repair enzyme was from Kangwei Century (product number CW3045), and the barcode and sequencing adapter were from the Oxford Nanopore official kit SQK-NBD.
[0018] In step 1), the end repair time is 1 hour; in step 2), the barcode ligation time is 2 hours. In step 4), after the cfDNA library is purified by magnetic beads, 20-50 ng is used for sequencing.
[0019] This invention also provides the application of the library preparation method in cell-free DNA (cfDNA) sequencing, wherein the application is a non-disease diagnostic application.
[0020] This invention offers the following technical advantages: Addressing the issues of low efficiency and insufficient data output in conventional nanopore library construction caused by short and low-concentration cfDNA fragments, this invention systematically optimizes the reaction times of two key steps—end repair and barcode ligation—extending them to 1 hour and 2 hours respectively, thereby significantly improving library construction success rate and sequencing efficiency. After library construction using this method, the nanopore occupancy rate can be stably maintained above 80%, effectively increasing the amount of sequencing data and providing a reliable technical solution for efficient and high-precision nanopore sequencing analysis of cfDNA. Attached Figure Description
[0021] Figure 1 The pore occupancy of the ONT nanopores is shown 15 hours after sequencing.
[0022] Figure 2 The cfDNA sequencing read length and the number of corresponding reads for volunteers.
[0023] Figure 3 The barcode fragment was compared with the original cfDNA fragment after ligation using the Agilent 4200 fragment analyzer.
[0024] Figure 4 This study describes the analysis of DNA fragment lengths after different durations of barcode ligation using an Agilent 4200 fragment analyzer.
[0025] Figure 5 The well occupancy is the result of sequencing 1.5 hours after library preparation according to ONT sequencing standards.
[0026] Figure 6 The well occupancy is the result of sequencing 1.5 hours after library construction according to the steps of this invention. Detailed Implementation
[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.
[0028] Example 1: cfDNA nanopore library construction and sequencing verification using the method of the present invention.
[0029] All cfDNA used in this invention was extracted from volunteer plasma using the Kangwei Century Magnetic Bead Free Nucleic Acid Extraction Kit. Nanopore library construction used the Oxford Nanopore Company's official SQK-NBD kit, which included the necessary barcodes and adapters. The ligase used was from Kangwei Century (catalog number CW2701), and the end-repair enzyme used was from Kangwei Century (catalog number CW3045).
[0030] Two mL of plasma from each of three volunteers was collected, and cfDNA was extracted. Library construction was then performed according to the optimized method of this invention, including the following steps:
[0031] 1) End repair and A-tail addition: Take the extracted cfDNA, add end repair enzyme and buffer to perform end repair, repair the extracted DNA double strand into a flat 5' end with a phosphate group; add an adenine deoxynucleotide to the 3' end of the end-repaired DNA fragment, i.e., an A-tail.
[0032] 2) Barcode ligation: Using ligase, barcodes are ligated to the A tail of the repaired DNA fragments. Specific barcodes are ligated to the ends of each DNA fragment.
[0033] 3) Adapter ligation: Mix the sequencing adapters for the nanopore sequencer with the DNA fragments obtained in the previous step, and perform adapter ligation under the action of DNA ligase to connect the sequencing adapters to the DNA fragments.
[0034] 4) Purification: After the ligation reaction, the DNA library obtained in the previous step is purified using magnetic beads. The product obtained after elution is the free DNA library.
[0035] In step 1), the end repair time is 1 hour; in step 2), the barcode ligation time is 2 hours. Then, adapter ligation (1.5 hours) and purification are performed according to the standard kit procedure, followed by library sequencing.
[0036] Experimental results:
[0037] The total extracted cfDNA amounts were 12.72 ng, 7.68 ng, and 16.08 ng, respectively. After mixing, end repair and barcode ligation were performed, yielding a product of 53.12 ng. Further adapter ligation and purification yielded a final library of 35.28 ng. All of this was used for nanopore sequencing, and the results are as follows... Figure 1-2 As shown.
[0038] from Figure 1 It can be seen that the well occupancy rate remained at 86.5% 15 hours after sequencing, and did not decrease significantly.
[0039] from Figure 2 It can be seen that the main peaks of the sequencing fragments in the three samples are located in the 130–190 bp range, which is consistent with the cfDNA length characteristics; each sample produced abundant sequencing reads, which meet the needs of downstream analysis.
[0040] Example 2: Determination and Comparison of Optimization Conditions (End-of-Line Repair and Barcode Connection Time)
[0041] Plasma cfDNA was extracted from one volunteer using the same kit, and the concentration was determined to be 0.423 ng / μL using Qubit assay. The effect of the optimized conditions of this invention on the intermediate product fragment length was compared with that of the standard kit procedure using the SQK-NBD kit.
[0042] The optimized version of this invention consists of: 1 hour of end-point repair + 2 hours of Barcode connection (specific steps are the same as in Example 1).
[0043] Standard procedure control group: Strictly follow the instructions of the SQK-NBD kit, end repair for 15 minutes + barcode ligation for 15 minutes.
[0044] Experimental results are as follows Figure 3-4 As shown:
[0045] Figure 3 Capillary electrophoresis results showed that, compared with the standard control group, the main peak of the product in the optimized group of this invention (1 hour of end repair + 2 hours of barcode ligation) shifted significantly, consistent with the expected increase in fragment length after successful barcode ligation. The products obtained from the control group (1 hour of end repair + 15 minutes of barcode ligation, 1 hour of end repair + 1 hour of barcode ligation, and the standard library preparation procedure (15 minutes of end repair + 15 minutes of barcode ligation) were the same length as the cfDNA fragments used in library preparation, indicating no change and suggesting that the barcode was not ligated.
[0046] Figure 4 The results showed that extending the barcode ligation time to 3–4 hours did not result in any further shift in the main peak of the product, and the peak area remained largely consistent with the nucleic acid concentration. This indicates that for cfDNA library preparation, the optimal reaction time for the barcode ligation step is 2 hours, and extending it further does not provide any additional benefit. Using 3–4 hours to ligate the barcode yields products with fragment sizes consistent with those obtained using 2 hours, both effectively increasing the length of the cfDNA fragment.
[0047] Based on previous explorations, the core optimization conditions of this invention are determined to be: on the basis of the standard procedure of the SQK-NBD reagent kit, the end repair time is extended to 1 hour and the barcode connection time is extended to 2 hours.
[0048] Example 3: Comparison of sequencing performance between the optimized library preparation method of this invention and the standard procedure.
[0049] The same cfDNA sample was sequenced after library construction using the following two different procedures:
[0050] Standard procedure control group: SQK-NBD kit was used, strictly following the instructions (end repair 15 minutes + barcode ligation 15 minutes, followed by standard adapter ligation).
[0051] The optimized group of this invention uses the same SQK-NBD kit, but adopts the optimized conditions of this invention (1 hour of end repair + 2 hours of barcode ligation, followed by standard adapter ligation). The specific steps are the same as in Example 1.
[0052] The nanopore occupancy rates of both methods during the sequencing process were recorded and compared. The results are as follows: Figure 5-6 As shown.
[0053] Figure 5 The results showed that, in the standard procedure control group, well occupancy was only about 20% 1.5 hours after library preparation and sequencing.
[0054] Figure 6 The results show that after sequencing the optimized library using this invention for 1.5 hours, the well occupancy rate is significantly improved.
[0055] In summary, after library construction using the method of this invention, the nanopore occupancy rate can be stably maintained at over 80%, effectively increasing the amount of sequencing data and providing a reliable technical solution for efficient and high-precision nanopore sequencing analysis of cfDNA.
[0056] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.
Claims
1. A method for preparing a free DNA library suitable for nanopore sequencing, comprising the following steps: 1) End repair and A-tail addition: Take the extracted cfDNA, add end repair enzyme and buffer to perform end repair, repair the extracted DNA double strand into a blunt 5' end with a phosphate group; add an adenine deoxynucleotide to the 3' end of the end-repaired DNA fragment, i.e., an A-tail; 2) Barcode ligation: Using ligase, barcodes are ligated to the A tail of the repaired DNA fragments. Specific barcodes are ligated to the ends of each DNA fragment. 3) Adapter ligation: Mix the sequencing adapter with the DNA fragment obtained in the previous step, and perform adapter ligation under the action of DNA ligase to connect the sequencing adapter to the DNA fragment; 4) Purification: After the ligation reaction, the DNA library obtained in the previous step is purified using magnetic beads. The product obtained after elution is the free DNA library. Its features are, In step 1), the end repair time is 1 hour; in step 2), the Barcode connection time is 2 hours. The barcodes and sequencing adapters required for the above steps are both from the Oxford Nanopore official kit SQK-NBD.
2. The application of the library preparation method as described in claim 1 in cell-free DNA (cfDNA) sequencing, wherein the application is not a disease diagnosis application.