A rapid library construction method and reagent for trace fragmented DNA based on a semiconductor platform

Abstract

The present application relates to a rapid library construction method and reagent for trace fragment DNA based on a semiconductor platform. The ligation reagent and the rapid library construction method are designed based on a semiconductor sequencing platform. In the face of samples with characteristics such as trace and fragmentation, such as NIPT, the rapid library construction method can shorten the library construction time while maintaining the quality of the library, and can reduce the number of sample tube transfers during library construction, reducing the risk of errors.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a rapid library construction method and reagents for micro-fragmented DNA based on a semiconductor platform. Background Technology

[0002] Semiconductor sequencing platforms have the advantages of not requiring optical systems, rapid sequencing, relatively low equipment cost, relatively low sequencing throughput, and flexible application, making them play an increasingly important role in non-invasive prenatal genetic testing and precision medicine.

[0003] Non-invasive prenatal genetic testing (NIPT) detects fetal chromosomal abnormalities by analyzing cell-free DNA in the peripheral blood plasma of pregnant women. With its non-invasiveness, high accuracy, safety, early detection capability, and convenience, it has become an important technology in the field of prenatal screening. Cell-free plasma DNA is characterized by fragmentation (length less than 200 bp), trace amounts, and an increase in cffDNA with gestational age. According to publicly available data from Qiagen, the amount of cell-free DNA in 1 mL of plasma is approximately 7.35 ng. Typically, NIPT uses 200–600 μL of plasma, yielding approximately 1.47–4.41 ng of DNA.

[0004] Library construction is required before sequencing. Semiconductor sequencing platform-based library construction involves adding adapters adapted for semiconductor sequencing to both ends of the sample DNA fragments. Typically, a P1 adapter is added to one end, and an A adapter to the other, with both P1 and A adapters having blunt 3' ends. During adapter ligation, the P1 and A adapters are randomly ligated to both ends of the blunt-ended sample DNA fragments. Based on this principle, the highest theoretical ligation efficiency for semiconductor platform-based library construction is only 50%, which results in significant sample DNA loss and low library conversion rates for micro-volume DNA (0.1–10 ng). Conventional semiconductor platform micro-volume DNA library construction methods include the following steps: 1) end repair; 2) magnetic bead purification; 3) adapter ligation; 4) magnetic bead purification; 5) amplification; and 6) magnetic bead purification. Fragment screening is usually added during purification to further reduce the amount of raw DNA input during library construction. However, multi-step magnetic bead purification is cumbersome, time-consuming, and increases the risk of transfection errors. Therefore, the industry urgently needs a rapid, efficient method for constructing libraries on semiconductor sequencing platforms that can handle trace amounts of DNA (especially trace amounts of DNA requiring fragment screening). Summary of the Invention

[0005] The purpose of this invention is to disclose a rapid library construction method and reagents for micro-fragmented DNA based on a semiconductor platform, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0006] The first aspect of the present invention is to provide a ligation reagent.

[0007] A second aspect of the present invention is to provide a rapid library construction method for a small amount of fragmented DNA.

[0008] The ligation reagent of the first aspect of the present invention includes a buffer salt solution, MgSO4, PEG, DTT, ATP, dNTP, P1 adapter, tag X adapter and T4 DNA ligase, wherein the buffer salt solution is Tris-HCl or phosphate solution, and the P1 adapter and the tag X adapter are both short adapters of 20-35 nt.

[0009] Using the ligation reagent in the ligation reaction system can effectively ensure the efficiency of micro-DNA ligation. The resulting ligation product can reduce non-specific amplification in subsequent amplification processes. Therefore, using the ligation reagent can eliminate the need for a magnetic bead purification step between the adapter ligation and amplification steps during library construction without compromising library quality.

[0010] In a further application embodiment, the P1 connector consists of two forward and reverse sequence segments, namely:

[0011] 5'-CCTCTCTATGGGGCAGTCGGTGAT-3' (SEQ ID No: 1);

[0012] 5'-ATCACCGACTGCCCATAGAGA-3' (SEQ ID No: 2).

[0013] In a further application embodiment, the tag X connector consists of two forward and reverse sequence segments, namely:

[0014] 5'-CCTGCGTGTCTCCGACTCAGNNNNNNNNNNGAT-3' (SEQ ID No: 3);

[0015] 5'-ATCNNNNNNNNNNCTGAGTCGGAGACACGCA-3' (SEQ ID No: 4).

[0016] In a further application embodiment, the final concentrations of each component of the ligation reagent are as follows: buffer salt solution 50-100 mmol / L, MgSO4 5-10 mmol / L, PEG 5-10%, DTT 5-10 mmol / L, ATP 0.25-0.75 mmol / L, dNTP 0.5-1 mmol / L, P1 adapter 0.04-0.06 μmol / L, tag X adapter 0.04-0.06 μmol / L, and T4 DNA ligase 1-2.5 U.

[0017] The rapid database construction method according to the second aspect of the present invention includes the following steps:

[0018] 1) Obtain sample DNA and perform end repair and / or fragment screening;

[0019] 2) Use the ligation reagent described in the first aspect of the present invention to perform adapter ligation reaction on sample DNA to obtain ligation products;

[0020] 3) The ligation product and amplification reagent are subjected to amplification reaction, purified, and a library is obtained.

[0021] Since the ligation reagent described in the first aspect of the present invention is used when ligating the connector in step 2), the ligation product can be directly used for the amplification reaction in step 3), omitting the intermediate purification step, effectively shortening the library construction time, reducing the number of tube transfers, and thus reducing the risk of errors.

[0022] In a further application implementation, the reaction conditions for the connector connection reaction in step 2) are: 20~25℃ for 20~30 minutes, and 60~75℃ for 5~20 minutes.

[0023] In a further application implementation, the amplification reagents for the amplification reaction in step 3) include buffer salt solution, MgCl2, KCl, BSA, dNTP, glycerol, DNA polymerase, and a mixture of amplification primers, wherein the buffer salt solution is Tris-SO4 or Tris-HCl.

[0024] Furthermore, the final concentrations of each component of the amplification reagent are as follows: buffer salt solution 10-15 mmol / L, MgCl2 1-2 mmol / L, KCl 25-50 mmol / L, BSA 50-100 μg / mL, dNTP 150-200 μmol / L, glycerol 2.5-10% (V / V), DNA polymerase 1-2 U, and amplification primer mixture 0.2-1 μmol / L.

[0025] Using ligation products for amplification requires reducing the size of the ligation reaction system. This necessitates adjusting PEG concentration, ATP concentration, adapter and tag concentration, using short adapters with high ligation efficiency, and modifying the ligation reaction program to ensure ligation efficiency in a small system. Similarly, since the ligation products are directly used for amplification, the amplification system is affected by the original ligation products, requiring adjustments to the amplification system to further reduce non-specific amplification.

[0026] In a further application embodiment, the amplification reaction conditions are as follows: 72°C for 5-10 minutes; 95°C for 2-5 minutes; 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds, for a total of 10-15 cycles; 72°C for 1-5 minutes; and storage at 4°C.

[0027] In a further application implementation, the purification in step 3) is magnetic bead purification; preferably, the purification coefficient of magnetic bead purification is 0.8×~1.2×.

[0028] In a further application implementation, the library described in step 3) is used in a semiconductor sequencing platform.

[0029] The advantages of this invention compared to existing technologies include: the ligation reagent and the rapid library construction method are designed based on a semiconductor sequencing platform. When dealing with fragmented and trace samples such as NIPT plasma cell-free DNA, the rapid library construction method can shorten the library construction time while maintaining library quality, and can reduce the number of sample transfections during the library construction process, thus reducing the risk of errors. Attached Figure Description

[0030] Figure 1 This is a flowchart comparing the conventional database creation method and the rapid database creation method shown in Example 1;

[0031] Figure 2 This is a distribution map of library fragments obtained through the conventional process in Example 2;

[0032] Figure 3 This is a distribution map of library fragments obtained from simplified process 1 in Example 2;

[0033] Figure 4 This is a distribution diagram of library fragments obtained from simplified process 2 in Example 2;

[0034] Figure 5 This is a distribution map of library fragments obtained from simplified process 3 in Example 2;

[0035] Figure 6 This is a distribution map of library fragments obtained through the conventional process in Example 3;

[0036] Figure 7This is a distribution map of library fragments obtained from simplified process 4 in Example 3;

[0037] Figure 8 This is a distribution map of library fragments obtained from simplified process 5 in Example 3;

[0038] Figure 9 This is a distribution diagram of library fragments obtained from simplified process 6 in Example 3;

[0039] Figure 10 This is a distribution map of library fragments obtained from simplified process 7 in Example 3;

[0040] Figure 11 This is a distribution map of library fragments obtained from simplified process 8 in Example 3;

[0041] Figure 12 This is a distribution diagram of library fragments obtained from simplified process 9 in Example 3. Detailed Implementation

[0042] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0043] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0044] Example 1: A rapid library construction method for micro-fragmented DNA based on a semiconductor platform.

[0045] The rapid library construction method includes the following reagents and steps:

[0046] (1) End repair

[0047] Prepare the end-repair reaction solution (10×), as shown in Table 1.

[0048]

[0049] The end-repair reaction system comprises 43.5 μL of extracted plasma DNA, 5 μL of the end-repair reaction solution, and 1.5 μL of end-repair mixed enzyme. After mixing, the end-repair reaction system is incubated at room temperature for 30 minutes. The end-repair mixed enzyme comprises T4 DNA polymerase and T4 polynucleotide kinase, mixed at an enzyme activity ratio of 5:4.

[0050] (2) Segment selection

[0051] Magnetic beads were used to screen the end-repair products to obtain enriched fragmented DNA.

[0052] Fragment selection can be performed before or after end-repair processing, and the order of processing does not affect the library quality of this method.

[0053] (3) Connector connection

[0054] Prepare the ligation reaction solution (10×), as shown in Table 2.

[0055]

[0056] The total volume of the ligation reaction system is 20 μL, including 16.8 μL of fragmented DNA, 2 μL of the ligation reaction solution, 0.4 μL of T4 DNA ligase at a concentration of 5 U / μL, 0.4 μL of P1 adapter at a concentration of 2.5 μmol / L, and 0.4 μL of tag X adapter at a concentration of 2.5 μmol / L.

[0057] The P1 adapter is a connector suitable for sequencing on a semiconductor platform, and its forward and reverse nucleotide sequences are shown in SEQ ID No:1 and SEQ ID No:2, respectively. The tag X adapter is also a connector suitable for sequencing on a semiconductor platform, and its forward and reverse nucleotide sequences are shown in SEQ ID No:3 and SEQ ID No:4, respectively, where N is 8-10 specific bases (i.e., the tag).

[0058] Place this reaction system in a PCR instrument and run the following reaction program: 25°C for 30 minutes, then 70°C for 10 minutes (with a hot cap at 85°C).

[0059] (4) PCR amplification:

[0060] Prepare PCR reaction solution (2×) as shown in Table 3.

[0061]

[0062] The total volume of the PCR reaction system was 40 μL, including 20 μL of ligation product and 20 μL of the PCR reaction solution. The reaction system was placed in a PCR instrument. The reaction program was as follows: 72°C for 10 minutes; 95°C for 2 minutes; 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds, 13 cycles; 72°C for 2 minutes; stored at 4°C (heat-sealed 105°C).

[0063] (5) Magnetic bead purification

[0064] PCR amplification products were purified using magnetic beads to obtain libraries for sequencing. The libraries were then analyzed to obtain library concentration, fragment distribution, and sequencing-related indicators to assess library quality.

[0065] A flowchart comparing the conventional database creation method with the rapid database creation method provided in this embodiment is shown below. Figure 1 As shown.

[0066] Example 2: Comparison of results between conventional database construction methods and three direct simplified database construction methods.

[0067] The standard library construction process is as follows: end repair, purification (fragment screening), adapter ligation, purification, amplification, and purification. To verify the necessity of the purification step between the adapter ligation and amplification steps, libraries were constructed from the same cell-free plasma DNA according to the library construction process shown in Table 4 in this embodiment. The differences between the processes in Table 4 are the ligation reaction system in step three and the amplification reaction system in step four, as well as whether purification is performed between steps three and four; the P1 adapter and the tag X adapter both use short adapters as shown in SEQ ID No:1 to SEQ ID No:4.

[0068]

[0069] The obtained library was subjected to concentration determination and fragment distribution analysis. The library concentration obtained by the standard procedure was 62.16 nmol / L, and the fragment distribution was as follows: Figure 1 As shown; the library concentration obtained by simplified procedure 1 is 69.67 nmol / L, and the distribution of library fragments is as follows. Figure 2 As shown; the simplified procedure 2 yielded a library concentration of 64.21 nmol / L, and the library fragment distribution is as follows. Figure 3 As shown; the simplified procedure 3 yielded a library concentration of 25.39 nmol / L, and the library fragment distribution is as follows. Figure 4 As shown in the results, directly skipping the purification step between steps three and four leads to significant differences in the distribution of library fragments obtained through the simplified procedure. Simply ligating the adapters and skipping purification to proceed directly to PCR amplification does not achieve the construction of a high-quality library, even with increased ligation system and amplification volume, the results of conventional library construction are not achieved.

[0070] Example 3: Comparison of results between conventional database construction methods and six simplified database construction methods.

[0071] In this embodiment, the same cell-free plasma DNA was constructed according to both the standard procedure and simplified procedures 4-9 provided in Table 5. The difference between simplified procedures 4-9 and simplified procedures 1-3 is as follows:

[0072] (1) Adjustment of PEG, ATP and T4 DNA ligase in the adapter ligation reaction system, and adjustment of Tris-HCl, MgCl2, BSA and DNA polymerase in the amplification reaction system;

[0073] (2) Changes in the reaction conditions for the adapter connection and the purification coefficient of the magnetic beads after amplification.

[0074]

[0075]

[0076] The obtained library was subjected to concentration determination and fragment distribution analysis. The library concentration obtained by the standard procedure was 76.57 nmol / L, and the fragment distribution was as follows: Figure 5 As shown; the simplified procedure 4 yielded a library concentration of 85.46 nmol / L, and the library fragment distribution is as follows. Figure 6 As shown; the simplified procedure 5 yielded a library concentration of 148.49 nmol / L, and the library fragment distribution is as follows. Figure 7 As shown; the simplified procedure 6 yielded a library concentration of 66.85 nmol / L, and the library fragment distribution is as follows. Figure 8 As shown; the simplified procedure 7 yielded a library concentration of 49.53 nmol / L, and the distribution of the library fragments is as follows. Figure 9 As shown; the simplified procedure 8 yielded a library concentration of 86.49 nmol / L, and the library fragment distribution is as follows. Figure 10 As shown; the simplified procedure 9 yielded a library concentration of 30.32 nmol / L, and the library fragment distribution is as follows. Figure 11 As shown in the distribution graph, the main peaks of libraries 4-9 in the simplified procedure are consistent with those in the conventional procedure. This indicates that adjusting the ligation reaction conditions, ligation reaction system, and amplification system reduces the dimers caused by adapter self-ligation after mixing the ligation product with the PCR system, significantly reduces specific amplification, and significantly improves the fragment distribution of the libraries. However, since the residual adapter dimers in the simplified procedure are still higher than in the normal procedure, the reaction system and reaction conditions for adapter ligation and amplification reagents still need to be adjusted.

[0077] Example 4: A rapid library construction method for constructing micro-fragmented DNA.

[0078] Simplified procedures 10-19 were obtained by adjusting simplified procedure 4 in Example 3. In this example, the same plasma free DNA was constructed according to the conventional procedure in Table 6 and simplified procedures 10-19 respectively. The fragment distribution of the library was detected and sequencing was performed in the same run. The difference between simplified procedures 10-19 is the adapter concentration in the ligation reaction, the primer concentration in the PCR amplification reaction, and the amount of purification magnetic beads used after PCR amplification.

[0079]

[0080]

[0081] The obtained libraries were subjected to concentration determination and sequencing. The library concentration, adapter dimer ratio, fetal concentration, GC content, and percentage of valid sequencing data were analyzed. The results are shown in Table 7.

[0082]

[0083] According to the test results in Table 7, the simplified procedure has no significant impact on the sequencing GC content and the proportion of valid sequencing data, and the library concentration is much higher than the sequencing concentration. The simplified procedures 10, 12 and 14 have higher library concentrations and lower proportions of adapter dimer molecules. The fetal concentration, GC content and proportion of valid sequencing data of the library sequencing are comparable to those of the conventional procedure.

[0084] Example 5: Compatibility of rapid library construction method with different amounts of DNA.

[0085] In this embodiment, libraries were constructed and sequenced for different amounts of cell-free plasma DNA according to the conventional procedure in Table 6 and the simplified procedure 10, respectively. The sequencing data were analyzed, and the results are shown in Tables 8 and 9.

[0086]

[0087]

[0088]

[0089] Tables 8 and 9 show the library concentrations and corresponding sequencing metrics for different input amounts of DNA (0.9–8.1 ng) in the conventional and simplified procedures. According to the results in Tables 8 and 9, the library concentrations constructed using the simplified procedure 10 are significantly higher than the sequencing concentrations. The sequencing metrics fluctuate within the normal range compared to the conventional procedure, showing no significant differences, and the results are comparable.

[0090] The simplified procedure 12 and simplified procedure 14 were subjected to the same comparative verification, and the results showed that the library quality was comparable to that of the conventional procedure. This indicates that the rapid library construction method and reagents of the present invention are compatible with different input amounts of DNA samples (0.9~8.1 ng).

[0091] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A rapid library building method for micro-fragmented DNA for semiconductor sequencing platforms, characterized by, The method comprises the following steps: 1) obtaining DNA in a plasma sample, performing end repair and / or fragment screening; 2) performing a linker ligation reaction on the sample DNA in step 1) using a ligation reagent to obtain a ligation product; the total volume of the ligation reagent is 20 μL, and the ligation reagent comprises Tris-HCl with a pH of 8 and a concentration of 75 mmol / L, MgSO4 with a concentration of 6 mmol / L, 6% PEG8000, DTT with a concentration of 7.5 mmol / L, ATP with a concentration of 0.6 mmol / L, dNTP with a concentration of 0.7 mmol / L, P1 linkers and tag X linkers with a concentration of 0.04 or 0.05 μmol / L, and 2 U of T4 DNA ligase; the P1 linkers are composed of two positive and negative sequence fragments, and the nucleotide sequences of the two positive and negative sequence fragments are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively; the tag X linkers are composed of two positive and negative sequence fragments, and the nucleotide sequences of the two positive and negative sequence fragments are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively; the reaction conditions of the linker ligation reaction are as follows: 25°C for 30 minutes and 70°C for 10 minutes; 3) performing an amplification reaction on the ligation product and an amplification reagent; the total volume of the amplification reagent is 40 μL, and the amplification reagent comprises Tris-HCl with a pH of 8 and a concentration of 15 mmol / L, MgCl2 with a concentration of 1.25 mmol / L, KCl with a concentration of 50 mmol / L, BSA with a concentration of 60 μg / mL, dNTP with a concentration of 200 μmol / L, glycerol with a volume ratio of 0.05, 1.5 U of DNA polymerase, an amplification primer mixture with a concentration of 0.3 or 0.4 μmol / L; the reaction conditions of the amplification reaction are as follows: 72°C for 10 minutes; 95°C for 2 minutes; 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds, a total of 13 cycles; 72°C for 2 minutes; 4°C storage; magnetic bead purification with a purification coefficient of 1x to obtain a library.

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