NUCLEIC ACID SEQUENCING METHOD

Abstract

The present invention provides a method for sequencing a nucleic acid using an immersion reaction protocol. The immersion reaction protocol comprises sequentially immersing a solid support having nucleic acid molecules immobilized thereon in different reaction containers to realize nucleic acid sequencing.

Claims

1-22. (canceled)

23. A method for sequencing a nucleic acid using a contact reaction protocol, the contact reaction protocol comprising the following steps: a) providing a first batch of solid supports having nucleic acid molecules immobilized thereon, b) contacting the nucleic acid molecules immobilized on the solid supports with one or more reaction solutions to generate a signal representing one or more nucleotides that bind to the nucleic acid molecules on the solid supports, wherein each of the one or more reaction solutions is placed in a separate reaction container, wherein, after removing the contact between the solid supports and the reaction solutions, contacting the solid supports with one or more washing solutions to remove the reaction solutions remaining on the solid supports, c) detecting the signal on the solid supports, and f) optionally, repeating the steps b) to c), wherein the solid supports are unencapsulated, wherein the first batch of solid supports consists of one or more solid supports.

24. The method according to claim 23, wherein the method further comprises the following steps after step c) and before step f): d) contacting the solid supports with one or more regeneration solutions each placed in a separate reaction container to eliminate the signal on the solid supports, and e) removing the contact between the solid supports and the regeneration solutions, and contacting the solid supports with one or more washing solutions to remove the regeneration solutions remaining on the solid supports, and wherein in step f), optionally, repeating the steps b) to e) or the steps b) to c).

25. The method according to claim 23, wherein the solid supports can be selected from beads, chips, glasses, sensors, electrodes, or silicon wafers.

26. The method according to claim 23, which further comprises: after contacting the first batch of solid supports with the one or more reaction solutions of the step b) and before the end of the step c) or e), contacting a second batch of solid supports having nucleic acid molecules immobilized thereon with the one or more reaction solutions of the step b), and then operating the second batch of solid supports according to the steps b) to f); optionally iteratively repeating this process for N−1 times, wherein, 1≤N≤t.sub.cycle/t.sub.speed-limit, and N is an integer value, wherein, t.sub.cycle is a total time from the step b) to the step c) or from the step b) to the step e), and t.sub.speed-limit is the time of the longest procedure in each contacting and washing in the step b), detecting in the step c), contacting in the step d), and washing in the step e).

27. The method according to claim 26, wherein the time interval, tN, between contacting a N.sup.th batch of solid supports with the one or more reaction solutions of the step b) and contacting a (N+1).sup.th batch of solid supports with the one or more reaction solutions of the step b) meets the following conditions: $t_N\ge t_{\mathrm{speed}-\mathrm{limit}},\mathrm{and}\underset{N=1}{\overset{N}{.Math.}}{t}_N\le t_{\mathrm{cycle}}-t_{\mathrm{speed}-\mathrm{limit}}.$

28. The method according to claim 27, wherein each batch of solid supports from the (N+1).sup.th batch of solid supports consists of one or more solid supports.

29. The method according to claim 23, wherein the signal comprises a fluorescence signal.

30. The method according to claim 23, wherein the sequencing comprises sequencing-by-ligation, sequencing-by-synthesis or sequencing by combinatorial probe-anchor synthesis (cPAS).

31. The method according to claim 30, wherein the sequencing is sequencing-by-ligation, and wherein the one or more reaction solutions of the step b) comprise a solution containing anchor probes, labeled sequencing probes, ligase, or a mixture thereof, provided that the solid supports are in contact with each of the anchor probes, the labeled sequencing probes and the ligase, and wherein the signal on the solid supports is generated by the labeled sequencing probe that complementarily bind to the nucleic acid molecules on the solid supports, the labeled sequencing probes are linked via the ligase to the anchor probes that complementarily bind to the same nucleic acid molecules, optionally, the regeneration solutions of the step d) comprise reagents that are capable of removing labels from the labeled sequencing probes or reagents that are capable of removing the labeled sequencing probes from the nucleic acid molecule.

32. The method according to claim 30, wherein the sequencing is sequencing-by-ligation, further comprising: before the step b), contacting the solid supports having the nucleic acid molecules immobilized thereon with the anchor probes, so that the anchor probes hybridize to the nucleic acid molecule on the solid supports.

33. The method according to claim 32, wherein the one or more reaction solutions of the step b) comprise a solution containing the labeled sequencing probes, the ligase, or a mixture thereof, provided that the solid supports are in contact with each of the labeled sequencing probes and the ligase, and wherein the signal on the solid supports is generated by the labeled sequencing probes that complementarily bind to the nucleic acid molecules on the solid supports, the labeled sequencing probes are linked via the ligase to the anchor probes that complementarily bind to the same nucleic acid molecule, optionally, the regeneration solutions of the step d) comprise reagents that are capable of removing labels from the labeled sequencing probes or reagents that are capable of removing the labeled sequencing probes from the nucleic acid molecule.

34. The method according to claim 30, wherein the sequencing is sequencing-by-synthesis, and wherein the one or more reaction solutions of the step b) comprise a solution containing a polymerase, sequencing primers, a labeled nucleotide, or a mixture thereof, provided that the solid supports are in contact with each of the polymerase, the sequencing primers, and the labeled nucleotide, and wherein the signal on the solid supports is generated by the labeled nucleotide that complementarily binds to the nucleic acid molecules on the solid supports, the labeled nucleotide is polymerized to a 3′ end of the sequencing primers via the polymerase using the nucleic acid molecule on the solid supports as a template, optionally, the regeneration solutions of the step d) contain reagents capable of removing a label from the labeled nucleotide, optionally, the labeled nucleotide further comprises a 3′ blocking group.

35. The method according to claim 30, wherein the sequencing is sequencing-by-synthesis, and wherein the nucleic acid molecules are immobilized on the solid supports by hybridization of the sequencing primers immobilized on the solid supports.

36. The method according to claim 30, wherein the sequencing is sequencing-by-synthesis, further comprising: before the step b), contacting the solid supports having the nucleic acid molecules immobilized thereon with the sequencing primers, so that the sequencing primers hybridize to the nucleic acid molecules on the solid supports.

37. The method according to claim 35, wherein the one or more reaction solutions of the step b) comprise a solution containing a polymerase, a labeled nucleotide, or a mixture thereof, provided that the solid supports are in contact with each of the polymerase and the labeled nucleotide, and wherein the signal on the solid supports is generated by the labeled nucleotide that complementarily binds to the nucleic acid molecule on the solid supports, the labeled nucleotide is polymerized to a 3′ end of the sequencing primers via the polymerase using the nucleic acid molecule on the solid supports as a template, optionally, the regeneration solutions of the step d) comprise reagents capable of removing a label from the labeled nucleotide, optionally, the labeled nucleotide further comprises a 3′ blocking group.

38. The method according to claim 24, further comprising: adding humectants to the one or more reaction solutions of the step b), the regeneration solutions of the step d) and/or the washing solutions of the step e), and/or adding reagents to the one or more reaction solutions in the step b), the regeneration solutions in the step d) and/or the washing solutions in the step e) so as to retain the reaction solutions and/or the washing solutions remaining on the solid supports thereon when removing the contact between the solid supports and the reaction solutions and/or the washing solutions.

39. The method according to claim 23, further comprising: optimizing a motion speed of the solid supports between the reaction containers so as to minimize the time that the solid supports are exposed to air.

40. The method according to claim 23, further comprising: using a temperature control device to adjust the temperature in each of the reaction containers.

41. The method according to claim 24, wherein during optionally repeating the steps b) to c) or the steps b) to e), the one or more reaction solutions of the step b) and/or the regeneration solutions of the step d) are replaced or not upon each repeating, optionally, when the one or more reaction solutions in the step b) have a reaction temperature of below about 55° C., the time interval for replacing the one or more reaction solutions in the step b) is less than about 8 hours, optionally, when the one or more reaction solutions in the step b) have a reaction temperature of below about 45° C., the time interval for replacing the one or more reaction solutions in the step b) is less than about 24 hours.

42. An apparatus for sequencing a nucleic acid molecule by using the contact reaction protocol according to claim 23, the apparatus comprising: a) one or more reaction containers, each containing one or more reaction solutions for contacting a nucleic acid molecule to generate a signal representing one or more nucleotides that bind to the nucleic acid molecules; b) one or more reaction containers containing one or more washing reagents; c) one or more reaction containers containing one or more reaction solutions for eliminating the signal from solid supports; d) device for detecting the signal; and e) temperature control device for controlling the temperature of the reaction container in above a) to c).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0105] FIG. 1 shows a flowchart (showing a sequencing cycle) of two exemplary embodiments of the immersion reaction protocol of the present invention, one of which uses a protecting reagent that protects the nucleic acid and label during the detection.

[0106] FIG. 2 shows a comparison of sequencing data between the immersion reaction protocol of the present invention and BGISEQ-500 SE50.

[0107] FIG. 3 shows sequencing results of the improved immersion reaction protocol of the present invention.

[0108] FIG. 4 shows the improvement of the drying of the upper end of the chip by adding a certain amount of surfactant to the polymerization reagent. The left side of the figure shows the lower end of the chip, and the right side of the figure shows the upper end of the chip. Condition 1: adding 5% to 10% glycerol (v/v) in the first reaction solution+quickly moving chip; Condition 2: adding 10% glycerol (v/v) in the first reaction solution+quickly moving chip+adding 0.05% to 1% Tween-20 (v/v) in the first reaction solution.

EXAMPLES

[0109] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that are commercially available.

Example 1: Nucleic Acid Sequencing with Immersion Reaction Protocol

[0110] According to the manufacturer's instructions, a MGIeasy™ DNA library preparation kit (Shenzhen Huada Zhizao Technology Co., Ltd.) was used to extract DNA from an E. coli standard strain as raw materials to prepare a library for sequencing, which was loaded on a sequencing chip. According to the manufacturer's instructions, the reagents in BGISEQ-500 high-throughput kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., article number: PF-UM-PEV30) were used, and the flowchart in FIG. 1A (an exemplary embodiment of the immersion reaction protocol described herein) was followed to perform 10 cycles of sequencing of the nucleic acid molecule on the obtained chip, in which 3 chips were simultaneously immersed.

[0111] The obtained sequencing data were uploaded to BGI Online (see https://www.bgionline.cn/) for analysis, or the software of the BGISEQ-500 sequencer was used to analyze the quality of the sequencing data and a visual report was output, as shown in FIG. 2.

[0112] The MGIEasy™ DNA library preparation kit was used to extract DNA from an E. coli standard strain as raw materials to prepare a library for sequencing, which was loaded on a sequencing chip, and the sequencing reagents in the BGISEQ-500 high-throughput kit (SE50 V3.0) were used, to perform a sequencing reaction as control on a BGISEQ-500 sequencer.

[0113] In FIG. 2, Q30 referred to the occurrence of one error in 1000 bases as sequenced, in which the larger the number was, the better the sequencing quality was; ESR was a sequencing indicator of the BGISEQ-500 sequencer and referred to the effective DNA nanosphere ratio, in which the larger the number was, the better the sequencing quality was; MappingRate referred to the comparison rate of the sequencing data relative to the reference genome, in which the larger the number was, the better the sequencing quality was; BarcodeSplitRate referred to the barcode split rate, in which the larger the number was, the better the sequencing quality was.

[0114] As compared with the control group of using the BGISEQ-500 sequencer to sequence the same chip, the quality of the sequencing data obtained by the immersion reaction protocol of the present invention was basically similar to the quality of those obtained by the BGISEQ-500 sequencer (the difference was within 5%, and the BarcodeSplitRate was better than that of the BGISEQ-500 sequencer).

Example 2: Comparison of Different Immersion Reaction Protocols

[0115] Using an experimental procedure similar to Example 1, the chip was subjected to 15 sequencing cycles using different immersion reaction protocols.

[0116] Firstly, the chip was sequenced using the following immersion protocol: [0117] Immersion protocol 1: the same experimental procedure as in Example 1 was performed on the chip for 15 sequencing cycles; [0118] Immersion protocol 2: on the basis of Immersion protocol 1, 5-10% glycerol (v/v) was added to the polymerization reaction reagent and the chip was quickly moved at a speed of 20 mm/s during the experiment;

[0119] As a control, the chip was sequenced using a BGISEQ-500 sequencer.

[0120] The decrease rate of the fluorescence signal intensity detected in the 15th sequencing cycle relative to the fluorescence signal intensity detected in the 5th sequencing cycle was calculated For these three kinds of sequencing procedures, respectively. The results were shown in FIG. 3, where A represented the fluorescence signal for detecting base A; C represented the fluorescence signal for detecting base C; G represented the fluorescence signal for detecting base G; T represented the fluorescence signal for detecting base T; AVG represented the average value of the fluorescence signals for detecting the four bases.

[0121] We found that after adding glycerol and increasing the chip lifting speed to 20 mm/s, the decrease rate of the fluorescence signal of the 15th cycle relative to that of the 5th cycle dropped to about 10%, which showed a noticeable improvement in comparison with the decrease rate of more than 20% for the Immersion protocol 1 (see FIG. 3).

[0122] In addition, based on 5% to 10% glycerol (v/v)+quickly moving chip (Condition 1), 0.05% to 1% Tween-20 (v/v, Condition 2) was further added to the sequencing reagent. Using the same experimental procedure as in Example 1, after performing a plurality of sequencing cycles on the chip, the analysis software of the BGISEQ-500 sequencer was used to obtain a heat map of the sequencing signal values on the chip. The results were shown in FIG. 4. It was found that compared with the chip sequenced under Condition 1, the drying phenomenon at the upper end of the chip sequenced under Condition 2 was significantly alleviated, and the data qualities at the upper end of the chip and the other regions of the chip were not significantly different (if the surfactant was not added, the signal value at the upper end of the chip decreased significantly after 30 cycles; while after a certain amount of Tween-20 was added, the signal value at the upper end of the chip was not significantly different from those at other regions, which indicated the uniformity was better) (as shown in FIG. 4).

Example 3: Nested Batch Processing of the Immersion Reaction Protocol of the Present Invention

[0123] According to an embodiment of the immersion reaction protocol of the present invention, a nested batch processing of chips was applied in the experimental procedure of Example 1. When the fluorescence labeling detection was performed after the polymerization reaction of the three chips of the first group was completed, the other three chips of the second group were immersed in a sequencing reaction reagent (i.e., a first reaction solution) to start the sequencing reaction. In the experiment, the polymerization reagent was replaced every 10 sequencing cycles, and the regeneration reagent was replaced every 20 sequencing cycles. By comparison, the cost was 25% of the same kind of sequencing of BGISEQ-500 sequencer. Finally, a total of 60 complete sequencing cycles for all of the two groups of chips were completed in 2 hours. In contrast, on the current BGISEQ-500 sequencer or illumina HiSeq-2500 platform, it took more than 10 hours to finish 60 complete sequencing cycles. Therefore, in the same sequencing time, the sequencing throughput of the present invention was more than five times that of the BGISEQ-500 sequencer or HiSeq-2500 platform, and the throughput could be further increased and the cost could be further reduced by increasing the number of batches for the nested batch processing.

Example 4: Reuse of Sequencing Reagents Did not Affect Sequencing Quality

[0124] The sequencing reagents that had been used in Example 1 were again used to perform the sequencing procedure described in Example 1, and the sequencing data were analyzed as described in Example 1 and compared with the unused reagents. The comparison results were shown in Table 1 below, showing that the used reagents could perform sequencing normally.

TABLE-US-00001 TABLE 1 Comparison of the used and unused reagents: Sequencing reagents after Unused sequencing Conditions 60 sequencing cycles reagents Number of cycles 50 50 Total reads number (M) 185.14 185.98 Reads number on 184.17 184.75 alignment (M) Q30% 92.17 91.35 Effective reads rate, % 84.39 84.33 Comparison rate, % 99.47 99.34 Sequencing error rate, % 0.1 0.17

Example 5: Study on the Stability of Reagents Used in the Immersion Reaction Protocol of the Present Invention

[0125] The sequencing reagents (the sequencing reagents referred to Example 1, including polymerization reagent, washing reagent 1, washing reagent 2, and regeneration reagent) were separately placed in 45° C. and 55° C. water baths and processed for different times, including 4 hours, 8 hours, and 24 hours.

[0126] The heat-treated reagents were then used to perform nucleic acid sequencing on a BGISEQ-500 sequencer, and the qualities of the sequencing data were compared by using the software provided by the BGISEQ-500 sequencer to determine the reagent stability.

[0127] The analysis results of the sequencing data qualities were shown in Table 2. It could be seen that the sequencing reagents were still stable after being treated at 55° C. for 4 hours or at 45° C. for 8 hours, and the sequencing qualities were essentially similar to that of the control group of using the reagents that were not subjected to the heat treatment. Therefore, based on this result, the replacement cycle of the sequencing reagents in the immersion reaction protocol could be determined to be 4 hours at 55° C., or 10 hours at 45° C.

TABLE-US-00002 TABLE 2 Stability analysis of sequencing reagents in immersion reaction protocol: Mapping Sequencing Q30% ESR % Rate % error rate, % Sequencing reagents heat-treated at 45° C. Control 92 78.6 98.4 0.41 Heat-treatment, 4 h 89.2 78 90.2 0.59 Heat-treatment, 8 h 90.1 92.3 98.3 0.43 Heat-treatment, 24 h 85.5 80.1 96.8 0.68 Sequencing reagents heat-treated at 55° C. Control 92.2 78.4 97.8 0.41 Heat-treatment, 4 h 92.1 79.6 95.2 0.39 Heat-treatment, 8 h 89.8 77.5 91.5 0.66 Heat-treatment, 24 h 79.7 79.2 79.3 1.39