DOUBLE-STRANDED NUCLEIC ACID MOLECULES AND METHOD FOR REMOVING GLASS ADAPTOR IN DNA LIBRARY BY MEANS OF SAME

Abstract

The present invention relates to a method for removing free adapters in a DNA library using a double-stranded nucleic acid molecule and a restriction enzyme, and more specifically, to a method for removing free adapters in a DNA library for next generation sequencing (NGS) using a double-stranded nucleic acid molecule including a type IIs restriction enzyme recognition site and a complementary sequence thereof, and a type IIs restriction enzyme.

Claims

1. A double-stranded nucleic acid molecule comprising: a sense strand containing a type IIs restriction enzyme recognition site and a random nucleotide sequence of a length of 3 to 30 nucleotides (nt); and an anti-sense strand containing a complementary sequence to the sense strand and a 3′-end A tail.

2. The double-stranded nucleic acid molecule of claim 1, wherein a random nucleotide sequence of a length of 1 to 10 nt is further contained in the 3′-end of the sense strand or the 5′-end of the anti-sense strand.

3. The double-stranded nucleic acid molecule of claim 2, wherein the random nucleotide sequence further contained in the 3′-end of the sense strand consists of only cytosine (C), guanine (G), or a combination thereof.

4. The double-stranded nucleic acid molecule of claim 2, wherein a base sequence of the random nucleotide further contained in the 5′-end of the anti-sense strand consists of only thymine (T), cytosine (C), guanine (G) or a combination thereof.

5. The double-stranded nucleic acid molecule of claim 1, wherein the 3′-end of the sense strand and the 5′-end of the anti-sense strand are connected to each other by a loop to have a hairpin structure.

6. The double-stranded nucleic acid molecule of claim 1, wherein the type IIs restriction enzyme is selected from the group consisting of MmeI, FokI, Alw26I, BbvI, BsrI, EarI, HphI, MboI, SfaNI, AlwI, BsaI, BbsI, BbuI, BsmAI, BsmI, BspMI, Esp3I, HgaI, MboII, PleI, SfaNi, Mn1I, CspCI, AloI, PpiI, PsrI, BplI, FalI, Bsp24I, BsaXI, HaeIV, CjeI, CjePI, Hin4I, BaeI, AlfI, BcgI, Bs1FI, and Tth111I.

7. The double-stranded nucleic acid molecule of claim 1, wherein the sense strand comprises a type IIs restriction enzyme recognition site and a random nucleotide of a length of 6 to 10 nt, and the base sequence of the random nucleotide is constituted so that a melting temperature (Tm) value of the double-stranded nucleic acid molecule is 20° C. or higher.

8. The double-stranded nucleic acid molecule of claim 1, wherein the sense strand consists of a base sequence defined by SEQ ID NO: 1 and the anti-sense strand consists of a base sequence defined by SEQ ID NO: 2.

9. The double-stranded nucleic acid molecule of claim 5, wherein the double-stranded nucleic acid molecule consists of a base sequence defined by SEQ ID NO: 3.

10. A composition comprising the double-stranded nucleic acid molecule according to claim 1, DNA ligase, and a type IIs restriction enzyme.

11. The composition of claim 10, wherein the DNA ligase is T4 ligase.

12. The composition of claim 10, wherein the composition is used for improving sample detection ability in an indexed DNA library in next generation sequencing (NGS).

13. The composition of claim 10, wherein the composition is used for removing free adapters which are not ligated to an insert in the process of constructing the indexed DNA library of next generation sequencing.

14. A kit for removing free adapters in an indexed DNA library of next generation sequencing, comprising the double-stranded nucleic acid molecule according to claim 1, DNA ligase and a type IIs restriction enzyme.

15. A method for removing free adapters in a DNA library for next generation sequencing comprising steps of: (a) treating and reacting the double-stranded nucleic acid molecule according to any one of claims 1 to 8 and DNA ligase in a DNA library for next generation sequencing constructed by linking adapters for next generation sequencing to both ends of a double-stranded DNA fragment of a genomic DNA to be analyzed; and (b) treating the resulting reaction products with a type IIs restriction enzyme.

16. The method of claim 15, wherein the adapter comprises an index sequence, a random barcode sequence or both thereof.

17. The method of claim 15, wherein the type IIs restriction enzyme is selected from the group consisting of MmeI, FokI, Alw26I, BbvI, BsrI, EarI, HphI, MboI, SfaNI, AlwI, BsaI, BbsI, BbuI, BsmAI, BsmI, BspMI, Esp3I, HgaI, MboII, PleI, SfaNi, MnII, CspCI, AloI, PpiI, PsrI, Bp1I, FalI, Bsp24I, BsaXI, HaeIV, CjeI, CjePI, Hin4I, BaeI, AlfI, BcgI, Bs1FI, and Tth111I.

18. The method of claim 15, further comprising: washing with a buffer or performing bead clean-up after step (b).

19. Use of the double-stranded nucleic acid molecule according to claim 1, DNA ligase and a type IIs restriction enzyme for preparing a agent for removing free adapters in an indexed DNA library for next generation sequencing.

Description

DESCRIPTION OF DRAWINGS

[0091] FIG. 1 is a diagram schematically illustrating the definition of an indexed DNA library.

[0092] FIG. 2 is a diagram schematically illustrating a concept of index hopping.

[0093] FIG. 3 is a diagram schematically illustrating a concept of UMI mix.

[0094] FIG. 4 illustrates four regions of a free adapter that may act as a primer in a PCR process during a process of constructing a DNA library.

[0095] FIG. 5 is a diagram schematically illustrating an operation method of a double-stranded nucleic acid molecule according to the present invention.

[0096] FIGS. 6A and 6B are diagrams schematically illustrating examples of the double-stranded nucleic acid molecule according to the present invention.

[0097] FIG. 7 is a diagram for confirming a result without performing bead clean-up in order to confirm whether an adapter is fragmented by treatment with a restriction enzyme MmeI.

[0098] FIG. 8 is a diagram schematically illustrating which region each band represents in the adapter in the result of FIG. 7.

[0099] FIG. 9 is a result of confirming whether non-specific decomposition appears after treatment with MmeI to a PCR product in order to confirm whether non-specific cutting occurs by treatment with a restriction enzyme.

[0100] FIG. 10 is a diagram illustrating an experiment method for confirming how much a UMI bias is caused by free adapters in an NGS sequencing process of a sample DNA and whether such a UMI bias may be removed by the double-stranded nucleic acid molecule according to the present invention and the restriction enzyme.

[0101] FIG. 11 is a diagram illustrating an experiment method for confirming how much a UMI bias is caused by free adapters in an NGS sequencing process of a sample DNA and whether such a UMI bias may be removed by the double-stranded nucleic acid molecule according to the present invention and the restriction enzyme.

MODES FOR THE INVENTION

[0102] Hereinafter, the present invention will be described in detail by the following Examples. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

Experiment Method

1. Preparation of Double-Stranded Nucleic Acid Molecule and Nucleic Acid Molecule Having Hairpin Structure

[0103] A sense strand of SEQ ID NO: 1 in which a random base sequence TTGTGC (SEQ ID NO: 20) providing a structure in which a restriction enzyme may operate was bound to a 3′ end of a recognition site sequence GTCGGA (SEQ ID NO: 25) of a type IIs restriction enzyme Mme1 and an anti-sense strand of SEQ ID NO: 2 in which TT was additionally bound to a 5′ end of a base sequence complementary to the sense strand and A was additionally added to the 3′ end were prepared. The sense strand and the anti-sense strand were mixed with the same moles, denatured at 50° C., and then cooled down slowly at room temperature to form a double-stranded nucleic acid molecule. The double-stranded nucleic acid molecule was referred to as cutting linker_FR (FIG. 6A).

[0104] In addition, a single-stranded nucleic acid molecule consisting of a base sequence of SEQ ID NO: 3 was prepared, denatured at 50° C., and then cooled down slowly, and induced to form an intra structure to form a nucleic acid molecule having a hairpin structure. The nucleic acid molecule having the hairpin structure was referred to as cutting linker_hairpin (FIG. 6B).

2. Ligation of Free Adapter and Double-Stranded Nucleic Acid Molecule

[0105] The adapter was treated with the prepared double-stranded nucleic acid molecule at a ratio of 1:10, and was treated with T4 DNA ligase to induce ligation thereof.

[0106] Specifically, experimental materials listed in Table 2 below were mixed and incubated at 25° C. for 2 hours, and then heat-inactivated at 65° C. for 20 minutes to terminate the reaction.

TABLE-US-00002 TABLE 2 Product Reagent Volume (ul) information Adaptor (3 uM) 2.5 IDT order production Cutting linker_FR or 25 IDT order production Cutting linker_hairpin T4 ligase 1 NEB (Cat.M0202) T4 ligase buffer (10X) 4 TDW 7.5 Total 40

3. Treatment of Restriction Enzyme

[0107] A reaction mixture reacted according to Experimental Method 2 was treated with a type IIs restriction enzyme, MmeI to induce a restriction enzyme reaction.

[0108] Specifically, experimental materials listed in Table 3 below were mixed and incubated at 37° C. for 1 hour, and then heat-inactivated at 65° C. for 20 minutes to terminate the reaction.

TABLE-US-00003 TABLE 3 Product Reagent Volume (ul) information Reaction mixture 40 (Experimental Method 2) Mmel (2 units/ul) 5 NEB (Cat.RO637) SAM (1.6 mM) 1.6 Cutsmart buffer (10X) 1 TDW 2.4 Total 50

4. Bead Clean-Up

[0109] By using 2.0× accubead (HiAccuBead (AccuGene (Cat. ACN01.50)), clean-up was performed according to manufacturer's instructions.

Experimental Results

1. Confirmation of Fragmentation of Free Adapter by Cutting Linker_FR or Cutting Linker_Hairpin, and Restriction Enzyme

[0110] It was confirmed whether free adapters in a DNA library may be removed by the double-stranded nucleic acid molecule prepared in the experimental method, T4 DNA ligase and a type IIs restriction enzyme MmeI. Specifically, the adapter, the double-stranded nucleic acid molecule according to the present invention, the DNA ligase, and Mme1 were mixed, respectively, to induce a reaction through the process described in the experimental method, and then the result was confirmed through tapestation (Agilent).

[0111] First, in order to confirm whether the adapter was fragmented by treatment of Mme1, the result was confirmed without performing bead clean-up, and 2 ng of each sample was loaded.

[0112] The results thereof were illustrated in FIGS. 7 and 8.

[0113] Referring to FIGS. 7 and 8, adapter fragments estimated to be cut by the double-stranded nucleic acid molecule according to the present invention and the restriction enzyme MmeI were confirmed. As shown in lanes 3 and 4 of FIG. 7, when the double-stranded nucleic acid molecule according to the present invention was ligated to the adapter, it was confirmed that a band of {circumflex over (2)} was shifted to form a band {circumflex over (1)}, and when Mme1 was treated thereto, as shown in lanes 1 and 2, the band {circumflex over (1)}disappeared and a cutting fragment {circumflex over (3)} was confirmed (see FIG. 8). A band of {circumflex over (3)} is a product derived by cutting and fragmenting the adapter and may be removed through a clean-up process.

[0114] Finally, in order to confirm whether non-specific cutting by Mmel occurred, it was confirmed whether non-specific decomposition appeared after Mmel was treated in a PCR product (no Mmel recognition site). As a result, as illustrated in FIG. 9, no fragmentation was observed in no Mmel recognition site.

2. Removal of UMI Bias Through Cutting of Free Adapter in NGS Sequencing Process

[0115] Through the above-described experimental results, it was confirmed that cutting linker_FR and cutting linker_hairpin provided by the present invention may efficiently cut the free adapter.

[0116] Therefore, in a subsequent experiment, how much the UMI bias was caused by the free adapter in the NGS sequencing process of the sample DNA, and whether such a UMI bias may be removed by the cutting linker_FR or cutting linker_hairpin and the restriction enzyme.

[0117] An experimental procedure was as follows (see FIG. 10):

[0118] 1. It was confirmed how many UMI biases have been induced in an adapter in a process of fixing an UMI base sequence to ATGCATG (SEQ ID NO: 26) and spiking in an adapter having NNNCNNN (SEQ ID NO: 27) therein to actually construct a DNA library.

[0119] 2. It was confirmed whether the UMI biases caused by the free adapter were removed by each of two types of cutting linkers (FR and hairpin).

[0120] 3. For the reproducibility of the results, two independent experiments were performed, and the mean and STDEV were expressed in graphs.

[0121] In the process of constructing the DNA library, an adapter with ATGCATG (SEQ ID NO: 26) was ligated to an input DNA, and when there was no UMI bias, all DNA libraries need to have a UMI type ATGCATG (SEQ ID NO: 26). On the contrary, when there was a UMI bias, the DNA library will have a random UMI type other than ATGCATG (SEQ ID NO: 26).

[0122] Based on this fact, % of reads with UMI of the sequence ATGCATG (SEQ ID NO: 26) in each experimental group was calculated as a result and illustrated in Table 4 below and FIG. 11.

TABLE-US-00004 TABLE 4 Mock_free Mock_free Mock_free Mock only adapter adapter_linker FR adapter_linker FR AVER STDEV AVER STDEV AVER STDEV AVER STDEV Mock UMI 96.4 1.07 70.18 4.95 97.74 0.08 95.25 0.28 read (%)

[0123] In Table 4 and FIG. 11, in the case of Mock only, after sequencing a library by ligating only an adapter having a fixed UMI (ATGCATG) (SEQ ID NO: 26), % of reads with the fixed UMI among all reads was obtained. In the case of Mock_free adapter, the fixed UMI was inactivated at 65° C. after ligation was induced, and then a free UMI (NNNANNN) (SEQ ID NO: 28) adapter was spiked in. At this time, the Mock_free adapter did not participate in the ligation process to a free UMI adapter insert, and remained as a free adapter corresponding to the UMI bias. At this time, after finally constructing and sequencing the library, when mock UMI % was confirmed in the same manner, it can be confirmed that the mock UMI % has dropped to 70.18%. These results show that UMI may be introduced up to 30% by functioning as a primer in the PCR process rather than ligation.

[0124] At this time, using the double-stranded nucleic acid molecule provided by the present invention and the restriction enzyme, the free adapter was induced to be ligated and then spiked in. If the free adapter was not removed by the double-stranded nucleic acid molecule and the restriction enzyme, according to the experimental results, it was confirmed that the proportion of reads with mock UMI should have been shown as about 70%, but actually shown as 97.74 and 95.25%, which was recovered back to a control level in which mock UMI was added (Mock_free adapter_linker_FR, Mock_free adapter_linker_hairpin).

[0125] Through the results, it was confirmed that most of the free adapters have been removed by the double-stranded nucleic acid molecule provided by the present invention and the restriction enzyme, and most of the UMI biases may be removed by using the double-stranded nucleic acid molecule of the present invention.

INDUSTRIAL APPLICABILITY

[0126] According to the double-stranded nucleic acid molecules, the composition, and the method provided by the present invention, only the free adapters in an indexed DNA library for next generation sequencing may be selectively removed to prevent problems of index hopping, UMI mix, and the like caused by the free adapters during the next generation sequencing. Therefore, industrial applicability is very high.