Rapid library building method and reagent for trace fragmented DNA based on semiconductor platform

Abstract

The invention relates to a rapid library building method and reagent for trace fragmented DNA based on a semiconductor platform. The connection reagent and the rapid library building method are designed based on a semiconductor sequencing platform. For samples with fragmented and trace characteristics, such as NIPT, the rapid library building method can shorten the library building time on the premise of keeping the library quality, and can reduce the sample transfer times and reduce the error risk in the library building process.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a method and reagent for rapid library construction of trace fragmented DNA based on a semiconductor platform. Background Art

[0002] Semiconductor sequencing platforms have the characteristics of no need for optical systems, rapid sequencing, relatively low equipment costs, relatively small sequencing throughput and flexible application. These characteristics make semiconductor sequencing platforms play an increasingly important role in non-invasive prenatal genetic testing and precision medicine.

[0003] Non-invasive prenatal genetic testing (NIPT) detects the chromosome status of the fetus by testing the plasma DNA of the pregnant woman's peripheral blood. It has become an important technology in the field of prenatal screening due to its non-invasiveness, high accuracy, safety, early detection capability and convenience. Plasma free DNA has the characteristics of fragmentation (less than 200 bp in length), trace amount, and cffDNA increases with the increase of gestational age. According to the data disclosed by Qiagen, the amount of free DNA in 1 mL of plasma is about 7.35 ng. Generally, the amount of plasma used in NIPT is 200~600 μL, and the amount of DNA obtained is about 1.47~4.41 ng.

[0004] Before sequencing, the sample library needs to be constructed. The library construction based on the semiconductor sequencing platform is to add adapters adapted to semiconductor sequencing to both ends of the sample DNA fragment, usually a P1 adapter is added to one end and an A adapter is added to the other end, where the 3' ends of the P1 adapter and the A adapter are both blunt ends. When connecting the adapters, the P1 adapter and the A adapter are randomly connected to the two ends of the blunt-ended sample DNA fragment. Based on this principle, the highest theoretical connection efficiency of the semiconductor platform library construction is only 50%, which is large for the construction of trace DNA (0.1~10 ng) library, and the library conversion rate is low. The conventional semiconductor platform trace DNA library construction method includes the following steps: 1) end repair; 2) magnetic bead purification; 3) adapter connection; 4) magnetic bead purification; 5) amplification; 6) magnetic bead purification. Generally, a fragment screening step is added to the purification, which will further reduce the amount of original DNA input in the library construction process. In addition, the multi-step magnetic bead purification operation is cumbersome and time-consuming, which increases the risk of transfer errors. Therefore, the industry urgently needs a fast, efficient, and semiconductor sequencing platform library construction method that can target trace amounts of DNA (especially trace amounts of DNA that require fragment screening). Summary of the invention

[0005] The purpose of the present invention is to disclose a method and reagent for rapid library construction of trace fragmented DNA based on a semiconductor platform, so as 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 provides a linking reagent.

[0007] The second aspect of the present invention is to provide a method for rapidly building a library of trace fragmented DNA.

[0008] The ligation reagents in the first aspect of the present invention include a buffered saline solution, MgSO4, PEG, DTT, ATP, dNTP, a P1 linker, a tag X linker and T4 DNA ligase, wherein the buffered saline solution is Tris-HCl or a phosphate solution, and the P1 linker and the tag X linker are both short linkers of 20 to 35 nt.

[0009] The use of the ligation reagent in the ligation reaction system can play a beneficial effect of ensuring the efficiency of trace DNA ligation. The ligation product thus obtained can reduce non-specific amplification in the subsequent amplification system. Therefore, the use of the ligation reagent can omit the magnetic bead purification step between the adapter ligation step and the amplification step in the process of library construction without reducing the construction quality of the library.

[0010] In a further application embodiment, the P1 linker is composed of two forward and reverse sequence fragments, respectively: 5'-CCTCTCTATGGGGCAGTCGGTGAT-3' (SEQ ID No: 1); 5'-ATCACCGACTGCCCATAGAGA-3' (SEQ ID No: 2).

[0011] In a further application embodiment, the tag X adapter is composed of two forward and reverse sequence fragments, respectively: 5'-CCTGCGTGTCTCCGACTCAGNNNNNNNNNNGAT-3' (SEQ ID No: 3); 5'-ATCNNNNNNNNNNCTGAGTCGGAGACACGCA-3' (SEQ ID No: 4).

[0012] In a further application embodiment, the final concentrations of the components of the ligation reagent are respectively 50-100 mmol / L of buffered saline, 5-10 mmol / L of MgSO4, 5-10% of PEG, 5-10% of DTT, 0.25-0.75 mmol / L of ATP, 0.5-1 mmol / L of dNTP, 0.04-0.06 μmol / L of P1 linker, 0.04-0.06 μmol / L of tag X linker and 1-2.5 U of T4 DNA ligase.

[0013] The rapid library construction method described in the second aspect of the present invention comprises the following steps: 1) Obtain sample DNA and perform end repair and / or fragment screening; 2) using the ligation reagent described in the first aspect of the present invention to perform a linker ligation reaction on the sample DNA to obtain a ligation product; 3) Perform amplification reaction on the ligation product and amplification reagent, purify, and obtain the library.

[0014] Since the ligation reagent described in the first aspect of the present invention is used in step 2) to connect the adapter, 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, and reducing the number of tube transfers, thereby reducing the risk of errors.

[0015] In a further application embodiment, the reaction conditions of the linker connection reaction in step 2) are: 20-25° C. for 20-30 minutes, and 60-75° C. for 5-20 minutes.

[0016] In a further application embodiment, the amplification reagents of the amplification reaction in step 3) include a buffered saline solution, MgCl2, KCl, BSA, dNTP, glycerol, DNA polymerase, and an amplification primer mixture, and the buffered saline solution is Tris-SO4 or Tris-HCl.

[0017] Furthermore, the final concentrations of the components of the amplification reagent are respectively 10~15 mmol / L of buffered saline, 1~2 mmol / L of MgCl2, 25~50 mmol / L of KCl, 50~100 μg / mL of BSA, 150~200 μmol / L of dNTP, 2.5~10% (V / V) of glycerol, 1~2 U of DNA polymerase and 0.2~1 μmol / L of amplification primer mixture.

[0018] When using the ligation product for amplification reaction, it is necessary to reduce the ligation reaction system. Therefore, it is necessary to adjust the PEG concentration, ATP concentration, adapter and label concentration, use a short adapter with high ligation efficiency and adjust the ligation reaction program to ensure the ligation efficiency in the small system. Similarly, because the ligation product is directly used in the amplification reaction, the amplification system is affected by the original ligation product, and the amplification system needs to be adjusted to further reduce nonspecific amplification.

[0019] In a further application embodiment, the reaction conditions of the amplification reaction are 72°C for 5 to 10 minutes; 95°C for 2 to 5 minutes; 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds, for a total of 10 to 15 cycles; 72°C for 1 to 5 minutes; and stored at 4°C.

[0020] In a further application embodiment, the purification in step 3) is magnetic bead purification; preferably, the purification coefficient of the magnetic bead purification is 0.8×~1.2×.

[0021] In a further application embodiment, the library in step 3) is used in a semiconductor sequencing platform.

[0022] Compared with the prior art, the present invention provides the following beneficial effects: the ligation reagent and the rapid library construction method are designed based on a semiconductor sequencing platform. When facing samples with fragmented and trace characteristics such as NIPT plasma free DNA, the rapid library construction method can shorten the library construction time while maintaining the library quality, and the number of sample transfers can be reduced during the library construction process, reducing the risk of errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart comparing the conventional library construction method and the rapid library construction method in Example 1; Figure 2 is the library fragment distribution diagram obtained by the conventional process in Example 2; Figure 3 This is the library fragment distribution diagram obtained by simplifying process 1 in Example 2; Figure 4 This is the library fragment distribution diagram obtained by simplifying process 2 in Example 2; Figure 5 This is the library fragment distribution diagram obtained by simplifying process 3 in Example 2; Figure 6 is the library fragment distribution diagram obtained by the conventional process in Example 3; Figure 7 This is the library fragment distribution diagram obtained by simplifying process 4 in Example 3; Figure 8 This is the library fragment distribution diagram obtained by simplifying process 5 in Example 3; Fig. 9 This is the library fragment distribution diagram obtained by simplifying process 6 in Example 3; Fig.10 is the library fragment distribution diagram obtained by simplifying process 7 in Example 3; Fig.11 is the library fragment distribution diagram obtained by simplifying process 8 in Example 3; Fig.12 This is the library fragment distribution diagram obtained by simplifying process 9 in Example 3. DETAILED DESCRIPTION

[0024] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0025] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0026] Example 1: A rapid library construction method for trace fragmented DNA based on a semiconductor platform.

[0027] The rapid library construction method comprises the following reagents and steps: (1) End repair Prepare end repair reaction solution (10×), the formula is shown in Table 1.

[0028]

[0029] The end repair reaction system includes 43.5 μL of extracted plasma DNA, 5 μL of the end repair reaction solution and 1.5 μL of the end repair mixed enzyme. After mixing, the end repair reaction system is incubated at room temperature for 30 minutes. The end repair mixed enzyme includes T4 DNA polymerase and T4 polynucleotide kinase, which are mixed at an enzyme activity ratio of 5:4.

[0030] (2) Fragment screening Magnetic beads are used to screen the fragments of the end-repair products to obtain enriched fragmented DNA.

[0031] The fragment screening process can be performed before or after the end repair process, and the order of the processes does not affect the quality of the library of this method.

[0032] (3) Connector connection Prepare ligation reaction solution (10×), the formula is shown in Table 2.

[0033]

[0034] The total volume of the ligation reaction system was 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 label X adapter at a concentration of 2.5 μmol / L.

[0035] The P1 adapter is a adapter suitable for semiconductor platform sequencing, and the nucleotide sequences of the forward and reverse sequence fragments thereof are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. The tag X adapter is also a adapter suitable for semiconductor platform sequencing, and the nucleotide sequences of the forward and reverse sequence fragments thereof are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively, wherein N is 8 to 10 specific bases (i.e., tags).

[0036] The reaction system was placed in a PCR instrument with the following reaction program: 25°C for 30 minutes, 70°C for 10 minutes (heat cover 85°C).

[0037] (4) PCR amplification: Prepare PCR reaction solution (2×), the formula is shown in Table 3.

[0038]

[0039] The total amount of the PCR reaction system is 40 μL, including 20 μL of the ligation product and 20 μL of the PCR reaction solution. The reaction system is placed in a PCR instrument. The reaction program is: 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; 4°C storage (heat cover 105°C).

[0040] (5) Magnetic bead purification The PCR amplification products were purified using magnetic beads to obtain the library to be sequenced. The libraries were tested separately to obtain library concentration, library fragment distribution and sequencing-related indicators to evaluate the library quality.

[0041] The schematic diagram of the process comparison between the conventional library construction method and the rapid library construction method provided in this embodiment is as follows Figure 1 shown.

[0042] Example 2: Comparison of results between conventional library construction method and three direct simplified library construction methods.

[0043] The conventional library construction process is: end repair, purification (fragment screening), adapter ligation, purification, amplification, purification. In order to verify the necessity of the purification step between the adapter ligation step and the amplification step, the same plasma free DNA was constructed according to the library construction process shown in Table 4 in this example. The difference between the processes in Table 4 lies in the ligation reaction system in step 3 and the amplification reaction system in step 4, as well as whether purification is performed between steps 3 and 4; wherein the P1 adapter and the tag X adapter both use short adapters as shown in SEQ ID No: 1 to SEQ ID No: 4.

[0044]

[0045] The obtained library was subjected to concentration determination and fragment distribution detection. The library concentration obtained by the conventional process was 62.16nmol / L, and the library fragment distribution was as follows: Figure 1 As shown; the library concentration obtained by simplified process 1 is 69.67 nmol / L, and the library fragment distribution is as follows Figure 2 As shown; the library concentration obtained by simplified process 2 is 64.21 nmol / L, and the library fragment distribution is as follows Figure 3 As shown; the library concentration obtained by simplified process 3 is 25.39 nmol / L, and the library fragment distribution is as follows Figure 4 As shown in the figure. The test results show that directly eliminating the purification step between step 3 and step 4 will lead to a large difference in the distribution of library fragments obtained by simplifying the process. Simply connecting conventional adapters and skipping purification to directly enter PCR amplification cannot achieve the construction of high-quality libraries. Even if the connection system and amplification volume are enlarged, the effect of conventional library construction cannot be achieved.

[0046] Example 3: Comparison of results between conventional library construction method and six simplified library construction methods.

[0047] In this example, the same plasma free DNA was constructed according to the conventional process and simplified processes 4 to 9 provided in Table 5. The difference between simplified processes 4 to 9 and simplified processes 1 to 3 is: (1) Adjustment of PEG, ATP and T4 DNA ligase in the adapter ligation reaction system, and adjustment of the amount of Tris-HCl, MgCl2, BSA and DNA polymerase in the amplification reaction system; (2) Changes in adapter ligation reaction conditions and post-amplification magnetic bead purification coefficients.

[0048]

[0049]

[0050] The obtained library was subjected to concentration determination and fragment distribution detection. The library concentration obtained by the conventional process was 76.57nmol / L, and the library fragment distribution was as follows: Figure 5 As shown; the library concentration obtained by simplified process 4 is 85.46 nmol / L, and the library fragment distribution is as follows Figure 6 As shown; the library concentration obtained by simplified process 5 is 148.49 nmol / L, and the library fragment distribution is as follows Figure 7 As shown; the library concentration obtained by simplified process 6 is 66.85 nmol / L, and the library fragment distribution is as follows Figure 8 As shown; the library concentration obtained by simplified process 7 is 49.53 nmol / L, and the library fragment distribution is as follows Fig. 9 As shown; the library concentration obtained by simplified process 8 is 86.49 nmol / L, and the library fragment distribution is as follows Fig.10 As shown; the library concentration obtained by simplified process 9 is 30.32 nmol / L, and the library fragment distribution is as follows Fig.11 As shown. From the distribution diagram, it can be observed that the main peaks of the libraries of simplified processes 4 to 9 are consistent with those of the libraries of the conventional process, indicating that after adjusting the ligation reaction conditions, ligation reaction system and amplification system, the dimers caused by the self-ligation of the adapters after the ligation products are mixed with the PCR system are reduced, and the specific amplification is significantly reduced, which significantly improves the fragment distribution of the library. However, since the residual adapter dimers of the simplified process are still higher than those of the normal process, the reaction system and reaction conditions of the adapter ligation and amplification reagents still need to be adjusted.

[0051] Example 4: A rapid library construction method for constructing trace amounts of fragmented DNA.

[0052] Based on the simplified process 4 in Example 3, simplified processes 10 to 19 were obtained by adjustment. In this embodiment, the same plasma free DNA was constructed according to the conventional process and simplified processes 10 to 19 in Table 6, respectively, and the library was subjected to fragment distribution detection and sequencing in the same run. The difference between simplified processes 10 to 19 lies in the concentration of the linker in the ligation reaction, the concentration of the primers in the PCR amplification reaction, and the amount of purification magnetic beads after PCR amplification.

[0053]

[0054]

[0055] The obtained library was subjected to concentration measurement and sequencing, and the library concentration, adapter dimer ratio, fetal concentration, GC content, and ratio of effective sequencing data were analyzed. The test results are shown in Table 7.

[0056]

[0057] According to the test results in Table 7, the simplified process has no significant effect on the sequencing GC content and the proportion of valid sequencing data, and the library concentration is much greater than the sequencing concentration; the library concentrations of simplified processes 10, 12 and 14 are higher, the proportion of adapter dimer molecules is lower, and the library sequencing fetal concentration, GC content and the proportion of valid sequencing data are comparable to those of the conventional process.

[0058] Example 5: Compatibility of the rapid library construction method with different DNA amounts.

[0059] In this example, libraries of different amounts of plasma free DNA were constructed and sequenced according to the conventional process in Table 6 and the simplified process 10, and the sequencing data were analyzed. The analysis results are shown in Tables 8 and 9.

[0060]

[0061]

[0062]

[0063] Tables 8 and 9 show the library concentrations and corresponding sequencing indicators of the conventional process and simplified process for different input amounts of DNA (0.9-8.1 ng) for library construction. According to the test results of Tables 8 and 9, the library concentration of the simplified process 10 is much greater than the sequencing concentration, and the test data of various sequencing indicators fluctuate within the normal range compared with the conventional process, with no significant difference, and the effect is equivalent.

[0064] The same control verification was performed on simplified process 12 and simplified process 14, and the results showed that the library quality was equivalent to that of the conventional process, indicating that the rapid library construction method and reagents of the present invention are compatible with samples with different input amounts of DNA (0.9-8.1 ng).

[0065] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A ligation reagent, characterized in that The method comprises a buffered salt solution, MgSO4, PEG, DTT, ATP, dNTP, a P1 adapter, a label X adapter and T4 DNA ligase, wherein the buffered salt solution is Tris-HCl or a phosphate solution, and the P1 adapter and the label X adapter are both short adapters of 20 to 35 nt.

2. The linking reagent according to claim 1, characterized in that The P1 linker is composed of two forward and reverse sequence fragments, and the nucleotide sequences thereof are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively.

3. The linking reagent according to claim 1, characterized in that The tag X linker is composed of two forward and reverse sequence fragments, and the nucleotide sequences thereof are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively.

4. The linking reagent according to claim 1, characterized in that The final concentrations of the components of the ligation reagent are: buffer saline 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 linker 0.04-0.06 μmol / L, tag X linker 0.04-0.06 μmol / L and T4 DNA ligase 1-2.5 U.

5. A method for rapid library construction of trace fragmented DNA, characterized in that: Includes steps: 1) Obtain sample DNA and perform end repair and / or fragment screening; 2) using the ligation reagent according to any one of claims 1 to 4 to perform a linker ligation reaction on the sample DNA to obtain a ligation product; 3) Perform amplification reaction on the ligation product and amplification reagent, purify, and obtain the library.

6. The rapid library construction method according to claim 5, characterized in that: Step 2) The reaction conditions of the linker ligation reaction are: 20-25°C for 20-30 minutes, and 60-75°C for 5-20 minutes.

7. The rapid library construction method according to claim 5, characterized in that: Step 3) The amplification reagents for the amplification reaction include a buffered saline solution, MgCl2, KCl, BSA, dNTP, glycerol, DNA polymerase, and a mixture of amplification primers, wherein the buffered saline solution is Tris-SO4 or Tris-HCl.

8. The rapid library construction method according to claim 5, characterized in that: The reaction conditions of the amplification reaction are 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 stored at 4°C.

9. The rapid library construction method according to claim 5, characterized in that: Step 3) The purification is performed by magnetic bead purification; preferably, the purification coefficient of the magnetic bead purification is 0.8×~1.2×.

10. The rapid library construction method according to claim 5, characterized in that: Step 3) The library is used in a semiconductor sequencing platform.

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